| 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. |
- 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
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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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| 3. |
- 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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| 4. |
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| 5. |
- 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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| 6. |
- 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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| 7. |
- 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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| 8. |
- 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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| 9. |
- Malisauskas, Mantas, et al.
(författare)
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Lability landscape and protease resistance of human insulin amyloid : a new insight into its molecular properties
- 2010
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 396:1, s. 60-74
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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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| 10. |
- Olsson, Ulrika, et al.
(författare)
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Overlap between folding and functional energy landscapes for adenylate kinase conformational change
- 2010
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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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| 11. |
- 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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| 12. |
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| 13. |
- Ådén, Jörgen, 1980-, et al.
(författare)
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Extraordinary μs-ms backbone dynamics in Arabidopsis thaliana peroxiredoxin Q
- 2011
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier. - 0006-3002 .- 1878-2434. ; 1814:12, s. 1880-1890
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Tidskriftsartikel (refereegranskat)abstract
- Peroxiredoxin Q (PrxQ) isolated from Arabidopsis thaliana belongs to a family of redox enzymes called peroxiredoxins, which are thioredoxin- or glutaredoxin-dependent peroxidases acting to reduce peroxides and in particular hydrogen peroxide. PrxQ cycles between an active reduced state and an inactive oxidized state during its catalytic cycle. The catalytic mechanism involves a nucleophilic attack of the catalytic cysteine on hydrogen peroxide to generate a sulfonic acid intermediate with a concerted release of a water molecule. This intermediate is subsequently relaxed by the reaction of a second cysteine, denoted the resolving cysteine, generating an intramolecular disulfide bond and release of a second water molecule. PrxQ is recycled to the active state by a thioredoxin-dependent reduction. Previous structural studies of PrxQ homologues have provided the structural basis for the switch between reduced and oxidized conformations. Here, we have performed a detailed study of the activity, structure and dynamics of PrxQ in both the oxidized and reduced states. Reliable and experimentally validated structural models of PrxQ in both oxidation states were generated using homology based modeling. Analysis of NMR spin relaxation rates shows that PrxQ is monomeric in both oxidized and reduced states. As evident from R(2) relaxation rates the reduced form of PrxQ undergoes unprecedented dynamics on the slow μs-ms timescale. The ground state of this conformational dynamics is likely the stably folded reduced state as implied by circular dichroism spectroscopy. We speculate that the extensive dynamics is intimately related to the catalytic function of PrxQ.
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| 14. |
- Ådén, Jörgen, et al.
(författare)
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Modulation of a pre-existing conformational equilibrium tunes adenylate kinase activity
- 2012
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Ingår i: Journal of the American Chemical Society. - Washington : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 134:40, s. 16562-16570
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Tidskriftsartikel (refereegranskat)abstract
- Structural plasticity is often required for distinct microscopic steps during enzymatic reaction cycles. Adenylate kinase from Escherichia coli (AKeco) populates two major conformations in solution; the open (inactive) and closed (active) state, and the overall turn-over rate is inversely proportional to the life-time of the active conformation. Therefore, structural plasticity is intimately cou-pled to enzymatic turn-over in AKeco. Here we probe the open to closed conformational equilibrium in the absence of bound substrate with NMR spectroscopy and molecular dynamics simulations. The conformational equilibrium in absence of substrate, and in turn, the turn-over number can be modulated with mutational- and osmolyte-driven perturbations. Removal of one hydrogen bond between the ATP and AMP binding sub-domains results in a population shift towards the open conformation and a resulting increase of kcat. Addition of the osmolyte TMAO to AKeco results in population shift towards the closed conformation and a significant reduction of kcat. The Michaelis constants (KM) scale with the change in kcat, which follows from the influence of the population of the closed conformation for substrate binding affinity. Hence, kcat and KM are mutually dependent and, in the case of AKeco, any perturbation that modulates kcat is mirrored with a proportional response in KM. Thus, our results demonstrate that the equilibrium constant of a pre-existing conformational equilibrium directly affect enzymatic catalysis. From an evolutionary perspective our findings suggests that, for AKeco, there exists ample flexibility to obtain a specificity constant (kcat/KM) that commensurate with the exerted cellular selective pressure.
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| 15. |
- Ådén, Jörgen, 1980-
(författare)
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NMR studies of protein dynamics and structure
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Enzymes are extraordinary molecules that can accelerate chemical reactions by several orders of magnitude. With recent advancements in structural biology together with classical enzymology the mechanism of many enzymes has become understood at the molecular level. During the last ten years significant efforts have been invested to understand the structure and dynamics of the actual catalyst (i. e. the enzyme). There has been a tremendous development in NMR spectroscopy (both hardware and pulse programs) that have enabled detailed studies of protein dynamics. In many cases there exists a strong coupling between enzyme dynamics and function. Here I have studied the conformational dynamics and thermodynamics of three model systems: adenylate kinase (Adk), Peroxiredoxin Q (PrxQ) and the structural protein S16. By developing a novel chemical shift-based method we show that Adk binds its two substrates AMP and ATP with an extraordinarily dynamic mechanism. For both substrate-saturated states the nucleotide-binding subdomains exchange between open and closed states, with the populations of these states being approximately equal. This finding contrasts with the traditional view of enzyme-substrate complexes as static low entropy states. We are also able to show that the individual subdomains in Adk fold and unfold in a non-cooperative manner. This finding is relevant from a functional perspective, since it allows a change in hydrogen bonding pattern upon substrate-binding without provoking global unfolding of the entire enzyme (as would be expected from a two-state folding mechanism). We also studied the structure and dynamics of the plant enzyme PrxQ in both reduced and oxidized states. Experimentally validated structural models were generated for both oxidation states. The reduced state displays unprecedented μs-ms conformational dynamics and we propose that this dynamics reflects local and functional unfolding of an α-helix in the active site. Finally, we solved the structure of S16 from Aquifex aeolicus and propose a model suggesting a link between thermostability and structure for a mesophilic and hyperthermophilic protein pair. A connection between the increased thermostability in the thermophilic S16 and residual structure in its unfolded state was discovered, persistent at high denaturant concentrations, thereby affecting the difference in heat capacity difference between the folded and unfolded state. In summary, we have contributed to the understanding of protein dynamics and to the coupling between dynamics and catalytic activity in enzymes.
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| 16. |
- Ådén, Jörgen, 1980-, et al.
(författare)
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Structural topology and activation of an initial adenylate kinase-substrate complex
- 2013
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 52:6, s. 1055-1061
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Tidskriftsartikel (refereegranskat)abstract
- Enzymatic activity is ultimately defined by the structure, chemistry and dynamics of the Michaelis complex. There exist a large number of experimentally determined structures between enzymes and substrates or substrate analogues or inhibitors. However, transient, short-lived encounter and equilibrium structures also play fundamental roles during enzymatic reaction cycles. Such structures are inherently difficult to study with conventional experimental techniques. The enzyme adenylate kinase undergoes major conformational rearrangements in response to binding of its substrates ATP and AMP. ATP is sandwiched between two binding surfaces in the closed and active enzyme conformation. Thus, ade-nylate kinase harbors two spatially distant surfaces in the substrate free open conformation of which one is responsible for the initial interaction with ATP. Here, we have performed primarily nuclear magnetic resonance experiments on Escherichia coli adenylate kinase (AKeco) variants that enabled identification of the site responsible for the initial ATP interaction. This allowed a characterization of the structural topology of an initial equilibrium complex between AKeco and ATP. Based on the results it is suggested that the ATP binding mechanism to AKeco is a mixture between "induced fit" and "conformational selection" models. It is shown that ATP is activated in the initial enzyme bound complex since it displays an appreciable rate of non-productive ATP hydrolysis. In summary our results provide novel structural and functional insights into adenylate kinase catalysis.
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| 17. |
- Österberg, Sofia, et al.
(författare)
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Genetic dissection of a motility-associated c-di-GMP signalling protein of Pseudomonas putida
- 2013
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Ingår i: Environmental Microbiology Reports. - Hoboken : Wiley-Blackwell. - 1758-2229. ; 5:4, s. 556-565
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Tidskriftsartikel (refereegranskat)abstract
- Lack of the Pseudomonas putidaPP2258 protein or its overexpression results in defective motility on solid media. The PP2258 protein is tripartite, possessing a PAS domain linked to two domains associated with turnover of c-di-GMP - a cyclic nucleotide that controls the switch between motile and sessile lifestyles. The second messenger c-di-GMP is produced by diguanylate cyclases and degraded by phosphodiesterases containing GGDEF and EAL or HD-GYP domains respectively. It is common for enzymes involved in c-di-GMP signalling to contain two domains with potentially opposing c-di-GMP turnover activities; however, usually one is degenerate and has been adopted to serve regulatory functions. Only a few proteins have previously been found to have dual enzymatic activities - being capable of both synthesizing and hydrolysing c-di-GMP. Here, using truncated and mutant derivatives of PP2258, we show that despite a lack of complete consensus in either the GGDEF or EAL motifs, the two c-di-GMP turnover domains can function independently of each other, and that the diguanylate cyclase activity is regulated by an inhibitory I-site within its GGDEF domain. Thus, motility-associated PP2258 can be added to the short list of bifunctional c-di-GMP signalling proteins.
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