Conformational Sampling von Enzym Dynamik: Triosephosphate Isomerase

Triosephosphat Isomerase ist ein Enzym, welches fü die glykolytische Interkonversion von Dihydroxyacetonephosphat zu Glyceraldehyde-3-phosphat zuständig ist. TIM ist ein Dimer und jedes Monomer besteht aus 249 Aminosäuren. Das aktive Zentrum besteht aus drei verschiedenen Schleifen Loop-6, 7 und schleifenförmigen Loop-8. Basierend auf Loop-6-und Loop-7 Konformation wir beschreiben das Enzym als Open TIM und Closed TIM. Verschiedene NMR, Röntgenkristallographie und QM / MM Simulationstechniken haben Einblicke in einzelne Ereignisse eines, im Wesentlichen, dynamischen Prozesses. Mittels Mikrosekunde-langer atomistischen MD Simulationen untersuchten wir die Konformationsänderungen von zwei getrennten Schleifen (Loop-6 und Loop-7), welche die aktiven Stelle umwickeln, in Gegenwart von natürlichem Substrat, Reaktionszwischenprodukten und Inhibitor-Molekülen. Unsere Untersuchungen haben ergeben, dass loop-6 offene und geschlossene Konformationen sowohl Apo- als auch Holo TIM Strukturen bemustern. Wir beobachten auch, dass die Loop-6 N-Terminus und C-Terminus unabhängig sind. In unseren Simulationen haben wir auch festgestellt, dass Rückgrat Diederwinkel der Schleife-7 Rückstände G210 (G210-phi, G210-psi) und G211 (G211-phi) offenen und geschlossenen Zuständen sowohl Apo-und Holo TIM Strukturen bemustern. Dagegen Rückgrat Diederwinkel von G211 (G211-psi) und S212 (S212-phi) nehmen die geschlossene Konformation an nur wenn das Ligandenmolekül an das aktive Zentrum gebunden ist. Wie in der Kette-B 1R2R der Kristallstrukturen beobachtet, beobachten wir auch, dass Wassermoleküle auch Flip G211-psi und S212-phi Diederwinkel in geschlossene Konformation initiieren können.Triosephosphate Isomerase is a glycolytic enzyme catalyzing the interconversion of Dihydroxyacetone phosphate to Glyceraldehyde-3-phosphate. TIM is a dimer and each monomer is comprised of 249 amino acids. The active site is comprised of three distinct loops loop-6, loop-7 and loop-8. Based on loop-6 and loop-7 conformation we describe the enzyme as Open TIM and Closed TIM. Various NMR, X-ray crystallography and QM/MM simulation techniques have provided glimpses of individual events of what is essentially a dynamic process. We studied the conformational changes of two distinct loops (loop-6 and loop-7) enveloping the active site, in the presence of natural substrate, reaction intermediates and inhibitor molecules, by means of microsecond atomistic MD simulations. Our studies have revealed that loop-6 samples open and closed conformations in both apo and holo TIM structures. We also observe that loop-6 N-terminus and C-terminus move independently. In our simulations we have also observed that backbone dihedrals of loop-7 residues G210 (G210-phi, G210-psi) and G211 (G211-phi) sample open and closed states in both apo and holo TIM structures. Whereas backbone dihedral angles of G211 (G211-psi) and S212 (S212-phi) adopt closed conformation only when the ligand molecule is bound to the active site. As observed in chain-B of 1R2R crystal structures, we also observe that water molecules can also initiate flip of G211-psi and S212-phi dihedral angles into closed conformation

Dynamic Profiling of β-Coronavirus 3CL M^proProtease Ligand-Binding Sites

Data availability statement: The trajectories of Mpro simulations and models of the metastable states can be downloaded from 10.5281/zenodo.4782284.β-coronavirus (CoVs) alone has been responsible for three major global outbreaks in the 21st century. The current crisis has led to an urgent requirement to develop therapeutics. Even though a number of vaccines are available, alternative strategies targeting essential viral components are required as a backup against the emergence of lethal viral variants. One such target is the main protease (Mpro) that plays an indispensable role in viral replication. The availability of over 270 Mpro X-ray structures in complex with inhibitors provides unique insights into ligand–protein interactions. Herein, we provide a comprehensive comparison of all nonredundant ligand-binding sites available for SARS-CoV2, SARS-CoV, and MERS-CoV Mpro. Extensive adaptive sampling has been used to investigate structural conservation of ligand-binding sites using Markov state models (MSMs) and compare conformational dynamics employing convolutional variational auto-encoder-based deep learning. Our results indicate that not all ligand-binding sites are dynamically conserved despite high sequence and structural conservation across β-CoV homologs. This highlights the complexity in targeting all three Mpro enzymes with a single pan inhibitor.There was no funding for this wor

Conformational dynamics of Peb4 exhibit “mother’s arms” chain model: a molecular dynamics study

<p>Peb4 from <i>Campylobacter jejuni</i> is an intertwined dimeric, periplasmic holdase, which also exhibits peptidyl prolyl cis/trans isomerase (PPIase) activity. Peb4 gene deletion alters the outer membrane protein profile and impairs cellular adhesion and biofilm formation for <i>C. jejuni</i>. Earlier crystallographic study has proposed that the PPIase domains are flexible and might form a cradle for holding the substrate and these aspects of Peb4 were explored using sub-microsecond molecular dynamics simulations in solution environment. Our simulations have revealed that PPIase domains are highly flexible and undergo a large structural change where they move apart from each other by 8 nm starting at .5 nm. Further, this large conformational change renders Peb4 as a compact protein with crossed-over conformation, forms a central cavity, which can “cradle” the target substrate. As reported for other chaperone proteins, flexibility of linker region connecting the chaperone and PPIase domains is key to forming the “crossed-over” conformation. The conformational transition of the Peb4 protein from the X-ray structure to the crossed-over conformation follows the “mother’s arms” chain model proposed for the FkpA chaperone protein. Our results offer insights into how Peb4 and similar chaperones can use the conformational heterogeneity at their disposal to perform its much-revered biological function.</p

Cleaving DNA with DNA: Cooperative tuning of structure and reactivity driven by copper ions

International audienceA copper‐dependent self‐cleaving DNA (DNAzyme or deoyxyribozyme) previously isolated by in vitro selection has been analyzed by a combination of Molecular Dynamics (MD) simulations and advanced Electron Paramagnetic Resonance (Electron Spin Resonance) EPR/ESR spectroscopy, providing insights on the structural and mechanistic features of the cleavage reaction. The modeled 46‐nucleotide deoxyribozyme in MD simulations forms duplex and triplex sub‐structures that flank a highly conserved catalytic core. The DNA self‐cleaving construct can also form a bimolecular complex that has a distinct substrate and enzyme domains. The highly dynamic structure combined with an oxidative site‐specific cleavage of the substrate are two key‐aspects to elucidate. By combining EPR/ESR spectroscopy with selectively isotopically labeled nucleotides it has been possible to overcome the major drawback related to the “metal‐soup” scenario, also known as “super‐stoichiometric” ratios of cofactors versus substrate, conventionally required for the DNA cleavage reaction within those nucleic acids‐based enzymes. The focus on the endogenous paramagnetic center (Cu$^{2+}$) here described paves the way for analysis on mixtures where several different cofactors are involved. Furthermore, the insertion of cleavage reaction within more complex architectures is now a realistic perspective towards the applicability of EPR/ESR spectroscopic studies

Structural insights into dynamics of RecU-HJ complex formation elucidates key role of NTR and stalk region toward formation of reactive state

© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. Holliday junction (HJ) resolving enzyme RecU is involved in DNA repair and recombination. We have determined the crystal structure of inactive mutant (D88N) of RecU from Bacillus subtilis in complex with a 12 base palindromic DNA fragment at a resolution of 3.2 Å. This structure shows the stalk region and the essential N-terminal region (NTR) previously unseen in our DNA unbound structure. The flexible nature of the NTR in solution was confirmed using SAXS. Thermofluor studies performed to assess the stability of RecU in complex with the arms of an HJ indicate that it confers stability. Further, we performed molecular dynamics (MD) simulations of wild type and an NTR deletion variant of RecU, with and without HJ. The NTR is observed to be highly flexible in simulations of the unbound RecU, in agreement with SAXS observations. These simulations revealed domain dynamics of RecU and their role in the formation of complex with HJ. The MD simulations also elucidate key roles of the NTR, stalk region, and breathing motion of RecU in the formation of the reactive state.BBSRC UK grant BB/E017576/1; Indian Institute of Technology, Bomba