Differences in Thermal Structural Changes and Melting Between Mesophilic and Thermophilic Dihydrofolate Reductase Enzymes

A key aspect of life's evolution on Earth is the adaptation of proteins to be stable and work in a very wide range of temperature conditions. A detailed understanding of the associated molecular mechanisms would also help to design enzymes optimized for biotechnological processes. Despite important advances, a comprehensive picture of how thermophilic enzymes succeed in functioning under extreme temperatures remains incomplete. Here, we examine the temperature dependence of stability and of flexibility in the mesophilic monomeric Escherichia coli (Ec) and thermophilic dimeric Thermotoga maritima (Tm) homologs of the paradigm dihydrofolate reductase (DHFR) enzyme. We use all-atom molecular dynamics simulations and a replica-exchange scheme that allows to enhance the conformational sampling while providing at the same time a detailed understanding of the enzymes' behavior at increasing temperatures. We show that this approach reproduces the stability shift between the two homologs, and provides a molecular description of the denaturation mechanism by identifying the sequence of secondary structure elements melting as temperature increases, which is not straightforwardly obtained in the experiments. By repeating our approach on the hypothetical TmDHFR monomer, we further determine the respective effects of sequence and oligomerization in the exceptional stability of TmDFHR. We show that the intuitive expectation that protein flexibility and thermal stability are correlated is not verified. Finally, our simulations reveal that significant conformational fluctuations already take place much below the melting temperature. While the difference between the TmDHFR and EcDHFR catalytic activities is often interpreted via a simplified two-state picture involving the open and closed conformations of the key M20 loop, our simulations suggest that the two homologs' markedly different activity temperature dependences are caused by changes in the ligand-cofactor distance distributions in response to these conformational changes.

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Differences in Thermal Structural Changes and Melting Between Mesophilic and Thermophilic Dihydrofolate Reductase Enzymes

10.26434/chemrxiv.12369737.v1 ◽

2020 ◽

Author(s):

Irene Maffucci ◽

Damien Laage ◽

Guillaume Stirnemann ◽

Fabio Sterpone

Keyword(s):

Dihydrofolate Reductase ◽

Conformational Changes ◽

Structural Changes ◽

Molecular Mechanisms ◽

Thermotoga Maritima ◽

Conformational Sampling ◽

Detailed Understanding ◽

Wide Range ◽

The Difference ◽

Dynamics Simulations

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Insights into the roles of charged residues in substrate binding and mode of action of mannuronan C-5 epimerase AlgE4

Glycobiology ◽

10.1093/glycob/cwab025 ◽

2021 ◽

Author(s):

Margrethe Gaardløs ◽

Sergey A Samsonov ◽

Marit Sletmoen ◽

Maya Hjørnevik ◽

Gerd Inger Sætrom ◽

...

Keyword(s):

Optical Tweezers ◽

Conformational Changes ◽

Molecular Mechanisms ◽

Hydrophobic Interactions ◽

Substrate Binding ◽

Interdisciplinary Approach ◽

Product Formation ◽

Titration Calorimetry ◽

Binding Groove ◽

Dynamics Simulations

Abstract Mannuronan C-5 epimerases catalyse the epimerization of monomer residues in the polysaccharide alginate, changing the physical properties of the biopolymer. The enzymes are utilized to tailor alginate to numerous biological functions by alginate-producing organisms. The underlying molecular mechanisms that control the processive movement of the epimerase along the substrate chain is still elusive. To study this, we have used an interdisciplinary approach combining molecular dynamics simulations with experimental methods from mutant studies of AlgE4, where initial epimerase activity and product formation were addressed with NMR spectroscopy, and characteristics of enzyme-substrate interactions were obtained with isothermal titration calorimetry and optical tweezers. Positive charges lining the substrate-binding groove of AlgE4 appear to control the initial binding of poly-mannuronate, and binding also seems to be mediated by both electrostatic and hydrophobic interactions. After the catalytic reaction, negatively charged enzyme residues might facilitate dissociation of alginate from the positive residues, working like electrostatic switches, allowing the substrate to translocate in the binding groove. Molecular simulations show translocation increments of two monosaccharide units before the next productive binding event resulting in MG-block formation, with the epimerase moving with its N-terminus towards the reducing end of the alginate chain. Our results indicate that the charge pair R343-D345 might be directly involved in conformational changes of a loop that can be important for binding and dissociation. The computational and experimental approaches used in this study complement each other, allowing for a better understanding of individual residues’ roles in binding and movement along the alginate chains.

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Insights into histidine kinase activation mechanisms from the monomeric blue light sensor EL346

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1813586116 ◽

2019 ◽

Vol 116 (11) ◽

pp. 4963-4972 ◽

Cited By ~ 5

Author(s):

Igor Dikiy ◽

Uthama R. Edupuganti ◽

Rinat R. Abzalimov ◽

Peter P. Borbat ◽

Madhur Srivastava ◽

...

Keyword(s):

Blue Light ◽

Conformational Changes ◽

Histidine Kinase ◽

Structural Changes ◽

Molecular Mechanisms ◽

Catalytic Domain ◽

Response Regulator ◽

Phosphoryl Group ◽

Dark State ◽

Light Sensing

Translation of environmental cues into cellular behavior is a necessary process in all forms of life. In bacteria, this process frequently involves two-component systems in which a sensor histidine kinase (HK) autophosphorylates in response to a stimulus before subsequently transferring the phosphoryl group to a response regulator that controls downstream effectors. Many details of the molecular mechanisms of HK activation are still unclear due to complications associated with the multiple signaling states of these large, multidomain proteins. To address these challenges, we combined complementary solution biophysical approaches to examine the conformational changes upon activation of a minimal, blue-light–sensing histidine kinase from Erythrobacter litoralis HTCC2594, EL346. Our data show that multiple conformations coexist in the dark state of EL346 in solution, which may explain the enzyme’s residual dark-state activity. We also observe that activation involves destabilization of the helices in the dimerization and histidine phosphotransfer-like domain, where the phosphoacceptor histidine resides, and their interactions with the catalytic domain. Similar light-induced changes occur to some extent even in constitutively active or inactive mutants, showing that light sensing can be decoupled from activation of kinase activity. These structural changes mirror those inferred by comparing X-ray crystal structures of inactive and active HK fragments, suggesting that they are at the core of conformational changes leading to HK activation. More broadly, our findings uncover surprising complexity in this simple system and allow us to outline a mechanism of the multiple steps of HK activation.

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Molecular mechanism of depolarization-dependent inactivation in W366F mutant of Kv1.2

10.1101/316174 ◽

2018 ◽

Author(s):

H. X. Kondo ◽

N. Yoshida ◽

M. Shirota ◽

K. Kinoshita

Keyword(s):

Membrane Potential ◽

Potassium Channels ◽

Molecular Mechanism ◽

Conformational Changes ◽

Structural Changes ◽

Membrane Depolarization ◽

Potassium Ions ◽

Selective Filter ◽

Dependent Inactivation ◽

Dynamics Simulations

ABSTRACTVoltage-gated potassium channels play crucial roles in regulating membrane potential. They are activated by membrane depolarization, allowing the selective permeation of potassium ions across the plasma membrane, and enter a nonconducting state after lasting depolarization of membrane potential, a process known as inactivation. Inactivation in voltage-activated potassium channels occurs through two distinct mechanisms, N-type inactivation and C-type inactivation. C-type inactivation is caused by conformational changes in the extracellular mouth of the channel, while N-type inactivation is elicited by changes in the cytoplasmic mouth of the protein. The W434F-mutated Shaker channel is known as a nonconducting mutant and is in a C-type inactivation state at a depolarizing membrane potential. To clarify the structural properties of C-type inactivated protein, we performed molecular dynamics simulations of the wild-type and W366F (corresponding to W434F in Shaker) mutant of the Kv1.2-2.1 chimera channel. The W366F mutant was in a nearly nonconducting state with a depolarizing voltage and recovered from inactivation with a reverse voltage. Our simulations and 3D-RISM analysis suggested that structural changes in the selective filter upon membrane depolarization trap potassium ions around the entrance of the selectivity filter and prevent ion permeation. This pore restriction is involved in the molecular mechanism of C-type inactivation.

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Molecular mechanisms of substrate-controlled ring dynamics and sub-stepping in a nucleic-acid dependent hexameric motor

10.1101/069559 ◽

2016 ◽

Author(s):

Nathan D. Thomsen ◽

Michael R. Lawson ◽

Lea B. Witkowsky ◽

Song Qu ◽

James M. Berger

Keyword(s):

Nucleic Acid ◽

Conformational Changes ◽

Molecular Motors ◽

Structural Changes ◽

Molecular Mechanisms ◽

Atp Hydrolysis ◽

Ring Closure ◽

Structural Studies ◽

Saxs Data ◽

Structural Methods

ABSTRACTRing-shaped hexameric helicases and translocases support essential DNA, RNA, and protein-dependent transactions in all cells and many viruses. How such systems coordinate ATPase activity between multiple subunits to power conformational changes that drive the engagement and movement of client substrates is a fundamental question. Using the E. coli Rho transcription termination factor as a model system, we have employed solution and crystallographic structural methods to delineate the range of conformational changes that accompany distinct substrate and nucleotide cofactor binding events. SAXS data show that Rho preferentially adopts an open-ring state in solution, and that RNA and ATP are both required to cooperatively promote ring closure. Multiple closed-ring structures with different RNA substrates and nucleotide occupancies capture distinct catalytic intermediates accessed during translocation. Our data reveal how RNA-induced ring closure templates a sequential ATP-hydrolysis mechanism, provide a molecular rationale for how the Rho ATPase domains distinguishes between distinct RNA sequences, and establish the first structural snapshots of substepping events in a hexameric helicase/translocase.SIGNIFICANCEHexameric, ring-shaped translocases are molecular motors that convert the chemical energy of ATP hydrolysis into the physical movement of protein and nucleic acid substrates. Structural studies of several distinct hexameric translocases have provided insights into how substrates are loaded and translocated; however, the range of structural changes required for coupling ATP turnover to a full cycle of substrate loading and translocation has not been visualized for any one system. Here, we combine low-and high-resolution structural studies of the Rho helicase, defining for the first time the ensemble of conformational transitions required both for substrate loading in solution and for substrate movement by a processive hexameric translocase.

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Structural insights into the synthesis of FMN in prokaryotic organisms

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004715019641 ◽

2015 ◽

Vol 71 (12) ◽

pp. 2526-2542 ◽

Cited By ~ 16

Author(s):

Beatriz Herguedas ◽

Isaias Lans ◽

María Sebastián ◽

Juan A. Hermoso ◽

Marta Martínez-Júlvez ◽

...

Keyword(s):

Conformational Changes ◽

Structural Changes ◽

Substrate Inhibition ◽

Bifunctional Enzyme ◽

Computational Simulations ◽

Binding Studies ◽

Oligomeric Protein ◽

Catalytic Sites ◽

Dynamics Simulations ◽

Structural Insights

Riboflavin kinases (RFKs) catalyse the phosphorylation of riboflavin to produce FMN. In most bacteria this activity is catalysed by the C-terminal module of a bifunctional enzyme, FAD synthetase (FADS), which also catalyses the transformation of FMN into FAD through its N-terminal FMN adenylyltransferase (FMNAT) module. The RFK module of FADS is a homologue of eukaryotic monofunctional RFKs, while the FMNAT module lacks homologyto eukaryotic enzymes involved in FAD production. Previously, the crystal structure ofCorynebacterium ammoniagenesFADS (CaFADS) was determined in its apo form. This structure predicted a dimer-of-trimers organization with the catalytic sites of two modules of neighbouring protomers approaching each other, leading to a hypothesis about the possibility of FMN channelling in the oligomeric protein. Here, two crystal structures of the individually expressed RFK module ofCaFADS in complex with the products of the reaction, FMN and ADP, are presented. Structures are complemented with computational simulations, binding studies and kinetic characterization. Binding of ligands triggers dramatic structural changes in the RFK module, which affect large portions of the protein. Substrate inhibition and molecular-dynamics simulations allowed the conformational changes that take place along the RFK catalytic cycle to be established. The influence of these conformational changes in the FMNAT module is also discussed in the context of the full-lengthCaFADS protomer and the quaternary organization.

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Computational methods for exploring protein conformations

Biochemical Society Transactions ◽

10.1042/bst20200193 ◽

2020 ◽

Vol 48 (4) ◽

pp. 1707-1724

Author(s):

Jane R. Allison

Keyword(s):

Free Energy ◽

Energy Landscape ◽

Free Energy Landscape ◽

Conformational Sampling ◽

Conformational Ensemble ◽

Collective Variables ◽

Conformational States ◽

Simulation Techniques ◽

Wide Range ◽

Dynamics Simulations

Proteins are dynamic molecules that can transition between a potentially wide range of structures comprising their conformational ensemble. The nature of these conformations and their relative probabilities are described by a high-dimensional free energy landscape. While computer simulation techniques such as molecular dynamics simulations allow characterisation of the metastable conformational states and the transitions between them, and thus free energy landscapes, to be characterised, the barriers between states can be high, precluding efficient sampling without substantial computational resources. Over the past decades, a dizzying array of methods have emerged for enhancing conformational sampling, and for projecting the free energy landscape onto a reduced set of dimensions that allow conformational states to be distinguished, known as collective variables (CVs), along which sampling may be directed. Here, a brief description of what biomolecular simulation entails is followed by a more detailed exposition of the nature of CVs and methods for determining these, and, lastly, an overview of the myriad different approaches for enhancing conformational sampling, most of which rely upon CVs, including new advances in both CV determination and conformational sampling due to machine learning.

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How low-resolution structural data predict the conformational changes of a protein: a study on data-driven molecular dynamics simulations

Physical Chemistry Chemical Physics ◽

10.1039/c8cp02246a ◽

2018 ◽

Vol 20 (26) ◽

pp. 17790-17798 ◽

Cited By ~ 2

Author(s):

Ryuhei Harada ◽

Yasuteru Shigeta

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Conformational Changes ◽

Sampling Method ◽

Protein A ◽

Structural Data ◽

Data Driven ◽

Conformational Sampling ◽

Transition Pathways ◽

Dynamics Simulations

Parallel cascade selection molecular dynamics (PaCS-MD) is a conformational sampling method for generating transition pathways between a given reactant and a product.

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An equilibrium constant pH molecular dynamics method for accurate prediction of pH-dependence in protein systems: Theory and application

10.1101/2020.11.23.394015 ◽

2020 ◽

Author(s):

Ekaterina Kots ◽

Derek M. Shore ◽

Harel Weinstein

Keyword(s):

Molecular Dynamics ◽

Equilibrium Constant ◽

Conformational Changes ◽

Ph Dependence ◽

Md Simulations ◽

Water Soluble ◽

Conformational Sampling ◽

Ph Change ◽

Constant Ph ◽

Dynamics Simulations

ABSTRACTComputational modeling and simulation of biomolecular systems at their functional pH ranges requires an accurate approach to exploring the pH dependence of conformations and interactions. Here we present a new approach – the Equilibrium Constant pH (ECpH) method – to perform conformational sampling of protein systems in the framework of molecular dynamics simulations in an N, P, T-thermodynamic ensemble. The performance of ECpH is illustrated for two proteins with experimentally determined conformational responses to pH change: the small globular water-soluble bovine b-lactoglobulin (BBL), and the dimer transmembrane antiporter CLC-ec1 Cl−/H+. We show that with computational speeds comparable to equivalent canonical MD simulations we performed, the ECpH trajectories reproduce accurately the pH-dependent conformational changes observed experimentally in these two protein systems, some of which were not seen in the corresponding canonical MD simulations.Abstract FigureTable of Contents artwork

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Dynamin-2 R465W mutation induces long range perturbation in highly ordered oligomeric structures

Scientific Reports ◽

10.1038/s41598-020-75216-0 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Fernando Hinostroza ◽

Alan Neely ◽

Ingrid Araya-Duran ◽

Vanessa Marabolí ◽

Jonathan Canan ◽

...

Keyword(s):

Conformational Changes ◽

Protein Function ◽

Molecular Mechanisms ◽

Protein Complexes ◽

Coarse Grained ◽

Cell Physiology ◽

Missense Mutations ◽

Small Change ◽

Dynamics Simulations ◽

Dynamin 2

Abstract High order oligomers are crucial for normal cell physiology, and protein function perturbed by missense mutations underlies several autosomal dominant diseases. Dynamin-2 is one of such protein forming helical oligomers that catalyze membrane fission. Mutations in this protein, where R465W is the most frequent, cause dominant centronuclear myopathy, but the molecular mechanisms underpinning the functional modifications remain to be investigated. To unveil the structural impact of this mutation in dynamin-2, we used full-atom molecular dynamics simulations and coarse-grained models and built dimers and helices of wild-type (WT) monomers, mutant monomers, or both WT and mutant monomers combined. Our results show that the mutation R465W causes changes in the interactions with neighbor amino acids that propagate through the oligomer. These new interactions perturb the contact between monomers and favor an extended conformation of the bundle signaling element (BSE), a dynamin region that transmits the conformational changes from the GTPase domain to the rest of the protein. This extended configuration of the BSE that is only relevant in the helices illustrates how a small change in the microenvironment surrounding a single residue can propagate through the oligomer structures of dynamin explaining how dominance emerges in large protein complexes.

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