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Kovermann, Michael
Publications (8 of 8) Show all publications
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
Kahra, D., Kovermann, M. & Wittung-Stafshede, P. (2016). The C-Terminus of Human Copper Importer Ctr1 Acts as a Binding Site and Transfers Copper to Atox1. Biophysical Journal, 110(1), 95-102
Open this publication in new window or tab >>The C-Terminus of Human Copper Importer Ctr1 Acts as a Binding Site and Transfers Copper to Atox1
2016 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, no 1, p. 95-102Article in journal (Refereed) Published
Abstract [en]

Uptake of copper (Cu) ions into human cells is mediated by the plasma membrane protein Ctr1 and is followed by Cu transfer to cytoplasmic Cu chaperones for delivery to Cu-dependent enzymes. The C-terminal cytoplasmic tail of Ctr1 is a 13-residue peptide harboring an HCH motif that is thought to interact with Cu. We here employ biophysical experiments under anaerobic conditions in peptide models of the Ctr1 C-terminus to deduce Cu-binding residues, Cu affinity, and the ability to release Cu to the cytoplasmic Cu chaperone Atox1. Based on NMR assignments and bicinchoninic acid competition experiments, we demonstrate that Cu interacts in a 1:1 stoichiometry with the HCH motif with an affinity, K-D, of similar to 10(-14) M. Removing either the Cys residue or the two His residues lowers the Cu-peptide affinity, but site specificity is retained. The C-terminal peptide and Atox1 do not interact in solution in the absence of Cu. However, as directly demonstrated at the residue level via NMR spectroscopy, Atox1 readily acquires Cu from the Cu-loaded peptide. We propose that Cu binding to the Ctr1 C-terminal tail regulates Cu transport into the cytoplasm such that the metal ion is only released to high-affinity Cu chaperones.

National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-114887 (URN)10.1016/j.bpj.2015.11.016 (DOI)000367783900032 ()26745413 (PubMedID)
Available from: 2016-04-27 Created: 2016-01-29 Last updated: 2018-06-07Bibliographically approved
Wolf-Watz, M. & Kovermann, M. (2015). Dynamics of a Naturally Hidden State Restricts Adenylate Kinase Activity. Biophysical Journal, 108(2), 30A-30A
Open this publication in new window or tab >>Dynamics of a Naturally Hidden State Restricts Adenylate Kinase Activity
2015 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 108, no 2, p. 30A-30AArticle in journal, Meeting abstract (Other academic) Published
National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-112291 (URN)10.1016/j.bpj.2014.11.189 (DOI)000359471700146 ()
Note

Supplement: 1 Meeting Abstract: 145-Plat

Available from: 2015-12-04 Created: 2015-12-04 Last updated: 2018-06-07Bibliographically approved
Petzoldt, S., Kahra, D., Kovermann, M., Dingeldein, A. P., Niemiec, M. S., Ådén, J. & Wittung-Stafshede, P. (2015). Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro. Biometals, 28(3), 577-585
Open this publication in new window or tab >>Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro
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2015 (English)In: Biometals, ISSN 0966-0844, E-ISSN 1572-8773, Vol. 28, no 3, p. 577-585Article in journal (Refereed) Published
Abstract [en]

After Ctr1-mediated copper ion (Cu) entry into the human cytoplasm, chaperones Atox1 and CCS deliver Cu to P-1B-type ATPases and to superoxide dismutase, respectively, via direct protein-protein interactions. Although the two Cu chaperones are presumed to work along independent pathways, we here assessed cross-reactivity between Atox1 and the first domain of CCS (CCS1) using biochemical and biophysical methods in vitro. By NMR we show that CCS1 is monomeric although it elutes differently from Atox1 in size exclusion chromatography (SEC). This property allows separation of Atox1 and CCS1 by SEC and, combined with the 254/280 nm ratio as an indicator of Cu loading, we demonstrate that Cu can be transferred from one protein to the other. Cu exchange also occurs with full-length CCS and, as expected, the interaction involves the metal binding sites since mutation of Cu-binding cysteine in Atox1 eliminates Cu transfer from CCS1. Cross-reactivity between CCS and Atox1 may aid in regulation of Cu distribution in the cytoplasm.

Place, publisher, year, edition, pages
Springer, 2015
Keywords
Human copper transport, Atox1, Copper chaperone for superoxide dismutase, (SOD), Size exclusion chromatography, Proton-NMR
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-100334 (URN)10.1007/s10534-015-9832-1 (DOI)000354273900014 ()25673218 (PubMedID)
Available from: 2015-03-01 Created: 2015-03-01 Last updated: 2018-06-07Bibliographically approved
Hoffmann, A., Kovermann, M., Oberwinkler, T., Siedler, F., Socorro Cortina, N., Balbach, J. & Oesterhelt, D. (2015). Novel sulfated phosphoglycolipids from Natronomonas moolapensis. Chemistry and Physics of Lipids, 191, 8-15
Open this publication in new window or tab >>Novel sulfated phosphoglycolipids from Natronomonas moolapensis
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2015 (English)In: Chemistry and Physics of Lipids, ISSN 0009-3084, E-ISSN 1873-2941, Vol. 191, p. 8-15Article in journal (Refereed) Published
Abstract [en]

Polar lipid pattern determination is often used for the taxonomic classification of halophilic Archaea in addition to a genomic characterization. During the analysis of polar lipid extracts from the recently described haloarchaeon Natrononomonas moolapensis, an unknown glycolipid was detected. Fragmentation patterns observed from preliminary mass spectrometric analysis initially suggested the presence of a sulfo-hexosyl-phosphatidylglycerol. However, by NMR spectroscopy and enzymatic assays the existence of two isomeric molecules with different hexoses (1-(6-sulfo-D-glcp/galf-beta 1,2-glycero)phospho-2,3-diphytanylglycerol) could be shown. The structural origin from phosphatidylglycerol distinguishes these glycolipids within Archaea, because all other characterized haloarchaeal glycolipids consist of diphytanylglycerol directly linked to an oligoglycosyl moiety. Now the door is open to investigate the physical and functional consequences of these architectural differences of the head groups.

Place, publisher, year, edition, pages
Elsevier, 2015
Keywords
Natronomonas moolapensis strain 8.8.11(T), Halophilic archaea, Phosphatidylglycerol, Mass, spectrometry, NMR
National Category
Biochemistry and Molecular Biology Biophysics
Identifiers
urn:nbn:se:umu:diva-114643 (URN)10.1016/j.chemphyslip.2015.06.004 (DOI)000366954400002 ()26134137 (PubMedID)
Available from: 2016-01-26 Created: 2016-01-25 Last updated: 2018-06-07Bibliographically approved
Kovermann, M., Ådén, J., Grundström, C., Sauer-Eriksson, A. E., Sauer, U. H. & Wolf-Watz, M. (2015). Structural basis for catalytically restrictive dynamics of a high-energy enzyme state. Nature Communications, 6, Article ID 7644.
Open this publication in new window or tab >>Structural basis for catalytically restrictive dynamics of a high-energy enzyme state
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2015 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, article id 7644Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Macmillan Publishers Ltd., 2015
Keywords
Biological sciences, Biophysics, Biochemistry
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-106747 (URN)10.1038/ncomms8644 (DOI)000358857800018 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2015-08-06 Created: 2015-08-06 Last updated: 2018-06-07Bibliographically approved
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