| 1. |
- 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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| 2. |
- 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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| 3. |
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| 4. |
- 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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| 5. |
- 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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| 6. |
- 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
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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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| 7. |
- 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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| 8. |
- 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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| 9. |
- Mishra, Laxmi S., 1983-
(författare)
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FtsH metalloproteases and their pseudo-proteases in the chloroplast envelope of Arabidopsis thaliana
- 2021
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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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| 10. |
- Nam, Kwangho, et al.
(författare)
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Elucidating dynamics of Adenylate kinase from enzyme opening to ligand release
- 2024
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Ingår i: Journal of Chemical Information and Modeling. - : American Chemical Society (ACS). - 1549-9596 .- 1549-960X. ; 64:1, s. 150-163
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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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