| 1. |
- Frost, Stefan, et al.
(författare)
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Autoproteolysis and Intramolecular Dissociation of Yersinia YscU Precedes Secretion of Its C-Terminal Polypeptide YscU CC
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:11
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Tidskriftsartikel (refereegranskat)abstract
- Type III secretion system mediated secretion and translocation of Yop-effector proteins across the eukaryotic target cell membrane by pathogenic Yersinia is highly organized and is dependent on a switching event from secretion of early structural substrates to late effector substrates (Yops). Substrate switching can be mimicked in vitro by modulating the calcium levels in the growth medium. YscU that is essential for regulation of this switch undergoes autoproteolysis at a conserved N↑PTH motif, resulting in a 10 kDa C-terminal polypeptide fragment denoted YscUCC. Here we show that depletion of calcium induces intramolecular dissociation of YscUCC from YscU followed by secretion of the YscUCC polypeptide. Thus, YscUCC behaved in vivo as a Yop protein with respect to secretion properties. Further, destabilized yscU mutants displayed increased rates of dissociation of YscUCC in vitro resulting in enhanced Yop secretion in vivo at 30°C relative to the wild-type strain.These findings provide strong support to the relevance of YscUCC dissociation for Yop secretion. We propose that YscUCC orchestrates a block in the secretion channel that is eliminated by calcium depletion. Further, the striking homology between different members of the YscU/FlhB family suggests that this protein family possess regulatory functions also in other bacteria using comparable mechanisms.
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| 2. |
- Ho, Oanh, 1986-, et al.
(författare)
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Characterization of the Ruler Protein Interaction Interface on the Substrate Specificity SwitchProtein in the Yersinia Type III Secretion System
- 2017
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 292:8, s. 3299-3311
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Forskningsöversikt (refereegranskat)abstract
- Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. In Yersinia the switch to secretion of effector proteins is induced first after that intimate contact between the bacterium and its eukaryotic targetcell has been established and the T3SS proteins YscP and YscU are playing a central role in thisprocess. Here we identify the molecular details of the YscP binding site on YscU by means o fnuclear magnetic resonance (NMR) spectroscopy. The binding interface is centeredon the C-terminal domain of YscU. Disruptingthe YscU/YscP interaction by introducing point mutations at the interaction interface significantly reduced the secretion of effector proteins and HeLa cell cytotoxicity. Interestingly, the bindingof YscP to the slowly self-cleaving YscU variantP264A conferred significant protection againstauto-proteolysis. The YscP mediated inhibition of YscU auto-proteolysis suggest that the cleavage event may act as a timing switch in the regulationof early vs. late T3SS substrates. We also show that YscUC binds to the inner-rod protein YscI with a Kd of 3.8 μM and with one-to-one stoichiometry. The significant similarity between different members of the YscU, YscP, YscI families suggests that the protein-protein interactions discussed in this study are alsorelevant for other T3SS-containing Gram-negative bacteria.
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| 3. |
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| 4. |
- Weise, Christoph F, et al.
(författare)
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Negatively charged lipid membranes promote a disorder-order transition in the Yersinia YscU protein
- 2014
-
Ingår i: Biophysical Journal. - : Cell Press. - 0006-3495 .- 1542-0086. ; 107:8, s. 1950-1961
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Tidskriftsartikel (refereegranskat)abstract
- The inner membrane of Gram-negative bacteria is negatively charged, rendering positively charged cytoplasmic proteins in close proximity likely candidates for protein-membrane interactions. YscU is a Yersinia pseudotuberculosis type III secretion system protein crucial for bacterial pathogenesis. The protein contains a highly conserved positively charged linker sequence that separates membrane-spanning and cytoplasmic (YscUC) domains. Although disordered in solution, inspection of the primary sequence of the linker reveals that positively charged residues are separated with a typical helical periodicity. Here, we demonstrate that the linker sequence of YscU undergoes a largely electrostatically driven coil-to-helix transition upon binding to negatively charged membrane interfaces. Using membrane-mimicking sodium dodecyl sulfate micelles, an NMR derived structural model reveals the induction of three helical segments in the linker. The overall linker placement in sodium dodecyl sulfate micelles was identified by NMR experiments including paramagnetic relaxation enhancements. Partitioning of individual residues agrees with their hydrophobicity and supports an interfacial positioning of the helices. Replacement of positively charged linker residues with alanine resulted in YscUC variants displaying attenuated membrane-binding affinities, suggesting that the membrane interaction depends on positive charges within the linker. In vivo experiments with bacteria expressing these YscU replacements resulted in phenotypes displaying significantly reduced effector protein secretion levels. Taken together, our data identify a previously unknown membrane-interacting surface of YscUC that, when perturbed by mutations, disrupts the function of the pathogenic machinery in Yersinia.
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| 5. |
- Horvath, Istvan, et al.
(författare)
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Mechanisms of Protein Oligomerization : Inhibitor of Functional Amyloids Templates α-Synuclein Fibrillation
- 2012
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 134:7, s. 3439-3444
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Tidskriftsartikel (refereegranskat)abstract
- Small organic molecules that inhibit functional bacterial amyloid fibers, curli, are promising new antibiotics. Here we investigated the mechanism by which the ring-fused 2-pyridone FN075 inhibits fibrillation of the curli protein CsgA. Using a variety of biophysical techniques, we found that FN075 promotes CsgA to form off-pathway, non-amyloidogenic oligomeric species. In light of the generic properties of amyloids, we tested whether FN075 would also affect the fibrillation reaction of human α-synuclein, an amyloid-forming protein involved in Parkinson's disease. Surprisingly, FN075 stimulates α-synuclein amyloid fiber formation as measured by thioflavin T emission, electron microscopy (EM), and atomic force microscopy (AFM). NMR data on (15)N-labeled α-synuclein show that upon FN075 addition, α-synuclein oligomers with 7 nm radius form in which the C-terminal 40 residues remain disordered and solvent exposed. The polypeptides in these oligomers contain β-like secondary structure, and the oligomers are detectable by AFM, EM, and size-exclusion chromatography (SEC). Taken together, FN075 triggers oligomer formation of both proteins: in the case of CsgA, the oligomers do not proceed to fibers, whereas for α-synuclein, the oligomers are poised to rapidly form fibers. We conclude that there is a fine balance between small-molecule inhibition and templation that depends on protein chemistry.
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| 6. |
- Liu, Hebin, et al.
(författare)
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AML1/Runx1 recruits calcineurin to regulate granulocyte macrophage colony-stimulating factor by Ets1 activation.
- 2004
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 279:28, s. 29398-29408
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Tidskriftsartikel (refereegranskat)abstract
- Acute myeloid leukemia 1 (AML1), also denoted Runx1, is a transcription factor essential for hematopoiesis, and the AML1 gene is the most common target of chromosomal translocations in human leukemias. AML1 binds to sequences present in the regulatory regions of a number of hematopoiesis-specific genes, including certain cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) up-regulated after T cell receptor stimulation. Here we show that both subunits of the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin (CN), which is activated upon T cell receptor stimulation, interact directly with the N-terminal runt homology domain-containing part of AML1. The regulatory CN subunit binds AML1 with a higher affinity and in addition also interacts with the isolated runt homology domain. The related Runx2 transcription factor, which is essential for bone formation, also interacts with CN. A constitutively active derivative of CN is shown to activate synergistically the GM-CSF promoter/enhancer together with AML1 or Runx2. We also provide evidence that relief of the negative effect of the AML1 sites is important for Ca(2+) activation of the GM-CSF promoter/enhancer and that AML1 overexpression increases this Ca(2+) activation. Both subunits of CN interact with AML1 in coimmunoprecipitation analyses, and confocal microscopy analysis of cells expressing fluorescence-tagged protein derivatives shows that CN can be recruited to the nucleus by AML1 in vivo. Mutant analysis of the GM-CSF promoter shows that the Ets1 binding site of the promoter is essential for the synergy between AML1 and CN in Jurkat T cells. Analysis of the effects of inhibitors of the protein kinase glycogen synthase kinase-3beta and in vitro phosphorylation/dephosphorylation analysis of Ets1 suggest that glycogen synthase kinase-3beta-phosphorylated Ets1 is a target of AML1-recruited CN phosphatase at the GM-CSF promoter.
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| 7. |
- Orädd, Fredrik, 1994-
(författare)
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Determining the effects of regulatory parameters on the structural dynamics of P-type ATPase membrane transporters
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are macromolecular machines with roles in all cellular activities and structures. The functional properties of each protein is the result of its combination of 3D-structure and inherent dynamics, and a wealth of structural and dynamic mechanisms have evolved to regulate protein activity. P-type ATPases are membrane transport proteins that hydrolyze ATP to move cations across membranes. These proteins are involved in important biological functions such as Ca2+ signaling and Cu+ homeostasis, making proper regulation critical. Adenylate kinase (AdK) is a small, soluble protein that plays a role in energy homeostasis by interconverting ATP, AMP, and ADP, which are bound by two substrate binding domains. In this thesis, the effect of regulatory parameters on the structural dynamics of Cu+-ATPases and the sarcoplasmic/endoplasmic Ca2+-ATPase (SERCA) was investigated, together with the reaction dynamics of AdK.In Paper III, the human Cu+-ATPase ATP7B was simulated with (holo) and without (apo) Cu+ bound to the regulatory metal binding domains (MBDs, with MBD-1 closest to the core protein). In the holo state, the MBD chain was more dynamic and extended, and MBD-2 approached the membrane Cu+ entry site. In Paper IV, the stability of the interaction between MBD-2 and the Cu+-entry site was evaluated using MD simulations, showing that the interaction was stable in the cytosol-open E1 state, but not in the lumen-facing E2P state. An interaction site between MBD-3 and the cytoplasmic domains was also found, where MBD-3 might inhibit activity by interfering with functional motions. Finally, in Paper II, Cu+ entry into the membrane high-affinity Cu+-binding site was simulated, showing that a proposed initial binding site was transient and that the Cu+ ion could move deeper into the membrane domain. In Paper I, we used time-resolved X-ray solution scattering (TR-XSS) to show a simultaneous closing of the substrate binding domains in AdK, which included a partial unfolding and refolding event in the ATP-binding domain. Paper VI demonstrated that a novel time-resolved setup based on detector readout at the MAX IV beamline CoSAXS could trigger and detect AdK structural dynamics.In Paper V, TR-XSS experiments showed that the rate-limiting step in skeletal-muscle SERCA1a was an E1-to-E2P intermediate at both low and high Ca2+ concentrations. An inhibitory effect at high Ca2+ concentration was explained by a fraction of SERCA molecules stalling in the ATP-binding/phosphorylation step. In Paper VII, TR-XSS experiments showed that the housekeeping isoform SERCA2b, which is slower but has higher Ca2+ affinity than the other SERCA isoforms, shared the same rate-limiting step as the SERCA1a isoform, but with a longer rise-time. Deletion of the SERCA2b luminal extension (LE) shifted the rate-limiting step to ATP-binding/phosphorylation, possibly because of LE-stabilization of the ATP-bound structure. These papers demonstrated the capability of TR-XSS to detect changes in rate-limiting steps and to investigate how protein structural dynamics respond to mutations and inhibitory conditions.
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| 8. |
- Orädd, Fredrik, et al.
(författare)
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Tracking the ATP-binding response in adenylate kinase in real time
- 2021
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Ingår i: Science Advances. - : American Association for the Advancement of Science. - 2375-2548. ; 7:47
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Tidskriftsartikel (refereegranskat)abstract
- The biological function of proteins is critically dependent on dynamics inherent to the native structure. Such structural dynamics obey a predefined order and temporal timing to execute the specific reaction. Determination of the cooperativity of key structural rearrangements requires monitoring protein reactions in real time. In this work, we used time-resolved x-ray solution scattering (TR-XSS) to visualize structural changes in the Escherichia coli adenylate kinase (AdK) enzyme upon laser-induced activation of a protected ATP substrate. A 4.3-ms transient intermediate showed partial closing of both the ATP- and AMP-binding domains, which indicates a cooperative closing mechanism. The ATP-binding domain also showed local unfolding and breaking of an Arg131-Asp146 salt bridge. Nuclear magnetic resonance spectroscopy data identified similar unfolding in an Arg131Ala AdK mutant, which refolded in a closed, substrate-binding conformation. The observed structural dynamics agree with a “cracking mechanism” proposed to underlie global structural transformation, such as allostery, in proteins.
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| 9. |
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| 10. |
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| 11. |
- Aguilar, Ximena, 1978-, et al.
(författare)
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Macromolecular crowding extended to a heptameric system : the co-chaperonin protein 10
- 2011
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 50:14, s. 3034-3044
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Tidskriftsartikel (refereegranskat)abstract
- Experiments on monomeric proteins have shown that macromolecular crowding can stabilize toward heat perturbation and also modulate native-state structure. To assess the effects of macromolecular crowding on unfolding of an oligomeric protein, we here tested the effects of the synthetic crowding agent Ficoll 70 on human cpn10 (GroES in E. coli), a heptameric protein consisting of seven identical β-barrel subunits assembling into a ring. Using far-UV circular dichroism (CD), tyrosine fluorescence, nuclear magnetic resonance (NMR), and cross-linking experiments, we investigated thermal and chemical stability, as well as the heptamer-monomer dissociation constant, without and with crowding agent. We find that crowding shifts the heptamer-monomer equilibrium constant in the direction of the heptamer. The cpn10 heptamer is both thermally and thermodynamically stabilized in 300 mg/mL Ficoll 70 as compared to regular buffer conditions. Kinetic unfolding experiments show that the increased stability in crowded conditions, in part, is explained by slower unfolding rates. A thermodynamic cycle reveals that in presence of 300 mg/mL Ficoll the thermodynamic stability of each cpn10 monomer increases by over 30%, whereas the interfaces are stabilized by less than 10%. We also introduce a new approach to analyze the spectroscopic data that makes use of multiple wavelengths: this provides robust error estimates of thermodynamic parameters.
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| 12. |
- Bosco, Daryl A, et al.
(författare)
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Dissecting the microscopic steps of the cyclophilin a enzymatic cycle on the biological substrate HIV-capsid by NMR
- 2010
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 403:5, s. 723-38
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Tidskriftsartikel (refereegranskat)abstract
- Peptidyl-prolyl isomerases (PPIases) are emerging as key regulators of many diverse biological processes. Elucidating the role of PPIase activity in vivo has been challenging because mutagenesis of active site residues not only reduces the catalytic activity of these enzymes, but also dramatically affects substrate binding. Employing the cyclophilin A (CypA) PPIase together with its biologically relevant and natively folded substrate, the N-terminal domain of the HIV-1 capsid (CA(N)) protein, we demonstrate here how to dissect residue specific contributions to PPIase catalysis versus substrate binding utilizing NMR spectroscopy. Surprisingly, a number of CypA active-site mutants previously assumed to be strongly diminished in activity toward biological substrates based on a peptide assay only, catalyze the HIV capsid with wild-type activity, but with a change in the rate-limiting step of the enzymatic cycle. The results illustrate that a quantitative analysis of catalysis using the biological substrates is critical when interpreting the effects of PPIase mutations in biological assays.
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| 13. |
- Bäckström, Stefan, et al.
(författare)
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Crystallization and preliminary studies of the DNA-binding runt domain of AML1.
- 2001
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Ingår i: Acta Crystallogr D Biol Crystallogr. - 0907-4449. ; 57:Pt 2, s. 269-71
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Tidskriftsartikel (refereegranskat)abstract
- The acute myeloid leukaemia 1 (AML1) protein belongs to the Runx family of transcription factors and is crucial for haematopoietic development. The genes encoding Runx1 and its associated factor CBF beta are the most frequent targets for chromosomal rearrangements in acute human leukaemias. In addition, point mutations of Runx1 in acute leukaemias and in the familial platelet disorder FPD/AML cluster within the evolutionary conserved runt domain that binds both DNA and CBF beta. Here, the crystallization of the Runx1 runt domain is reported. Crystals belong to space groups C2 and R32 and diffract to 1.7 and 2.0 A resolution, respectively.
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| 14. |
- Bäckström, Stefan, et al.
(författare)
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The RUNX1 Runt domain at 1.25A resolution : a structural switch and specifically bound chloride ions modulate DNA binding.
- 2002
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Ingår i: J Mol Biol. - 0022-2836. ; 322:2, s. 259-72
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Tidskriftsartikel (refereegranskat)abstract
- The evolutionarily conserved Runt homology domain is characteristic of the RUNX family of heterodimeric eukaryotic transcription factors, including RUNX1, RUNX2 and RUNX3. The genes for RUNX1, also termed acute myeloid leukemia protein 1, AML1, and its dimerization partner core-binding factor beta, CBFbeta, are essential for hematopoietic development and are together the most common targets for gene rearrangements in acute human leukemias. Here, we describe the crystal structure of the uncomplexed RUNX1 Runt domain at 1.25A resolution and compare its conformation to previously published structures in complex with DNA, CBFbeta or both. We find that complex formation induces significant structural rearrangements in this immunoglobulin (Ig)-like DNA-binding domain. Most pronounced is the movement of loop L11, which changes from a closed conformation in the free Runt structure to an open conformation in the CBFbeta-bound and DNA-bound forms. This transition, which we refer to as the S-switch, and accompanying structural movements that affect other parts of the Runt domain are crucial for sustained DNA binding. The closed to open transition can be induced by CBFbeta alone; suggesting that one role of CBFbeta is to trigger the S-switch and to stabilize the Runt domain in a conformation enhanced for DNA binding.A feature of the Runt domain hitherto unobserved in any Ig-like DNA-binding domain is the presence of two specifically bound chloride ions. One chloride ion is coordinated by amino acid residues that make direct DNA contact. In a series of electrophoretic mobility-shift analyses, we demonstrate a chloride ion concentration-dependent stimulation of the DNA-binding activity of Runt in the physiological range. A comparable DNA-binding stimulation was observed for negatively charged amino acid residues. This suggests a regulatory mechanism of RUNX proteins through acidic amino acid residues provided by activation domains during cooperative interaction with other transcription factors.
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| 15. |
- Carius, Anke B., et al.
(författare)
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Dynamic pH‐induced conformational changes of the PsbO protein in the fluctuating acidity of the thylakoid lumen
- 2019
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Ingår i: Physiologia Plantarum. - : John Wiley & Sons. - 0031-9317 .- 1399-3054. ; 166:1, s. 288-299
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Tidskriftsartikel (refereegranskat)abstract
- The PsbO protein is an essential extrinsic subunit of photosystem II, the pigment–protein complex responsible for light‐driven water splitting. Water oxidation in photosystem II supplies electrons to the photosynthetic electron transfer chain and is accompanied by proton release and oxygen evolution. While the electron transfer steps in this process are well defined and characterized, the driving forces acting on the liberated protons, their dynamics and their destiny are all largely unknown. It was suggested that PsbO undergoes proton‐induced conformational changes and forms hydrogen bond networks that ensure prompt proton removal from the catalytic site of water oxidation, i.e. the Mn4CaO5 cluster. This work reports the purification and characterization of heterologously expressed PsbO from green algae Chlamydomonas reinhardtii and two isoforms from the higher plant Solanum tuberosum (PsbO1 and PsbO2). A comparison to the spinach PsbO reveals striking similarities in intrinsic protein fluorescence and CD spectra, reflecting the near‐identical secondary structure of the proteins from algae and higher plants. Titration experiments using the hydrophobic fluorescence probe ANS revealed that eukaryotic PsbO proteins exhibit acid–base hysteresis. This hysteresis is a dynamic effect accompanied by changes in the accessibility of the protein's hydrophobic core and is not due to reversible oligomerization or unfolding of the PsbO protein. These results confirm the hypothesis that pH‐dependent dynamic behavior at physiological pH ranges is a common feature of PsbO proteins and causes reversible opening and closing of their β‐barrel domain in response to the fluctuating acidity of the thylakoid lumen.
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| 16. |
- Dossena, Silvia, et al.
(författare)
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The molecular and functional interaction between ICLN and HSPCo38 modulates the regulation of cell volume
- 2011
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 286:47, s. 40659-40670
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Tidskriftsartikel (refereegranskat)abstract
- Identifying functional partners for protein/protein interactions can be a difficult challenge. We proposed the use of the operon structure of the Caenorhabditis elegans genome as a 'New Gene-Finding Tool' (Eichmueller et al, JBC, 2004, 279:7136) that could be functionally translated to the human system. Here we show the validity of this approach by studying the predicted functional interaction between ICln and HSPC038. In Caenorhabditis elegans, the gene encoding for the ICln homolog (icln-1) is embedded in an operon with two other genes, Nx (the human homolog of Nx is HSPC038) and Ny. ICln is a highly conserved, ubiquitously expressed multifunctional protein that plays a critical role in the regulatory volume decrease after cell swelling. Following hypotonic stress, ICln translocates from the cytosol to the plasma membrane, where it has been proposed to participate in the activation of the swelling induced chloride current (IClswell). Here we show that the interaction between human ICln and HSPC038 plays a role in volume regulation after cell swelling and that HSPC038 acts as an escort, directing ICln to the cell membrane after cell swelling and facilitating the activation of IClswell. Assessment of the NMR structure of HSPC038 showed the presence of a zinc finger motif. Moreover, NMR and additional biochemical techniques enabled us to identify the putative ICln/HSPC038 interacting sites, thereby explaining the functional interaction of both proteins on a molecular level.
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| 17. |
- Dulko-Smith, Beata, et al.
(författare)
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Mechanistic basis for a connection between the catalytic step and slow opening dynamics of adenylate kinase
- 2023
-
Ingår i: Journal of Chemical Information and Modeling. - : American Chemical Society (ACS). - 1549-9596 .- 1549-960X. ; 63:5, s. 1556-1569
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Tidskriftsartikel (refereegranskat)abstract
- Escherichia coli adenylate kinase (AdK) is a small, monomeric enzyme that synchronizes the catalytic step with the enzyme’s conformational dynamics to optimize a phosphoryl transfer reaction and the subsequent release of the product. Guided by experimental measurements of low catalytic activity in seven single-point mutation AdK variants (K13Q, R36A, R88A, R123A, R156K, R167A, and D158A), we utilized classical mechanical simulations to probe mutant dynamics linked to product release, and quantum mechanical and molecular mechanical calculations to compute a free energy barrier for the catalytic event. The goal was to establish a mechanistic connection between the two activities. Our calculations of the free energy barriers in AdK variants were in line with those from experiments, and conformational dynamics consistently demonstrated an enhanced tendency toward enzyme opening. This indicates that the catalytic residues in the wild-type AdK serve a dual role in this enzyme’s function─one to lower the energy barrier for the phosphoryl transfer reaction and another to delay enzyme opening, maintaining it in a catalytically active, closed conformation for long enough to enable the subsequent chemical step. Our study also discovers that while each catalytic residue individually contributes to facilitating the catalysis, R36, R123, R156, R167, and D158 are organized in a tightly coordinated interaction network and collectively modulate AdK’s conformational transitions. Unlike the existing notion of product release being rate-limiting, our results suggest a mechanistic interconnection between the chemical step and the enzyme’s conformational dynamics acting as the bottleneck of the catalytic process. Our results also suggest that the enzyme’s active site has evolved to optimize the chemical reaction step while slowing down the overall opening dynamics of the enzyme.
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| 18. |
- Eisenmesser, Elan Z, et al.
(författare)
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Intrinsic dynamics of an enzyme underlies catalysis
- 2005
-
Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 438, s. 117-21
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Tidskriftsartikel (refereegranskat)abstract
- A unique feature of chemical catalysis mediated by enzymes is that the catalytically reactive atoms are embedded within a folded protein. Although current understanding of enzyme function has been focused on the chemical reactions and static three-dimensional structures, the dynamic nature of proteins has been proposed to have a function in catalysis1, 2, 3, 4, 5. The concept of conformational substates has been described6; however, the challenge is to unravel the intimate linkage between protein flexibility and enzymatic function. Here we show that the intrinsic plasticity of the protein is a key characteristic of catalysis. The dynamics of the prolyl cis–trans isomerase cyclophilin A (CypA) in its substrate-free state and during catalysis were characterized with NMR relaxation experiments. The characteristic enzyme motions detected during catalysis are already present in the free enzyme with frequencies corresponding to the catalytic turnover rates. This correlation suggests that the protein motions necessary for catalysis are an intrinsic property of the enzyme and may even limit the overall turnover rate. Motion is localized not only to the active site but also to a wider dynamic network. Whereas coupled networks in proteins have been proposed previously3, 7, 8, 9, 10, we experimentally measured the collective nature of motions with the use of mutant forms of CypA. We propose that the pre-existence of collective dynamics in enzymes before catalysis is a common feature of biocatalysts and that proteins have evolved under synergistic pressure between structure and dynamics.
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| 19. |
- Esteban-Martin, Santiago, et al.
(författare)
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Correlated Inter-Domain Motions in Adenylate Kinase
- 2014
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Ingår i: PloS Computational Biology. - : PLOS. - 1553-734X .- 1553-7358. ; 10:7, s. e1003721-
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Tidskriftsartikel (refereegranskat)abstract
- Correlated inter-domain motions in proteins can mediate fundamental biochemical processes such as signal transduction and allostery. Here we characterize at structural level the inter-domain coupling in a multidomain enzyme, Adenylate Kinase (AK), using computational methods that exploit the shape information encoded in residual dipolar couplings (RDCs) measured under steric alignment by nuclear magnetic resonance (NMR). We find experimental evidence for a multi-state equilibrium distribution along the opening/closing pathway of Adenylate Kinase, previously proposed from computational work, in which inter-domain interactions disfavour states where only the AMP binding domain is closed. In summary, we provide a robust experimental technique for study of allosteric regulation in AK and other enzymes.
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| 20. |
- Gardino, Alexandra K, et al.
(författare)
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Transient Non-native Hydrogen Bonds Promote Activation of a Signaling Protein
- 2009
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Ingår i: Cell. - : Elsevier BV. - 0092-8674 .- 1097-4172. ; 139:6, s. 1109-18
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Tidskriftsartikel (refereegranskat)abstract
- SummaryPhosphorylation is a common mechanism for activating proteins within signaling pathways. Yet, the molecular transitions between the inactive and active conformational states are poorly understood. Here we quantitatively characterize the free-energy landscape of activation of a signaling protein, nitrogen regulatory protein C (NtrC), by connecting functional protein dynamics of phosphorylation-dependent activation to protein folding and show that only a rarely populated, pre-existing active conformation is energetically stabilized by phosphorylation. Using nuclear magnetic resonance (NMR) dynamics, we test an atomic scale pathway for the complex conformational transition, inferred from molecular dynamics simulations (Lei et al., 2009). The data show that the loss of native stabilizing contacts during activation is compensated by non-native transient atomic interactions during the transition. The results unravel atomistic details of native-state protein energy landscapes by expanding the knowledge about ground states to transition landscapes.
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| 21. |
- Gupta, Arun A., et al.
(författare)
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Formation of a Secretion-Competent Protein Complex by a Dynamic Wrap-around Binding Mechanism
- 2018
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 430:18, Part B, s. 3157-3169
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial virulence is typically initiated by translocation of effector or toxic proteins across host cell membranes. A class of gram-negative pathogenic bacteria including Yersinia pseudotuberculosis and Yersinia pestis accomplishes this objective with a protein assembly called the type III secretion system. Yersinia effector proteins (Yop) are presented to the translocation apparatus through formation of specific complexes with their cognate chaperones (Syc). In the complexes where the structure is available, the Yops are extended and wrap around their cognate chaperone. This structural architecture enables secretion of the Yop from the bacterium in early stages of translocation. It has been shown previously that the chaperone-binding domain of YopE is disordered in its isolation but becomes substantially more ordered in its wrap-around complex with its chaperone SycE. Here, by means of NMR spectroscopy, small-angle X-ray scattering and molecular modeling, we demonstrate that while the free chaperone-binding domain of YopH (YopHCBD) adopts a fully ordered and globular fold, it populates an elongated, wrap-around conformation when it engages in a specific complex with its chaperone SycH2. Hence, in contrast to YopE that is unstructured in its free state, YopH transits from a globular free state to an elongated chaperone-bound state. We demonstrate that a sparsely populated YopHCBD state has an elevated affinity for SycH2 and represents an intermediate in the formation of the protein complex. Our results suggest that Yersinia has evolved a binding mechanism where SycH2 passively stimulates an elongated YopH conformation that is presented to the type III secretion system in a secretion-competent conformation.
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| 22. |
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| 23. |
- Henzler-Wildman, Katherine A, et al.
(författare)
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Intrinsic motions along an enzymatic reaction trajectory.
- 2007
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 450, s. 838-44
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Tidskriftsartikel (refereegranskat)abstract
- The mechanisms by which enzymes achieve extraordinary rate acceleration and specificity have long been of key interest in biochemistry. It is generally recognized that substrate binding coupled to conformational changes of the substrate-enzyme complex aligns the reactive groups in an optimal environment for efficient chemistry. Although chemical mechanisms have been elucidated for many enzymes, the question of how enzymes achieve the catalytically competent state has only recently become approachable by experiment and computation. Here we show crystallographic evidence for conformational substates along the trajectory towards the catalytically competent 'closed' state in the ligand-free form of the enzyme adenylate kinase. Molecular dynamics simulations indicate that these partially closed conformations are sampled in nanoseconds, whereas nuclear magnetic resonance and single-molecule fluorescence resonance energy transfer reveal rare sampling of a fully closed conformation occurring on the microsecond-to-millisecond timescale. Thus, the larger-scale motions in substrate-free adenylate kinase are not random, but preferentially follow the pathways that create the configuration capable of proficient chemistry. Such preferred directionality, encoded in the fold, may contribute to catalysis in many enzymes.
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| 24. |
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| 25. |
- Kovachev, Petar Stefanov
(författare)
-
The role of RNA in prion aggregation and disease
- 2018
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- As humanity evolved to witness an exceptionally high standard of living, Alzheimer’s, cancer and diabetes gradually replaced infections as the main limiting factors in longevity. It is both disturbing and captivating that such degenerative conditions are caused by the most ubiquitous biomolecule – the protein. Indeed, proteins are not only the most functional, but also the least understood of the cellular biopolymers. It is then not surprising that many severe human ailments are associated with aberrant proteostasis. The key, causative mechanism of proteinopathy is protein aggregation. Naturally occurring and sometimes functional, aggregation is an auxiliary pathway in protein folding. In the context of a crowded cellular environment, folding and aggregation are the least and one of the least understood molecular processes, respectively. Unravelling one can help deconstruct the other and vice versa, but also can provide mechanistic insight on degenerative proteinopathies. A special class of proteins, which appear to propagate their own aggregation, occupy center-stage in the scientific field devoted to this goal. These proteins known as prions, can exist in at least two distinct forms. With the human prion, one of those is functional and benign and the other is infectious, aggregation prone, self-replicating and fatally pathogenic. As it happens, prion disease shares many of the descriptive features of other proteinaceous neuropathies. That, and the seductive idea that prions dwell in the twilight zone between folding and aggregation, have made the prion phenomenon a fixation for many molecular biologists. This thesis, although not the product of fixation, deals with one aspect of the prion process – the involvement of a molecular cofactor.Of all plausible adjuvants, RNAs have been proposed as likely participants in the prion process. Their prominent secondary structures and attractive polyanionic surfaces allow RNAs to freely engage in interactions, at times transmitting conformational information through induced fit effects. The present work summarizes the influence of various RNAs on the aggregation profiles of three prionogenic model systems. The produced results indicate a generic role for RNA in the molecular processes prion propagation and aggregation. Altogether, this study illustrates a previously overlooked RNA function, of potential relevance for protein-based disease.
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| 26. |
- Kovermann, Michael, et al.
(författare)
-
Protein dynamics and function from solution state NMR spectroscopy
- 2016
-
Ingår i: Quarterly reviews of biophysics (Print). - : Cambridge University Press. - 0033-5835 .- 1469-8994. ; 49
-
Forskningsöversikt (refereegranskat)abstract
- It is well-established that dynamics are central to protein function; their importance is implicitly acknowledged in the principles of the Monod, Wyman and Changeux model of binding cooperativity, which was originally proposed in 1965. Nowadays the concept of protein dynamics is formulated in terms of the energy landscape theory, which can be used to understand protein folding and conformational changes in proteins. Because protein dynamics are so important, a key to understanding protein function at the molecular level is to design experiments that allow their quantitative analysis. Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited for this purpose because major advances in theory, hardware, and experimental methods have made it possible to characterize protein dynamics at an unprecedented level of detail. Unique features of NMR include the ability to quantify dynamics (i) under equilibrium conditions without external perturbations, (ii) using many probes simultaneously, and (iii) over large time intervals. Here we review NMR techniques for quantifying protein dynamics on fast (ps-ns), slow (μs-ms), and very slow (s-min) time scales. These techniques are discussed with reference to some major discoveries in protein science that have been made possible by NMR spectroscopy.
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| 27. |
- Kovermann, Michael, et al.
(författare)
-
Structural basis for catalytically restrictive dynamics of a high-energy enzyme state
- 2015
-
Ingår i: Nature Communications. - : Macmillan Publishers Ltd.. - 2041-1723. ; 6
-
Tidskriftsartikel (refereegranskat)abstract
- An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or ‘invisible’ states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme’s catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes’ conformational dynamics and hence their catalytic power—a key aspect in rational design of enzymes catalysing novel reactions.
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| 28. |
- Kovermann, Michael, et al.
(författare)
-
Structural basis for ligand binding to an enzyme by a conformational selection pathway
- 2017
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 114:24, s. 6298-6303
-
Tidskriftsartikel (refereegranskat)abstract
- Proteins can bind target molecules through either induced fit or conformational selection pathways. In the conformational selection model, a protein samples a scarcely populated high-energy state that resembles a target-bound conformation. In enzymatic catalysis, such high-energy states have been identified as crucial entities for activity and the dynamic interconversion between ground states and high-energy states can constitute the rate-limiting step for catalytic turnover. The transient nature of these states has precluded direct observation of their properties. Here, we present a molecular description of a high-energy enzyme state in a conformational selection pathway by an experimental strategy centered on NMR spectroscopy, protein engineering, and X-ray crystallography. Through the introduction of a disulfide bond, we succeeded in arresting the enzyme adenylate kinase in a closed high-energy conformation that is on-pathway for catalysis. A 1.9-angstrom X-ray structure of the arrested enzyme in complex with a transition state analog shows that catalytic side-chains are properly aligned for catalysis. We discovered that the structural sampling of the substrate free enzyme corresponds to the complete amplitude that is associated with formation of the closed and catalytically active state. In addition, we found that the trapped high-energy state displayed improved ligand binding affinity, compared with the wild-type enzyme, demonstrating that substrate binding to the high-energy state is not occluded by steric hindrance. Finally, we show that quenching of fast time scale motions observed upon ligand binding to adenylate kinase is dominated by enzyme-substrate interactions and not by intramolecular interactions resulting from the conformational change.
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| 29. |
- Leeb, Sarah, 1989-
(författare)
-
How transient interactions in the crowded cytosol affect protein mobility and stability
- 2020
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Most biochemical reactions have evolved in crowded intracellular environments. However, the complexity of intracellular environments is often neglected in structural or functional studies of proteins. In these cases, reactions involving proteins are deliberately separated from the perturbations of co-solutes in order to simplify data acquisition and interpretation. Having acquired an enormous body of knowledge under these simplified dilute-buffer conditions, methodological progress of the past two decades has made the study of proteins inside living cells increasingly accessible and concomitantly kindled an interest to investigate proteins in their native habitat. Naturally, major questions that arose were to what extent ubiquitous transient interactions alter protein structure, function and thermodynamics and, not least, what role protein surfaces and their physicochemical properties play in determining the frequency and duration of these diffusive encounters.By looking at the rotational-tumbling rates of three structurally well-characterized proteins in live cells with nuclear magnetic resonance (NMR) relaxation, we expand on previous research performed in the bacterium Escherichia coli and establish the physicochemical principles that determine diffusive interactions in the mammalian cytosol of the human ovarian cancer cell line A2780. Just as in E. coli, net charge is the dominating factor in regulating protein interactivity, albeit with the impact on rotational retardation greatly diminished. We ascribe this to the generally lower macromolecular concentrations in the eukaryotic cytosol, and put forward a hypothesis in which less stringent rules regarding protein surface decoration in eukaryotes could have facilitated the development of multi-cellular organisms. Furthermore, by developing a model where a distribution of differently sized interaction partners is taken into account when examining rotational retardation, we reconcile transverse and longitudinal in-cell relaxation with theory, and are able to estimate the populations of the bound and free form of a set of reporter proteins. Looking at the populations of bound protein instead of a mean-field rotational retardation finally allows us to re-assess the guiding rules behind diffusive cytosolic interactions. Last, we outline a putative mechanism behind the in-cell destabilization of a variant of Superoxide dismutase 1 (SOD1barrel). By mimicking generic poly-anionic intracellular co-solutes with poly-acetic acid (NaPAc1200), we identify the positively charged N-terminal portion of the unfolded form of the protein as the interaction site with the highest affinity. Further examining the unfolded ensemble of SOD1barrel with a mutationally destabilized variant reveals a compact state, that remains almost unchanged upon binding to NaPAc1200. This suggests that NaPAc1200-mediated destabilization occurs mainly through mass action, in full accord with the postulated mechanism for in-cell protein destabilization.
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| 30. |
- Li, Yaozong, 1982-
(författare)
-
Understanding molecular mechanisms of protein tyrosine kinases by molecular dynamics and free energy calculations
- 2017
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: Insulin receptor kinase (IRK) and Insulin-like growth factor 1 receptor kinase (IGF-1RK) are two important members in the large class of tyrosine kinase receptors. They play pivotalroles in the regulation of glucose homeostasis, cell proliferation, differentiation, motility, andtransformation. Their dysfunctions are linked to diabetes, rheumatoid arthritis and many cancers.Although their regulatory mechanisms have been widely studied experimentally, the atomisticdetails are still poorly understood, especially for the influences caused by activation loop (A-loop)phosphorylation.Methods: Molecular dynamics (MD) and alchemical free energy simulations are carried out tounderstand mechanisms underlying the kinase proteins regulation and their thermodynamic basis.To capture a full picture about the entire kinase catalytic cycle, different functional steps areconsidered, i.e., conformational transition, substrate binding, phosphoryl transfer and productrelease. The effects of the A-loop phosphorylation on protein’s dynamics, structure, stability, andfree energy landscape are examined by various analysis methods, including principle componentanalysis (PCA), motion projection, dynamical network analysis and free energy perturbation.Results: The main findings are: 1) A-loop phosphorylation shifts the kinase conformationalpopulation to the active one by changing the electrostatic environments in the kinase apo form, 2)allosterically fine-tunes the orientation of the catalytic residues mediated by the >C-helix in thereactant and product binding states, and 3) thermodynamically favors the kinase catalysis presentedby a catalytic-cycle-mimic free energy landscape. An integrated view on the roles of A-loopphosphorylation in kinase allostery is developed by incorporating kinase’s dynamics, structuralinteractions, thermodynamics and free energy landscape. In addition, new soft-core potentials(Gaussian soft-core) and protocols are developed to conduct accurate and efficient alchemical freeenergy calculations.Conclusions: The entire catalytic cycle is examined by MD and free energy calculations andcomprehensive analyses are conducted. The findings from the studied kinases are general and canbe implemented to the other members in IRK family or even to more non-homologous familiesbecause of the conservation of the characteristic residues between their A-loop and >C-helix. Inaddition, the Gaussian soft-core potentials provide a new tool to perform alchemical free energycalculations in an efficient way.
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| 31. |
- Lidman, Martin, 1985-
(författare)
-
The role of the mitochondrial membrane system in apoptosis : the influence of oxidative stress on membranes and their interactions with apoptosis-regulating Bcl-2 proteins
- 2015
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Apoptosis is a crucial process in multicellular organisms in sculpting them, especially during embryogenesis. In addition, apoptosis is responsible for the clearance of harmful or damaged cells which can otherwise be detrimental to the organism. The Bcl-2 family proteins are key players in the regulation of the intrinsic pathway of the apoptotic machinery. This family consists of three subfamilies with B-cell CLL/lymphoma 2 (Bcl-2) protein itself representing anti-apoptotic members, the Bcl-2-associated X protein (Bax), and pro-apoptotic BH3-only signaling proteins. The interplay between pro- and anti-apoptotic proteins on the mitochondrial membranes is central to the balance between the life and death decision of whether the membrane should be permeabilized or not. The cytosolic Bax protein can upon cellular stress translocate to the mitochondrial membrane where it can either carry out its action of forming homo-oligomers that cause outer membrane permeabilization or be inhibited there by the anti-apoptotic membrane protein Bcl-2. Upon mitochondrial outer membrane permeabilization (MOMP) apoptogenic factors leak out from the intermembrane space (IMS) of the mitochondria, leading to caspase activation and ultimately cell death. A common stress signal initiating apoptosis is an increased formation of reactive oxygen species (ROS in the mitochondria, who can cause oxidative damage to lipid membranes. This membrane damage presumably influences the lipid landscape and the membrane features and hence the interactions of the Bcl-2 family proteins with each other and the mitochondrial outer membrane (MOM). To investigate the significance of membrane oxidation on the behavior of the Bcl-2 family proteins, especially Bax, synthetically produced oxidized phospholipids (OxPls) were incorporated in MOM-mimicking vesicles. Differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR) spectroscopy and circular dichroism (CD) spectroscopy revealed a major perturbation in membrane organization in the presence of OxPls. These changes in membrane properties increase the affinity of Bax to its target membrane and enable its partial penetration and formation of pores, as fluorescence leakage assays confirmed. However, in the absence of BH3-only proteins these pores are not sufficiently large for the release of apopototic factors such as cytochrome C (CytC). To understand the inhibition of Bax by the full-length Bcl-2 protein, suitable detergent solubilizing conditions were carefully chosen to enable the measurement of their direct binding to each other outside the membrane, by an antimycin A2 fluorescence assay. The observed protein-protein interaction was confirmed by surface plasmon resonance (SPR). An established protocol for the reconstitution of Bcl-2 into stable proteoliposomes now paves the way for structural studies of this key protein, in its membrane environment near physiological conditions; information essential for understanding its function, on a molecular level, and its potential as a cancer drug target.
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| 32. |
- Malisauskas, Mantas, et al.
(författare)
-
Lability landscape and protease resistance of human insulin amyloid : a new insight into its molecular properties
- 2010
-
Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 396:1, s. 60-74
-
Tidskriftsartikel (refereegranskat)abstract
- Amyloid formation is a universal behavior of proteins central to many important human pathologies and industrial processes. The extreme stability of amyloids towards chemical and proteolytic degradation is an acquired property compared to the precursor proteins and is a major prerequisite for their accumulation. Here we report a study on the lability of human insulin amyloid as a function of pH and amyloid ageing. Using a range of methods such as AFM, thioflavin-T fluorescence, circular dichroism and gas phase electrophoretic mobility macromolecule analysis we probed the propensity of human insulin amyloid to propagate or dissociate in a wide span of pHs and ageing in a low concentration regime. We generated a three-dimensional amyloid lability landscape in coordinates of pH and amyloid ageing, which displays three distinctive features: (i) a maximum propensity to grow near pH 3.8 and an age corresponding the inflection point of the growth phase; (ii) an abrupt cut-off between growth and disaggregation at pH 8-10; (iii) isoclines shifted towards older age during the amyloid growth phase at pH 4-9, reflecting the greater stability of aged amyloid. Thus, lability of amyloid strongly depends on the ionization state of insulin and on the structure and maturity of amyloid fibrils. The stability of insulin amyloid towards protease K was assessed by using real-time AFM and thioflavin-T fluorescence. We estimated that amyloid fibrils can be digested both from the free ends and within the length of the fibril with a rate of ca. 4 nm/min. Our results highlight that amyloid structures, depending on solution conditions, can be less stable than commonly perceived. These results have wide implications for understanding the propagation of amyloids via a seeding mechanism as well as for understanding their natural clearance and dissociation under solution conditions unfavorable for amyloid formation in biological systems and industrial applications.
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| 33. |
- Mishra, Laxmi S., 1983-
(författare)
-
FtsH metalloproteases and their pseudo-proteases in the chloroplast envelope of Arabidopsis thaliana
- 2021
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- By cleaving peptide bonds, proteases either activate or degrade proteins and maintain protein quality control in response to various developmental stimuli and environmental factors. My work has focused on elucidating the role of the filamentation temperature sensitive protein H (FtsH) proteases. FtsHs belong to a membrane-embedded class of proteases found in eubacteria, animals and plants, which are located in the organelles of endosymbiosis (mitochondria and chloroplasts). They possess an AAA+ (ATPase associated with various cellular activities) and a peptidase M41 domain containing the HEXXH consensus sequence in the Zn2+ metalloprotease domain. FtsH proteases are known to form ring-like homo- or hetero-hexameric complexes. Arabidopsis thaliana, the model plant used in this study, contains seventeen AtFtsH proteases, of which twelve are presumably proteolytically active and five presumably proteolytic inactive members, known as AtFtsHi (i for inactive). In AtFtsHi members, the HEXXH motif is either deleted (AtFtsHi3) or mutated (AtFtsHi1, 2, 4, 5). Twelve AtFtsHs (AtFtsH 1, 2, 5–9, 11, 12 and AtFtsHi 1-5) are targeted to the chloroplast, whereas the remaining three (AtFtsH 3, 4 and 10) are mitochondrial. In Paper I, we demonstrate that AtFtsH12 interacts with AtFtsHi1, 2, 4, 5 to form a heteromeric complex. Abundance of these AtFtsH12-AtFtsHi complexes alters the accumulation of TIC (translocon on the inner chloroplast membrane) complexes. Transgenic mi12 (miRNA) knockdown plants that express lower amounts of AtFtsH12 displayed a pale-seedling and an aberrant chloroplast phenotype. mi12 plants displayed lowered total chlorophyll (Chla+Chlb) amount compared to wild type (WT), complementation lines and native AtFtsH12 promoter overexpressor (ox12) lines. Our biochemical studies identified drastic modifications in the total proteome of mi12 seedlings. N-terminome analyses of mi12 seedlings showed undisturbed plastidic protein maturation. In Paper II, we have shown that single mutants depleted in AtFTSHI1, 2, 4 or 5 are embryo-lethal, suggesting the pseudo-proteases to have an indispensable role in seed germination. This study further identified “weak” Atftshi1, Atftshi4, Atftshi3-1(kd) and Atftshi3-2 homozygous mutants, which develop into plants with altered photosynthetic efficiency. Field experiments were performed to determine the Darwinian fitness of these homozygous as well as heterozygous AtFtsHi mutants. The results suggested AtFtsHi enzymes to be critical during early developmental stages. A complete Atftshi3 knockdown mutant (Atftshi3-1(kd)) was identified (described in Paper III), which is not embryo-lethal and tolerates drought better than WT plants. Atftshi3-1(kd) leaves were smaller with fewer and smaller stomatal aperture. Above ground, Atftshi3-1(kd) leaves displayed lowered stomatal conductance and increased WUEi (intrinsic water-use efficiency), while below ground, the root-associated bacterial community showed a typical drought stress response. Upregulated transcripts of the ABA-responsive genes in leaves of Atftshi3-1(kd) compared to WT indicate the drought tolerance to be controlled independently of ABA. To conclude, AtFtsHi pseudo-proteases affect various stages of plant development and abiotic stress management, especially drought.
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| 34. |
- Nam, Kwangho, et al.
(författare)
-
Elucidating dynamics of Adenylate kinase from enzyme opening to ligand release
- 2024
-
Ingår i: Journal of Chemical Information and Modeling. - : American Chemical Society (ACS). - 1549-9596 .- 1549-960X. ; 64:1, s. 150-163
-
Tidskriftsartikel (refereegranskat)abstract
- This study explores ligand-driven conformational changes in adenylate kinase (AK), which is known for its open-to-close conformational transitions upon ligand binding and release. By utilizing string free energy simulations, we determine the free energy profiles for both enzyme opening and ligand release and compare them with profiles from the apoenzyme. Results reveal a three-step ligand release process, which initiates with the opening of the adenosine triphosphate-binding subdomain (ATP lid), followed by ligand release and concomitant opening of the adenosine monophosphate-binding subdomain (AMP lid). The ligands then transition to nonspecific positions before complete dissociation. In these processes, the first step is energetically driven by ATP lid opening, whereas the second step is driven by ATP release. In contrast, the AMP lid opening and its ligand release make minor contributions to the total free energy for enzyme opening. Regarding the ligand binding mechanism, our results suggest that AMP lid closure occurs via an induced-fit mechanism triggered by AMP binding, whereas ATP lid closure follows conformational selection. This difference in the closure mechanisms provides an explanation with implications for the debate on ligand-driven conformational changes of AK. Additionally, we determine an X-ray structure of an AK variant that exhibits significant rearrangements in the stacking of catalytic arginines, explaining its reduced catalytic activity. In the context of apoenzyme opening, the sequence of events is different. Here, the AMP lid opens first while the ATP lid remains closed, and the free energy associated with ATP lid opening varies with orientation, aligning with the reported AK opening and closing rate heterogeneity. Finally, this study, in conjunction with our previous research, provides a comprehensive view of the intricate interplay between various structural elements, ligands, and catalytic residues that collectively contribute to the robust catalytic power of the enzyme.
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| 35. |
- Nam, Kwangho, et al.
(författare)
-
Perspectives on computational enzyme modeling : from mechanisms to design and drug development
- 2024
-
Ingår i: ACS Omega. - : American Chemical Society (ACS). - 2470-1343. ; 9:7, s. 7393-7412
-
Forskningsöversikt (refereegranskat)abstract
- Understanding enzyme mechanisms is essential for unraveling the complex molecular machinery of life. In this review, we survey the field of computational enzymology, highlighting key principles governing enzyme mechanisms and discussing ongoing challenges and promising advances. Over the years, computer simulations have become indispensable in the study of enzyme mechanisms, with the integration of experimental and computational exploration now established as a holistic approach to gain deep insights into enzymatic catalysis. Numerous studies have demonstrated the power of computer simulations in characterizing reaction pathways, transition states, substrate selectivity, product distribution, and dynamic conformational changes for various enzymes. Nevertheless, significant challenges remain in investigating the mechanisms of complex multistep reactions, large-scale conformational changes, and allosteric regulation. Beyond mechanistic studies, computational enzyme modeling has emerged as an essential tool for computer-aided enzyme design and the rational discovery of covalent drugs for targeted therapies. Overall, enzyme design/engineering and covalent drug development can greatly benefit from our understanding of the detailed mechanisms of enzymes, such as protein dynamics, entropy contributions, and allostery, as revealed by computational studies. Such a convergence of different research approaches is expected to continue, creating synergies in enzyme research. This review, by outlining the ever-expanding field of enzyme research, aims to provide guidance for future research directions and facilitate new developments in this important and evolving field.
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| 36. |
- Nam, Kwangho, et al.
(författare)
-
Protein dynamics : the future is bright and complicated!
- 2023
-
Ingår i: Structural Dynamics. - : American Crystallographic Association. - 2329-7778. ; 10:1
-
Tidskriftsartikel (refereegranskat)abstract
- Biological life depends on motion, and this manifests itself in proteins that display motion over a formidable range of time scales spanning from femtoseconds vibrations of atoms at enzymatic transition states, all the way to slow domain motions occurring on micro to milliseconds. An outstanding challenge in contemporary biophysics and structural biology is a quantitative understanding of the linkages among protein structure, dynamics, and function. These linkages are becoming increasingly explorable due to conceptual and methodological advances. In this Perspective article, we will point toward future directions of the field of protein dynamics with an emphasis on enzymes. Research questions in the field are becoming increasingly complex such as the mechanistic understanding of high-order interaction networks in allosteric signal propagation through a protein matrix, or the connection between local and collective motions. In analogy to the solution to the "protein folding problem,"we argue that the way forward to understanding these and other important questions lies in the successful integration of experiment and computation, while utilizing the present rapid expansion of sequence and structure space. Looking forward, the future is bright, and we are in a period where we are on the doorstep to, at least in part, comprehend the importance of dynamics for biological function.
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| 37. |
- Nors Perdersen, Martin, et al.
(författare)
-
Direct Correlation Between Ligand-Induced α-Synuclein Oligomers and Amyloid-like Fibril Growth.
- 2015
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 5, s. 10422-
-
Tidskriftsartikel (refereegranskat)abstract
- Aggregation of proteins into amyloid deposits is the hallmark of several neurodegenerative diseases such as Alzheimer's and Parkinson's disease. The suggestion that intermediate oligomeric species may be cytotoxic has led to intensified investigations of pre-fibrillar oligomers, which are complicated by their transient nature and low population. Here we investigate alpha-synuclein oligomers, enriched by a 2-pyridone molecule (FN075), and the conversion of oligomers into fibrils. As probed by leakage assays, the FN075 induced oligomers potently disrupt vesicles in vitro, suggesting a potential link to disease related degenerative activity. Fibrils formed in the presence and absence of FN075 are indistinguishable on microscopic and macroscopic levels. Using small angle X-ray scattering, we reveal that FN075 induced oligomers are similar, but not identical, to oligomers previously observed during alpha-synuclein fibrillation. Since the levels of FN075 induced oligomers correlate with the amounts of fibrils among different FN075:protein ratios, the oligomers appear to be on-pathway and modeling supports an 'oligomer stacking model' for alpha-synuclein fibril elongation.
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| 38. |
- Oanh, Ho Ngoc Hoang, 1986-
(författare)
-
Regulation of the multi-functional protein YscU in assembly of the Yersinia type III secretion injectisome
- 2017
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Yersinia pseudotuberculosis is a Gram-negative zoonotic pathogenic bacterium causing gastroenteritis in human and animals. It shares a conserved virulence plasmid encoding for a needle-like secretion machinery, or type III secretion system, which can be found in other pathogenic Gram-negative bacteria. The type III secretion system (T3SS) is a macromolecular assembly that enables pathogenic effector proteins (or Yersinia outer proteins, Yops) to be transported into eukaryotic host cells. This export machinery is assembled in a highly ordered stepwise mechanism. The activation of T3SS is also dependent on calcium concentration, temperature, and pH of the growth media as mimic factors for host cell’s contact. The T3SS-associated inner-membrane protein, YscU, of Yersinia is proposed to function as a substrate specificity switch protein and forms basal structure of T3SS. YscU has four α helical transmembrane domain and a soluble cytoplasmic domain YscUC which undergoes auto-proteolysis at a conserved N↑PTH motif. The auto-proteolysis process, which is required for the assembly of the injectisome and secretion of Yops, results in a 10-kDa C-terminal polypeptide fragment, denoted YscUCC and 6-kDa N-terminal fragment YscUCN. In this thesis, we showed that YscUC dissociation was important for Yops secretion and resulted in unfolded YscUCN and oligomeric YscUCC. By combination in vivo and in vitro methods, growth media conditions as calcium, temperature, and pH were indicated to control secretion by regulation of YscUC dissociation. The calcium-binding isotherm to YscUC was fit best with a one-site binding model resulting in Kd 800 µM, which is identical to calcium level that blocks secretion in vivo. YscU is also the key protein for the T3SS pH dependence, demonstrated by thermal unfolding profile and secondary structure of protein were altered between pH 7.4 and 6.0. In addition, bacterial inner membrane was proposed to assist the YscUCN folding, monitored by using lipid bilayer as a mimic environment in nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. This binding is important for Yops secretion and YscUC is anchored to bacterial membrane upon dissociation. The other substrate specificity switch protein YscP has function as a “molecular ruler” controlling length of the secretion needle. Previous genetic experiments have suggested that YscP and YscU interact physically, when mutation at defined residues on yscU (suppressor mutants) rescued Yops secretion in null-yscP mutant. In this research, direct binding of YscU and YscP was proved as weak but important interaction with Kd 430 mM by application of NMR and the binding interface of YscP was centred on the last helix of YscUC. Furthermore, we found that the YscP interaction could inhibit YscU auto-proteolysis. Studying the dissociation kinetic of suppressor YscUC variants at temperature 30 and 37oC provides strong support to a model where YscU is a temperature sensor for T3SS and YscUC dissociation is required for Yops secretion. Interestingly, the NPTH motif is conserved through most of YscU family members, meaning that role of dissociation may be conserved also in other bacterial injectisomes. To this end, the dissociation of YscU can be used as a therapeutic target in drug discovery. We attempted to identify the small-molecules that can hinder YscU dissociation. The small compound methyl(5-methyl-2-phenyl-1,3-thiazolidin-4-yl)acetate was found to be able to inhibit dissociation and to crystalize full YscUC, which has never been successfully done before. Finally, we found that the inner-rod protein YscI is binding to YscUC with a 1:1 stoichiometry as shown with pull-down assays and isothermal titration calorimetry. Taken together we have made several discoveries that expand the functional palette of YscU and all these functions were shown to have biological relevance with Yops secretion levels. In light of the strong sequence conservation between T3SS utilizing pathogenic bacteria the findings are likely to be general characters.
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| 39. |
- Ojeda-May, Pedro, et al.
(författare)
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Dynamic Connection between Enzymatic Catalysis and Collective Protein Motions
- 2021
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 60:28, s. 2246-2258
-
Tidskriftsartikel (refereegranskat)abstract
- Enzymes employ a wide range of protein motions to achieve efficient catalysis of chemical reactions. While the role of collective protein motions in substrate binding, product release, and regulation of enzymatic activity is generally understood, their roles in catalytic steps per se remain uncertain. Here, molecular dynamics simulations, enzyme kinetics, X-ray crystallography, and nuclear magnetic resonance spectroscopy are combined to elucidate the catalytic mechanism of adenylate kinase and to delineate the roles of catalytic residues in catalysis and the conformational change in the enzyme. This study reveals that the motions in the active site, which occur on a time scale of picoseconds to nanoseconds, link the catalytic reaction to the slow conformational dynamics of the enzyme by modulating the free energy landscapes of subdomain motions. In particular, substantial conformational rearrangement occurs in the active site following the catalytic reaction. This rearrangement not only affects the reaction barrier but also promotes a more open conformation of the enzyme after the reaction, which then results in an accelerated opening of the enzyme compared to that of the reactant state. The results illustrate a linkage between enzymatic catalysis and collective protein motions, whereby the disparate time scales between the two processes are bridged by a cascade of intermediate-scale motion of catalytic residues modulating the free energy landscapes of the catalytic and conformational change processes.
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| 40. |
- Olsson, Ulrika, et al.
(författare)
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Overlap between folding and functional energy landscapes for adenylate kinase conformational change
- 2010
-
Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 1:8, s. 111-
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Tidskriftsartikel (refereegranskat)abstract
- Enzyme function is often dependent on fluctuations between inactive and active structural ensembles. Adenylate kinase isolated from Escherichia coli (AK(e)) is a small phosphotransfer enzyme in which interconversion between inactive (open) and active (closed) conformations is rate limiting for catalysis. AK(e) has a modular three-dimensional architecture with two flexible substrate-binding domains that interact with the substrates AMP, ADP and ATP. Here, we show by using a combination of biophysical and mutagenic approaches that the interconversion between open and closed states of the ATP-binding subdomain involves partial subdomain unfolding/refolding in an otherwise folded enzyme. These results provide a novel and, possibly general, molecular mechanism for the switch between open and closed conformations in AK(e).
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| 41. |
- Palm, Maria E, et al.
(författare)
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Cisplatin binds human copper chaperone Atox1 and promotes unfolding in vitro.
- 2011
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 108:17, s. 6951-6
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Tidskriftsartikel (refereegranskat)abstract
- Cisplatin (cisPt), Pt(NH(3))(2)Cl(2), is a cancer drug believed to kill cells via DNA binding and damage. Recent work has implied that the cellular copper (Cu) transport machinery may be involved in cisPt cell export and drug resistance. Normally, the Cu chaperone Atox1 binds Cu(I) via two cysteines and delivers the metal to metal-binding domains of ATP7B; the ATP7B domains then transfer the metal to the Golgi lumen for loading on cuproenzymes. Here, we use spectroscopic methods to test if cisPt interacts with purified Atox1 in solution in vitro. We find that cisPt binds to Atox1's metal-binding site regardless of the presence of Cu or not: When Cu is bound to Atox1, the near-UV circular dichroism signals indicate Cu-Pt interactions. From NMR data, it is evident that cisPt binds to the folded protein. CisPt-bound Atox1 is however not stable over time and the protein begins to unfold and aggregate. The reaction rates are limited by slow cisPt dechlorination. CisPt-induced unfolding of Atox1 is specific because this effect was not observed for two unrelated proteins that also bind cisPt. Our study demonstrates that Atox1 is a candidate for cisPt drug resistance: By binding to Atox1 in the cytoplasm, cisPt transport to DNA may be blocked. In agreement with this model, cell line studies demonstrate a correlation between Atox1 expression levels, and cisplatin resistance.
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| 42. |
- Patrick, Joan, 1987-
(författare)
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Functional dynamics of glycosyltransferases : Solution-state NMR studies of peripheral membrane proteins involved in glycolipid biosynthesis in bacteria
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antibiotic resistance is an existential threat enabled by bacterial adaptation and fuelled by inappropriate use of medication. The ensuing shortage of effective treatments has led to a rise in deaths linked to resistant bacterial pathogens. Disrupting cell wall biosynthesis can undermine bacterial defences, so new insights into the dynamic function of the enzymes involved could facilitate new therapies.Glycosyltransferases (GTs), enzymes forming glycosidic bonds, build molecules by transferring a sugar group from a donor to an acceptor. In Gram-negative bacteria, an enzymatic assembly line constructs membrane-anchored virulence factor lipopolysaccharide (LPS), which dominates the outer membrane, forming a protective layer. In mycobacteria, phosphatidyl-myo-inositol mannosides (PIMs) ensure the stability and impermeability of the inner membrane, and are constructed by a similar array of enzymes. In this thesis, bacterial GTs that work at the cytoplasmic leaflet of the inner membrane were investigated.PimA is an essential mycobacterial enzyme involved in constructing PIMs. It exists in multiple conformations, implying that it undergoes complex conformational changes, including a fold-switch. Associated motions were characterised with NMR dynamics experiments, revealing donor substrate-dependent population shifts and dynamic changes. At least four different states co-exist in solution, regardless of whether or not the enzyme is bound to substrate.WaaG performs one step in the biosynthesis of LPS in bacteria including E. coli and P. aeruginosa. As it is not an essential enzyme, EcWaaG-deficient E. coli survive, but are more vulnerable to antibiotics. 19F NMR was employed to detect conformational and dynamic changes in EcWaaG. Upon interaction with bicelle-bound lipids and its donor substrate, UDP-glucose, EcWaaG was shown to experience a dynamic change, while a part of the protein was shown to experience slow conformational change. Hydrolysis of the donor substrate was quantified using 31P NMR. WaaG from P. aeruginosa was also investigated, focusing on the functional mechanism. NMR experiments determined that only UDP-GalNAc was hydrolysed by PaWaaG. When the active site was mutated to resemble that of EcWaaG, it was shown by 31P NMR that the mutated enzyme instead hydrolysed the donor substrate of EcWaaG, UDP-glucose. However, PaWaaG cannot be substituted for EcWaaG in vivo, underlining the importance of the interaction with the lipid-bound acceptor substrate.Both WaaG and PimA function adjacent to membrane. As larger objects give rise to broader signals, solution-state NMR imposes constraints on the detection of protein-lipid interactions. Small membrane mimetics like lipid bicelles can be used to mimic a membrane, but while they permit detection of effects on protein signals, detecting the effects on lipid signals requires further optimization, as further concentration-dependent challenges arise in multi-component experiments. Thus, lipid dynamics in bicelles designed to exist at low concentrations were characterized using 1H and 13C NMR. Upon binding spin-labelled PimA, paramagnetic relaxation enhancement of the lipids could be observed.This thesis thus widens the toolkit available to study membrane-associated proteins. It demonstrates that, far from being static structures, biomolecules like lipids and proteins are highly flexible objects whose function can only be understood if dynamics are taken into account.
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| 43. |
- Perdersen, Martin Nors, et al.
(författare)
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Direct Correlation Between Ligand-Induced alpha-Synuclein Oligomers and Amyloid-like Fibril Growth
- 2015
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Ingår i: Scientific Reports. - : Springer. - 2045-2322. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- Aggregation of proteins into amyloid deposits is the hallmark of several neurodegenerative diseases such as Alzheimer's and Parkinson's disease. The suggestion that intermediate oligomeric species may be cytotoxic has led to intensified investigations of pre-fibrillar oligomers, which are complicated by their transient nature and low population. Here we investigate alpha-synuclein oligomers, enriched by a 2-pyridone molecule (FN075), and the conversion of oligomers into fibrils. As probed by leakage assays, the FN075 induced oligomers potently disrupt vesicles in vitro, suggesting a potential link to disease related degenerative activity. Fibrils formed in the presence and absence of FN075 are indistinguishable on microscopic and macroscopic levels. Using small angle X-ray scattering, we reveal that FN075 induced oligomers are similar, but not identical, to oligomers previously observed during alpha-synuclein fibrillation. Since the levels of FN075 induced oligomers correlate with the amounts of fibrils among different FN075: protein ratios, the oligomers appear to be on-pathway and modeling supports an 'oligomer stacking model' for alpha-synuclein fibril elongation.
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| 44. |
- Phoeurk, Chanrith, et al.
(författare)
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Milligram scale expression, refolding, and purification of Bombyx mori cocoonase using a recombinant E. coli system
- 2021
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Ingår i: Protein Expression and Purification. - : Elsevier. - 1046-5928 .- 1096-0279. ; 186
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Tidskriftsartikel (refereegranskat)abstract
- Silk is one of the most versatile biomaterials with signature properties of outstanding mechanical strength and flexibility. A potential avenue for developing more environmentally friendly silk production is to make use of the silk moth (Bombyx mori) cocoonase, this will at the same time increase the possibility for using the byproduct, sericin, as a raw material for other applications. Cocoonase is a serine protease utilized by the silk moth to soften the cocoon to enable its escape after completed metamorphosis. Cocoonase selectively degrades the glue protein of the cocoon, sericin, without affecting the silk-fiber made of the protein fibroin. Cocoonase can be recombinantly produced in E. coli, however, it is exclusively found as insoluble inclusion bodies. To solve this problem and to be able to utilize the benefits associated with an E. coli based expression system, we have developed a protocol that enables the production of soluble and functional protease in the milligram/liter scale. The core of the protocol is refolding of the protein in a buffer with a redox potential that is optimized for formation of native and intramolecular di-sulfide bridges. The redox potential was balanced with defined concentrations of reduced and oxidized glutathione. This E. coli based production protocol will, in addition to structure determination, also enable modification of cocoonase both in terms of catalytic function and stability. These factors will be valuable components in the development of alternate silk production methodology.
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| 45. |
- Qureshi, Abdul Aziz, 1984-
(författare)
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Establishing the mechanistic basis of sugar transport
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Sugar is a vital molecule required for cell viability and homeostasis. Sugar is important for metabolic energy, energy storage, signaling, structure and osmolyte regulation. Transport of sugar represents an important physiological process. Specific membrane transporter families have evolved to mediate the transport of sugar across biological membranes. In this thesis, we describe our work leading to a better mechanistic understanding of two sugar transporter families, namely glucose (GLUT) transporters and nucleotide-sugar (NST) transporters.Members of GLUT transporters, belonging to the Solute Carrier (SLC2) family, are involved in the uptake of various monosaccharides across the cellular membranes. Activity of different NSTs, belonging to the (SLC35) family, is crucial for the process of glycosylation by mediating the translocation of activated sugars from the cytoplasm into the lumen of either Golgi and/or ER organelles. GLUTs and NSTs families carry out transport processes fundamental to human physiology and pathophysiology. Despite the profound importance of GLUTs and NSTs in human health, comprehensive understanding of their architecture and mechanistic features with respect to determinants of substrate binding and allosteric coupling at the molecular level has remained elusive.In this thesis, we address key functional and structural properties of GLUT and NST mediated sugar transport. We combine crystal structures with robust binding and transport assays as well as computational approaches. The role of lipids in fine-tuning the activity of transporters is also exemplified by demonstrating the effect of lipid composition in the transport activity of GLUTs using in-vitro proteoliposome assays. Our work has not only enhanced the current understanding of GLUT and NST function, but also developed themes and methods that are likely relevant to many types of small molecule transporters.
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| 46. |
- Ravishankar, Harsha, 1987-
(författare)
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Characterization of ATP-dependent protein dynamics under native-like conditions
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are biological macromolecules capable of accelerating biochemical reactions. To accomplish this, proteins undergo changes in their molecular structure. Advances in structural biology have resulted in ever-increasing numbers of high-resolution protein structures. However, the majority of transient intermediate states will not amendable with traditional structural determination methods. Therefore, understanding how protein structural changes are correlated with the biological function necessitates development of methods that characterize the reaction in the native environment. P-type ATPase membrane transporters and the adenylate kinase (AK) are two ATP-dependent proteins that undergo extensive conformational change in their reaction cycles. While P-type ATPases maintain concentration gradients of ions across the cellular membranes, AK regulates cellular energy homeostasis by catalyzing interconversion of nucleotides. Resolving P-type ATPase and AK temporal and spatial structural dynamics is crucial to understand how these proteins are triggered by ATP for functionality. To pave way for time-resolved X-ray characterization of ATP-dependent conformational changes, it was necessary to identify optimal conditions for triggering protein reactions. Therefore, time-dependent Fourier-Transform Infra-Red (FTIR) spectroscopy of a recombinant Zn2+-transporting ATPase was used to optimize activation by photolysis of caged ATP. These conditions were then used to track structural dynamics of the Ca2+-transporting sarcoplasmic reticulum ATPase (SERCA) in skeletal muscle native membranes. Fast single-cycle dynamics were registered with the formation of an intermediate state at 1.5 ms followed by steady-state accumulation at 13 ms. The molecular dynamic (MD)-based structural refinement procedure showed that the 13-ms transient intermediate represented an ADP-sensitive, phosphorylated Ca2+-bound E1 state (Ca2E1P), with a domain arrangement that has so far eluded structural characterization.MD simulations of the identified SERCA transient intermediates further finetuned their positions in the reaction cycle. The 1.5-ms state was assigned to an ATP-bound state prior to phosphorylation, while the 13-ms state was stable in its Ca2E1P conformation. Because the simulations were performed in multicomponent lipid bilayers mimicking the native membrane, specific state-dependent lipid interactions were also identified. Finally, the wider applicability of the time-resolved X-ray method to study ATP-dependent protein dynamics was demonstrated by tracking AK structural dynamics. A transient intermediate at 5 ms was identified that showed closing of the ATP-binding domain prior to the NMP-binding domain, in the presence of both ATP and AMP substrates. This study provided conclusive experimental proof of the relative ordering of domain closure that had been predicted by several computational studies.In summary, the work presented in this thesis has contributed to developing the time-resolved X-ray method to study the structural dynamics of ATP-dependent proteins.
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| 47. |
- Rogne, Per, et al.
(författare)
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Molecular mechanism of ATP versus GTP selectivity of adenylate kinase
- 2018
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:12, s. 3012-3017
-
Tidskriftsartikel (refereegranskat)abstract
- Enzymatic substrate selectivity is critical for the precise control of metabolic pathways. In cases where chemically related substrates are present inside cells, robust mechanisms of substrate selectivity are required. Here, we report the mechanism utilized for catalytic ATP versus GTP selectivity during adenylate kinase (Adk) -mediated phosphorylation of AMP. Using NMR spectroscopy we found that while Adk adopts a catalytically competent and closed structural state in complex with ATP, the enzyme is arrested in a catalytically inhibited and open state in complex with GTP. X-ray crystallography experiments revealed that the interaction interfaces supporting ATP and GTP recognition, in part, are mediated by coinciding residues. The mechanism provides an atomic view on how the cellular GTP pool is protected from Adk turnover, which is important because GTP has many specialized cellular functions. In further support of this mechanism, a structure-function analysis enabled by synthesis of ATP analogs suggests that a hydrogen bond between the adenine moiety and the backbone of the enzyme is vital for ATP selectivity. The importance of the hydrogen bond for substrate selectivity is likely general given the conservation of its location and orientation across the family of eukaryotic protein kinases.
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| 48. |
- Rogne, Per, et al.
(författare)
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Nucleation of an Activating Conformational Change by a Cation−Π Interaction
- 2019
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 58:32, s. 3408-3412
-
Tidskriftsartikel (refereegranskat)abstract
- As a key molecule in biology, adenosine triphosphate (ATP) has numerous crucial functions in, for instance, energetics, post-translational modifications, nucleotide biosynthesis, and cofactor metabolism. Here, we have discovered an intricate interplay between the enzyme adenylate kinase and its substrate ATP. The side chain of an arginine residue was found to be an efficient sensor of the aromatic moiety of ATP through the formation of a strong cation−π interaction. In addition to recognition, the interaction was found to have dual functionality. First, it nucleates the activating conformational transition of the ATP binding domain and also affects the specificity in the distant AMP binding domain. In light of the functional consequences resulting from the cation−π interaction, it is possible that the mode of ATP recognition may be a useful tool in enzyme design.
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| 49. |
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| 50. |
- Rogne, Per, et al.
(författare)
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Real-time 31P NMR investigation on the catalytic behavior of the enzyme Adenylate kinase in the matrix of a switchable ionic liquid
- 2015
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Ingår i: ChemSusChem. - : John Wiley & Sons. - 1864-5631 .- 1864-564X. ; 8:2, s. 3764-3768
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Tidskriftsartikel (refereegranskat)abstract
- The integration of highly efficient enzymatic catalysis with the solvation properties of ionic liquids for an environmentally friendly and efficient use of raw materials such as wood requires fundamental knowledge about the influence of relevant ionic liquids on enzymes. Switchable ionic liquids (SIL) are promising candidates for implementation of enzymatic treatments of raw materials. One industrially interesting SIL is constituted by monoethanol amine (MEA) and 1,8-diazabicyclo-[5.4.0]-undec-7-ene (DBU) formed with sulfur dioxide (SO2) as the coupling media (DBU-SO2-MEASIL). It has the ability to solubilize the matrix of lignocellulosic biomass while leaving the cellulose backbone intact. Using a novel 31P NMR-based real-time assay we show that this SIL is compatible with enzymatic catalysis because a model enzyme, adenylate kinase, retains its activity in up to at least 25 wt % of DBU-SO2-MEASIL. Thus this SIL appears suitable for, for example, enzymatic degradation of hemicellulose.
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