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Carius, A. B., Rogne, P., Duchoslav, M., Wolf-Watz, M., Samuelsson, G. & Shutova, T. (2019). Dynamic pH‐induced conformational changes of the PsbO protein in the fluctuating acidity of the thylakoid lumen. Physiologia Plantarum: An International Journal for Plant Biology, 166(1), 288-299
Open this publication in new window or tab >>Dynamic pH‐induced conformational changes of the PsbO protein in the fluctuating acidity of the thylakoid lumen
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2019 (English)In: Physiologia Plantarum: An International Journal for Plant Biology, ISSN 0031-9317, E-ISSN 1399-3054, Vol. 166, no 1, p. 288-299Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
John Wiley & Sons, 2019
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-157462 (URN)10.1111/ppl.12948 (DOI)000466108300023 ()30793329 (PubMedID)
Funder
The Kempe FoundationsKnut and Alice Wallenberg Foundation, KAW2011.0055
Note

Special Issue: SI

Available from: 2019-03-21 Created: 2019-03-21 Last updated: 2019-06-10Bibliographically approved
Rogne, P., Andersson, D., Grundström, C., Sauer-Eriksson, E., Linusson, A. & Wolf-Watz, M. (2019). Nucleation of an Activating Conformational Change by a Cation−Π Interaction. Biochemistry, 58(32), 3408-3412
Open this publication in new window or tab >>Nucleation of an Activating Conformational Change by a Cation−Π Interaction
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2019 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 58, no 32, p. 3408-3412Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2019
National Category
Organic Chemistry Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-162099 (URN)10.1021/acs.biochem.9b00538 (DOI)000480827100002 ()31339702 (PubMedID)
Available from: 2019-08-14 Created: 2019-08-14 Last updated: 2019-09-02Bibliographically approved
Wolf-Watz, M., Rogne, P., Sauer-Eriksson, A. E., Sauer, U. H. & Hedberg, C. (2019). Positive and Negative Substrate Interference Supported by Coinciding Enzyme Residues. Paper presented at 63rd Annual Meeting of the Biophysical-Society, MAR 02-06, 2019, Baltimore, MD. Biophysical Journal, 116(3), 485A-485A
Open this publication in new window or tab >>Positive and Negative Substrate Interference Supported by Coinciding Enzyme Residues
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2019 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 116, no 3, p. 485A-485AArticle in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
CELL PRESS, 2019
National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-157776 (URN)10.1016/j.bpj.2018.11.2620 (DOI)000460779802439 ()
Conference
63rd Annual Meeting of the Biophysical-Society, MAR 02-06, 2019, Baltimore, MD
Available from: 2019-04-10 Created: 2019-04-10 Last updated: 2019-04-10Bibliographically approved
Gupta, A. A., Reinartz, I., Karunanithy, G., Spilotros, A., Jonna, V. R., Hofer, A., . . . Wolf-Watz, M. (2018). Formation of a Secretion-Competent Protein Complex by a Dynamic Wrap-around Binding Mechanism. Journal of Molecular Biology, 430(18, Part B), 3157-3169
Open this publication in new window or tab >>Formation of a Secretion-Competent Protein Complex by a Dynamic Wrap-around Binding Mechanism
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2018 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, no 18, Part B, p. 3157-3169Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
NMR spectroscopy, protein complex, binding mechanism
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-151495 (URN)10.1016/j.jmb.2018.07.014 (DOI)000444668100010 ()
Funder
Swedish Research Council, 2013-5954Knut and Alice Wallenberg FoundationCarl Tryggers foundation , CTS 15:210The Kempe Foundations
Available from: 2018-09-05 Created: 2018-09-05 Last updated: 2018-10-04Bibliographically approved
Rogne, P., Rosselin, M., Grundström, C., Hedberg, C., H. Sauer, U. & Wolf-Watz, M. (2018). Molecular mechanism of ATP versus GTP selectivity of adenylate kinase. Proceedings of the National Academy of Sciences of the United States of America, 115(12), 3012-3017
Open this publication in new window or tab >>Molecular mechanism of ATP versus GTP selectivity of adenylate kinase
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2018 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 12, p. 3012-3017Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
adenylate kinase, selectivity, ATP, GTP, mechanism
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-145883 (URN)10.1073/pnas.1721508115 (DOI)000427829500063 ()29507216 (PubMedID)
Available from: 2018-03-20 Created: 2018-03-20 Last updated: 2018-06-09Bibliographically approved
Ho, O., Rogne, P., Edgren, T., Wolf-Watz, H., Login, F. & Wolf-Watz, M. (2017). Characterization of the Ruler Protein Interaction Interface on the Substrate Specificity SwitchProtein in the Yersinia Type III Secretion System. Journal of Biological Chemistry, 292(8), 3299-3311
Open this publication in new window or tab >>Characterization of the Ruler Protein Interaction Interface on the Substrate Specificity SwitchProtein in the Yersinia Type III Secretion System
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2017 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 8, p. 3299-3311Article, review/survey (Refereed) Published
Abstract [en]

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.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-130048 (URN)10.1074/jbc.M116.770255 (DOI)000395538800021 ()
Available from: 2017-01-11 Created: 2017-01-11 Last updated: 2018-06-09Bibliographically approved
Kovermann, M., Grundström, C., Sauer-Eriksson, A. E., Sauer, U. H. & Wolf-Watz, M. (2017). Structural basis for ligand binding to an enzyme by a conformational selection pathway. Proceedings of the National Academy of Sciences of the United States of America, 114(24), 6298-6303
Open this publication in new window or tab >>Structural basis for ligand binding to an enzyme by a conformational selection pathway
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2017 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 24, p. 6298-6303Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
National Academy of Sciences, 2017
Keywords
enzymatic catalysis, ligand binding, structural biology, adenylate kinase
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-137382 (URN)10.1073/pnas.1700919114 (DOI)000403179300051 ()28559350 (PubMedID)
Available from: 2017-07-06 Created: 2017-07-06 Last updated: 2018-06-09Bibliographically approved
Tükenmez, H., Magnussen, H., Kovermann, M., Byström, A. & Wolf-Watz, M. (2016). Linkage between Fitness of Yeast Cells and Adenylate Kinase Catalysis. PLoS ONE, 11(9), Article ID e0163115.
Open this publication in new window or tab >>Linkage between Fitness of Yeast Cells and Adenylate Kinase Catalysis
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2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 9, article id e0163115Article in journal (Refereed) Published
Abstract [en]

Enzymes have evolved with highly specific values of their catalytic parameters kcat and KM. This poses fundamental biological questions about the selection pressures responsible for evolutionary tuning of these parameters. Here we are address these questions for the enzyme adenylate kinase (Adk) in eukaryotic yeast cells. A plasmid shuffling system was developed to allow quantification of relative fitness (calculated from growth rates) of yeast in response to perturbations of Adk activity introduced through mutations. Biophysical characterization verified that all variants studied were properly folded and that the mutations did not cause any substantial differences to thermal stability. We found that cytosolic Adk is essential for yeast viability in our strain background and that viability could not be restored with a catalytically dead, although properly folded Adk variant. There exist a massive overcapacity of Adk catalytic activity and only 12% of the wild type kcat is required for optimal growth at the stress condition 20°C. In summary, the approach developed here has provided new insights into the evolutionary tuning of kcat for Adk in a eukaryotic organism. The developed methodology may also become useful for uncovering new aspects of active site dynamics and also in enzyme design since a large library of enzyme variants can be screened rapidly by identifying viable colonies.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-125853 (URN)10.1371/journal.pone.0163115 (DOI)000383891900032 ()27642758 (PubMedID)
Funder
Swedish Research Council, 621-2013-5954Swedish Research Council, 621-2012-3576
Available from: 2016-09-20 Created: 2016-09-20 Last updated: 2018-06-07Bibliographically approved
Kovermann, M., Rogne, P. & Wolf-Watz, M. (2016). Protein dynamics and function from solution state NMR spectroscopy. Quarterly reviews of biophysics (Print), 49, Article ID e6.
Open this publication in new window or tab >>Protein dynamics and function from solution state NMR spectroscopy
2016 (English)In: Quarterly reviews of biophysics (Print), ISSN 0033-5835, E-ISSN 1469-8994, Vol. 49, article id e6Article, review/survey (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Cambridge University Press, 2016
National Category
Chemical Sciences Biophysics
Identifiers
urn:nbn:se:umu:diva-119440 (URN)10.1017/S0033583516000019 (DOI)000375229500001 ()27088887 (PubMedID)
Available from: 2016-04-19 Created: 2016-04-19 Last updated: 2018-06-07Bibliographically approved
Rogne, P. & Wolf-Watz, M. (2016). Urea-Dependent Adenylate Kinase Activation following Redistribution of Structural States. Biophysical Journal, 111(7), 1385-1395
Open this publication in new window or tab >>Urea-Dependent Adenylate Kinase Activation following Redistribution of Structural States
2016 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 111, no 7, p. 1385-1395Article in journal (Refereed) Published
Abstract [en]

Proteins are often functionally dependent on conformational changes that allow them to sample structural states that are sparsely populated in the absence of a substrate or binding partner. The distribution of such structural microstates is governed by their relative stability, and the kinetics of their interconversion is governed by the magnitude of associated activation barriers. Here, we have explored the interplay among structure, stability, and function of a selected enzyme, adenylate kinase (Adk), by monitoring changes in its enzymatic activity in response to additions of urea. For this purpose we used a 31P NMR assay that was found useful for heterogeneous sample compositions such as presence of urea. It was found that Adk is activated at low urea concentrations whereas higher urea concentrations unfolds and thereby deactivates the enzyme. From a quantitative analysis of chemical shifts, it was found that urea redistributes preexisting structural microstates, stabilizing a substrate-bound open state at the expense of a substrate-bound closed state. Adk is rate-limited by slow opening of substrate binding domains and the urea-dependent redistribution of structural states is consistent with a model where the increased activity results from an increased rate-constant for domain opening. In addition, we also detected a strong correlation between the catalytic free energy and free energy of substrate (ATP) binding, which is also consistent with the catalytic model for Adk. From a general perspective, it appears that urea can be used to modulate conformational equilibria of folded proteins toward more expanded states for cases where a sizeable difference in solvent-accessible surface area exists between the states involved. This effect complements the action of osmolytes, such as trimethylamine N-oxide, that favor more compact protein states.

National Category
Chemical Sciences Biophysics
Identifiers
urn:nbn:se:umu:diva-126432 (URN)10.1016/j.bpj.2016.08.028 (DOI)000385471500006 ()27705762 (PubMedID)
Available from: 2016-10-06 Created: 2016-10-06 Last updated: 2018-06-09Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-9098-7974

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