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Nam, K., Arattu Thodika, A. R., Grundström, C., Sauer, U. H. & Wolf-Watz, M. (2024). Elucidating dynamics of Adenylate kinase from enzyme opening to ligand release. Journal of Chemical Information and Modeling, 64(1), 150-163
Open this publication in new window or tab >>Elucidating dynamics of Adenylate kinase from enzyme opening to ligand release
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2024 (English)In: Journal of Chemical Information and Modeling, ISSN 1549-9596, E-ISSN 1549-960X, Vol. 64, no 1, p. 150-163Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2024
National Category
Organic Chemistry
Identifiers
urn:nbn:se:umu:diva-219745 (URN)10.1021/acs.jcim.3c01618 (DOI)38117131 (PubMedID)2-s2.0-85181026300 (Scopus ID)
Funder
NIH (National Institutes of Health)
Available from: 2024-01-24 Created: 2024-01-24 Last updated: 2024-01-24Bibliographically approved
Nam, K., Shao, Y., Major, D. T. & Wolf-Watz, M. (2024). Perspectives on computational enzyme modeling: from mechanisms to design and drug development. ACS Omega, 9(7), 7393-7412
Open this publication in new window or tab >>Perspectives on computational enzyme modeling: from mechanisms to design and drug development
2024 (English)In: ACS Omega, E-ISSN 2470-1343, Vol. 9, no 7, p. 7393-7412Article, review/survey (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2024
National Category
Biochemistry and Molecular Biology Biocatalysis and Enzyme Technology
Identifiers
urn:nbn:se:umu:diva-221555 (URN)10.1021/acsomega.3c09084 (DOI)001164706400001 ()38405524 (PubMedID)2-s2.0-85185273563 (Scopus ID)
Funder
Swedish Research Council, 2021-04513
Available from: 2024-03-06 Created: 2024-03-06 Last updated: 2024-03-06Bibliographically approved
Wolf-Watz, M., Jönsson, M., Ul Mushtaq, A. & Hober, S. (2023). Calcium-dependent protein folding in a designed molecular switch. Biophysical Journal, 122(3S1), 190a-190a, Article ID 928-Pos.
Open this publication in new window or tab >>Calcium-dependent protein folding in a designed molecular switch
2023 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 122, no 3S1, p. 190a-190a, article id 928-PosArticle in journal, Meeting abstract (Refereed) Published
Abstract [en]

Allosteric regulation of protein activity is a fundamental principle for the temporal and spatial control of cellular function. Transfer of such principles to rational design of proteins will pave the way for biotechnological applications were control of a given process with external cues is desirable. Through a semi-rational and directed evolution approach we have been able to design a calcium dependent molecular switch with distinct “on” and “off” states decided by the presence and absence of calcium, respectively. The design was established in the context of protein-protein interactions with antibodies which is a massive biotechnological application linked to purification of therapeutic antibodies. Our approach was to introduce a randomized calcium binding loop into the C2 domain of Streptococcal Protein G. The large ensemble of different sequences was displayed on the surface of E. coli and subjected to selective pressures for binding to a human FAB in the presence but not in the absence of calcium. From this directed evolution experiment we discovered evolved variants that contained calcium switches with distinct “on” and “off” behavior towards FAB binding. The molecular mechanism underlying the calcium switch was discovered from quantification of both structure and dynamics with NMR spectroscopy. We found the designed protein was partially disordered in the absence of calcium, and that the disordered segment corresponded to the calcium loop and part of the FAB interaction surface of the parental C2 domain. In presence of calcium both the calcium binding loop and the FAB surface became fully structured and as a consequence the FAB binding activity was restored. Therefore, the calcium-switch in our designed protein is dictated by a “coupled folding and binding” mechanism, a principle that has evolved over and over again under natural selection in for instance intrinsically disordered proteins.

Place, publisher, year, edition, pages
Elsevier, 2023
National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-205181 (URN)10.1016/j.bpj.2022.11.1164 (DOI)36782914 (PubMedID)2-s2.0-85148093031 (Scopus ID)
Available from: 2023-02-28 Created: 2023-02-28 Last updated: 2023-02-28Bibliographically approved
Tischlik, S., Oelker, M., Rogne, P., Sauer-Eriksson, A. E., Drescher, M. & Wolf-Watz, M. (2023). Insights into Enzymatic Catalysis from Binding and Hydrolysis of Diadenosine Tetraphosphate by E. coli Adenylate Kinase. Biochemistry, 62(15), 2238-2243
Open this publication in new window or tab >>Insights into Enzymatic Catalysis from Binding and Hydrolysis of Diadenosine Tetraphosphate by E. coli Adenylate Kinase
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2023 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 62, no 15, p. 2238-2243Article in journal (Refereed) Published
Abstract [en]

Adenylate kinases play a crucial role in cellular energy homeostasis through the interconversion of ATP, AMP, and ADP in all living organisms. Here, we explore how adenylate kinase (AdK) from Escherichia coli interacts with diadenosine tetraphosphate (AP4A), a putative alarmone associated with transcriptional regulation, stress, and DNA damage response. From a combination of EPR and NMR spectroscopy together with X-ray crystallography, we found that AdK interacts with AP4A with two distinct modes that occur on disparate time scales. First, AdK dynamically interconverts between open and closed states with equal weights in the presence of AP4A. On a much slower time scale, AdK hydrolyses AP4A, and we suggest that the dynamically accessed substrate-bound open AdK conformation enables this hydrolytic activity. The partitioning of the enzyme into open and closed states is discussed in relation to a recently proposed linkage between active site dynamics and collective conformational dynamics.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2023
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-212552 (URN)10.1021/acs.biochem.3c00189 (DOI)001022362500001 ()37418448 (PubMedID)2-s2.0-85165636090 (Scopus ID)
Funder
EU, Horizon 2020, 772027─SPICE-ERC-2017-COGEU, European Research CouncilSwedish Research Council, 2019-03771Swedish Research Council, 2021-04513
Available from: 2023-08-08 Created: 2023-08-08 Last updated: 2023-08-08Bibliographically approved
Dulko-Smith, B., Ojeda-May, P., Ådén, J., Wolf-Watz, M. & Nam, K. (2023). Mechanistic basis for a connection between the catalytic step and slow opening dynamics of adenylate kinase. Journal of Chemical Information and Modeling, 63(5), 1556-1569
Open this publication in new window or tab >>Mechanistic basis for a connection between the catalytic step and slow opening dynamics of adenylate kinase
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2023 (English)In: Journal of Chemical Information and Modeling, ISSN 1549-9596, E-ISSN 1549-960X, Vol. 63, no 5, p. 1556-1569Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2023
National Category
Theoretical Chemistry
Research subject
biological chemistry
Identifiers
urn:nbn:se:umu:diva-205159 (URN)10.1021/acs.jcim.2c01629 (DOI)000936859400001 ()36802243 (PubMedID)2-s2.0-85148862970 (Scopus ID)
Funder
Swedish Research Council, 2021-04513NIH (National Institutes of Health), R01GM132481
Available from: 2023-02-24 Created: 2023-02-24 Last updated: 2023-03-17Bibliographically approved
Nam, K. & Wolf-Watz, M. (2023). Protein dynamics: the future is bright and complicated!. Structural Dynamics, 10(1), Article ID 014301.
Open this publication in new window or tab >>Protein dynamics: the future is bright and complicated!
2023 (English)In: Structural Dynamics, E-ISSN 2329-7778, Vol. 10, no 1, article id 014301Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American Crystallographic Association, 2023
National Category
Structural Biology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-205737 (URN)10.1063/4.0000179 (DOI)000940173300001 ()36865927 (PubMedID)2-s2.0-85149439696 (Scopus ID)
Funder
Swedish Research Council, 2021-04513
Available from: 2023-03-16 Created: 2023-03-16 Last updated: 2023-09-05Bibliographically approved
Verma, A., Åberg-Zingmark, E., Sparrman, T., Ul Mushtaq, A., Rogne, P., Grundström, C., . . . Wolf-Watz, M. (2022). Insights into the evolution of enzymatic specificity and catalysis: from Asgard archaea to human adenylate kinases [Letter to the editor]. Science Advances, 8(44), Article ID eabm4089.
Open this publication in new window or tab >>Insights into the evolution of enzymatic specificity and catalysis: from Asgard archaea to human adenylate kinases
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2022 (English)In: Science Advances, E-ISSN 2375-2548, Vol. 8, no 44, article id eabm4089Article in journal, Letter (Refereed) Published
Abstract [en]

Enzymatic catalysis is critically dependent on selectivity, active site architecture, and dynamics. To contribute insights into the interplay of these properties, we established an approach with NMR, crystallography, and MD simulations focused on the ubiquitous phosphotransferase adenylate kinase (AK) isolated from Odinarchaeota (OdinAK). Odinarchaeota belongs to the Asgard archaeal phylum that is believed to be the closest known ancestor to eukaryotes. We show that OdinAK is a hyperthermophilic trimer that, contrary to other AK family members, can use all NTPs for its phosphorylation reaction. Crystallographic structures of OdinAK-NTP complexes revealed a universal NTP-binding motif, while 19F NMR experiments uncovered a conserved and rate-limiting dynamic signature. As a consequence of trimerization, the active site of OdinAK was found to be lacking a critical catalytic residue and is therefore considered to be "atypical." On the basis of discovered relationships with human monomeric homologs, our findings are discussed in terms of evolution of enzymatic substrate specificity and cold adaptation.

Place, publisher, year, edition, pages
American Association for the Advancement of Science (AAAS), 2022
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-201106 (URN)10.1126/sciadv.abm4089 (DOI)000918406800003 ()36332013 (PubMedID)2-s2.0-85141889911 (Scopus ID)
Funder
Swedish Research Council, 2017-04203Swedish Research Council, 2019-03771Swedish Research Council, 2016-03599Knut and Alice Wallenberg Foundation, 2016-03599The Kempe Foundations, SMK-1869Carl Tryggers foundation , 17.504NIH (National Institutes of Health), (R01GM132481
Note

The Protein Expertise Platform (PEP) at the Umeå University is acknowledged for providing reagents for protein production, and M. Lindberg at PEP is appreciated for preparation of plasmids. We acknowledge MAX IV Laboratory (Lund, Sweden) for time on BioMAX and DESY (Hamburg, Germany) for time on PETRA-3. All NMR experiments were performed at the Swedish NMR Center at Umeå University. We also acknowledge the Swedish National Infrastructure for Computing (SNIC) at the High Performance Computing Center North (HPC2N) and the National Energy Research Scientific Computing Center (NERSC) for computational resources.

Available from: 2022-11-19 Created: 2022-11-19 Last updated: 2023-09-05Bibliographically approved
Ojeda-May, P., Ul Mushtaq, A., Rogne, P., Verma, A., Ovchinnikov, V., Grundström, C., . . . Nam, K. (2021). Dynamic Connection between Enzymatic Catalysis and Collective Protein Motions. Biochemistry, 60(28), 2246-2258
Open this publication in new window or tab >>Dynamic Connection between Enzymatic Catalysis and Collective Protein Motions
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2021 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 60, no 28, p. 2246-2258Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2021
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-187197 (URN)10.1021/acs.biochem.1c00221 (DOI)000677482100003 ()34250801 (PubMedID)2-s2.0-85111203330 (Scopus ID)
Available from: 2021-09-08 Created: 2021-09-08 Last updated: 2021-09-08Bibliographically approved
Phoeurk, C., Ul Mushtaq, A., Rogne, P. & Wolf-Watz, M. (2021). Milligram scale expression, refolding, and purification of Bombyx mori cocoonase using a recombinant E. coli system. Protein Expression and Purification, 186, Article ID 105919.
Open this publication in new window or tab >>Milligram scale expression, refolding, and purification of Bombyx mori cocoonase using a recombinant E. coli system
2021 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 186, article id 105919Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier, 2021
Keywords
Cocoonase, Escherichia coli, Refolding, Serine protease, Silk moth (Bombyx mori)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-184200 (URN)10.1016/j.pep.2021.105919 (DOI)000671874800003 ()2-s2.0-85106960638 (Scopus ID)
Funder
Swedish Research Council, 2017–04203The Kempe Foundations, JCK-1417
Available from: 2021-06-14 Created: 2021-06-14 Last updated: 2023-09-05Bibliographically approved
Orädd, F., Ravishankar, H., Goodman, J., Rogne, P., Backman, L., Duelli, A., . . . Andersson, M. (2021). Tracking the ATP-binding response in adenylate kinase in real time. Science Advances, 7(47), Article ID eabi5514.
Open this publication in new window or tab >>Tracking the ATP-binding response in adenylate kinase in real time
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2021 (English)In: Science Advances, E-ISSN 2375-2548, Vol. 7, no 47, article id eabi5514Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American Association for the Advancement of Science, 2021
Keywords
Multidisciplinary
National Category
Natural Sciences Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-189986 (URN)10.1126/sciadv.abi5514 (DOI)000720347400008 ()34788091 (PubMedID)2-s2.0-85119418495 (Scopus ID)
Funder
Swedish Research Council, 2017-04203Swedish Research Council, 2020-03840
Available from: 2021-11-29 Created: 2021-11-29 Last updated: 2024-02-22Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-9098-7974

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