scholarly journals Conformational dynamics of active site loops 5, 6 and 7 of enzyme Triosephosphate Isomerase: A molecular dynamics study

2018 ◽  
Author(s):  
Sarath Chandra Dantu ◽  
Gerrit Groenhof
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Triosephosphate Isomerase ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
Glycolytic Enzyme ◽  
Dihedral Angles ◽  
Dihydroxyacetone Phosphate ◽  
Closed Conformation ◽  
N Terminus

AbstractTriosephosphate Isomerase is a glycolytic enzyme catalyzing the interconversion of Dihydroxyacetone phosphate to Glyceraldehyde-3-phosphate. 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 in solution and crystal environment. Our studies have revealed that loop-6 samples open and closed conformations in both apo and holo TIM structures. As seen in solution state NMR experiments, 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 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. Except, loop-5, which has a dominant effect on the conformational behaviour of loop-6 N-terminus, we do not observe any influence of either loop-6 or loop-7 on the conformational dynamics of the other.

The kinetic consequences of altering the catalytic residues of triosephosphate isomerase

1986 ◽  
Vol 317 (1540) ◽  
pp. 371-380 ◽  
Keyword(s):  
Active Site ◽  
Triosephosphate Isomerase ◽  
Glycolytic Enzyme ◽  
Enzyme Mechanism ◽  
Site Directed Mutagenesis ◽  
Dihydroxyacetone Phosphate ◽  
Free Energies ◽  
Wild Type ◽  
Basic Residue ◽  
And Function

The essential basic residue at the active site of the glycolytic enzyme triosephosphate isomerase is Glu-165, which is responsible for the abstraction of either the 1-pro-R proton of dihydroxyacetone phosphate or of the 2-proton of D-glyceraldehyde 3-phosphate, in the enolization steps that constitute the reaction catalysed by this enzyme. We have changed this residue to Asp by oligonucleotide-mediated site-directed mutagenesis, and have evaluated the free-energy profile for the mutant protein. Comparison of the detailed energetics of the wild-type and mutant enzymes shows that only the transition-state free energies have been seriously affected, each of the enolization steps having been slowed by a factor of about one thousand. Evidently the movement of a catalytic group by less than 1 Å (1 Å = 10 -1 nm = 10 -10 m) has dramatic effects on catalysis, and the nature of these effects can provide important information about enzyme mechanism and function.

scholarly journals Correlation of membrane protein conformational and functional dynamics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Raghavendar Reddy Sanganna Gari ◽  
Joel José Montalvo‐Acosta ◽  
George R. Heath ◽  
Yining Jiang ◽  
Xiaolong Gao ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Conformational Changes ◽  
Single Channel ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
High Temporal Resolution ◽  
Dependent Manner ◽  
Functional Dynamics ◽  
Ph Dependent ◽  
Open States

AbstractConformational changes in ion channels lead to gating of an ion-conductive pore. Ion flux has been measured with high temporal resolution by single-channel electrophysiology for decades. However, correlation between functional and conformational dynamics remained difficult, lacking experimental techniques to monitor sub-millisecond conformational changes. Here, we use the outer membrane protein G (OmpG) as a model system where loop-6 opens and closes the β-barrel pore like a lid in a pH-dependent manner. Functionally, single-channel electrophysiology shows that while closed states are favored at acidic pH and open states are favored at physiological pH, both states coexist and rapidly interchange in all conditions. Using HS-AFM height spectroscopy (HS-AFM-HS), we monitor sub-millisecond loop-6 conformational dynamics, and compare them to the functional dynamics from single-channel recordings, while MD simulations provide atomistic details and energy landscapes of the pH-dependent loop-6 fluctuations. HS-AFM-HS offers new opportunities to analyze conformational dynamics at timescales of domain and loop fluctuations.

scholarly journals Structure of an ancestral ADP-dependent kinase with fructose-6P reveals key residues for binding, catalysis, and ligand-induced conformational changes

2020 ◽  
pp. jbc.RA120.015376
Author(s):  
Sebastián M. Muñoz ◽  
Victor Castro-Fernandez ◽  
Victoria Guixe
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Substrate Specificities ◽  
Closed Conformation ◽  
Sugar Binding ◽  
Bifunctional Enzymes ◽  
Glucose Binding ◽  
Critical Residue ◽  
Key Residues ◽  
Human And Mouse

ADP-dependent kinases were first described in archaea, although their presence has also been reported in bacteria and eukaryotes (human and mouse). This enzyme family comprises three substrate specificities; specific phosphofructokinases (ADP-PFK), specific glucokinases (ADP-GK), and bifunctional enzymes (ADP-PFK/GK). Although many structures are available for members of this family, no one exhibits fructose-6P at the active site. Employing an ancestral enzyme, we obtain the first structure of an ADP-dependent kinase (AncMsPFK) with fructose-6P at its active site. Key residues for sugar-binding and catalysis were identified by alanine scanning, being D36 a critical residue for F6P binding and catalysis. However, this residue hinders glucose binding since its mutation to alanine converts the AncMsPFK enzyme into a specific ADP-GK. Residue K179 is critical for F6P binding, while residues N181 and R212 are also important for this sugar-binding, but to a lesser extent. This structure also provides evidence for the requirement of both substrates (sugar and nucleotide) to accomplish the conformational change leading to a closed conformation. This suggests that AncMsPFK mainly populates two states (open and closed) during the catalytic cycle, as reported for specific ADP-PFK. This situation differs from that described for specific ADP-GK enzymes, where each substrate independently causes a sequential domain closure, resulting in three conformational states (open, semi-closed and closed).

scholarly journals High-Speed AFM directly visualizes conformational dynamics of the HIV-Vif protein complex

2020 ◽  
Author(s):  
Yangang Pan ◽  
Luda S. Shlyakhtenko ◽  
Yuri L. Lyubchenko
Keyword(s):  
Conformational Changes ◽  
Protein Complex ◽  
High Speed ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
Time Lapse ◽  
Hiv Treatment ◽  
X Ray Crystallography ◽  
Host Cell Proteins ◽  
Virus Survival

AbstractViral infectivity factor (Vif) is a protein that is essential for the replication of the HIV-1 virus. The key function of Vif is to disrupt the antiviral activity of APOBEC3 proteins, which mutate viral nucleic acids. Inside the cell, Vif binds to the host cell proteins Elongin-C, Elongin-B, and CBF-β, forming a four-protein complex called VCBC. The structure of VCBC in complex with the Cullin5 (Cul5) protein has been solved by X-ray crystallography, and recently, using molecular dynamic (MD) simulations, the dynamics of VCBC and VCBC-Cul5 complexes were characterized. Here, we applied time-lapse high-speed atomic force microscopy (HS-AFM) to visualize the conformational changes of the VCBC complex. We determined the three most favorable conformations of the VCBC complex, which we identified as triangle, dumbbell, and globular structures. In addition, we characterized the dynamics of each of these structures. While our data show a very dynamic behavior for all these structures, we found the triangle and dumbbell structures to be the most dynamic. These findings provide insight into the structure and dynamics of the VCBC complex and support further research into the improvement of HIV treatment, as Vif is essential for virus survival in the cell.

scholarly journals Resistance from Afar: Distal Mutation V36M Allosterically Modulates the Active Site to Accentuate Drug Resistance in HCV NS3/4A Protease

2018 ◽  
Author(s):  
Ayşegül Özen ◽  
Kuan-Hung Lin ◽  
Keith P Romano ◽  
Davide Tavella ◽  
Alicia Newton ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Active Site ◽  
Md Simulations ◽  
Drug Susceptibility ◽  
Active Site Residues ◽  
Allosteric Coupling ◽  
Direct Acting ◽  
Distal Mutation ◽  
Active Site Mutations

AbstractHepatitis C virus rapidly evolves, conferring resistance to direct acting antivirals. While resistance via active site mutations in the viral NS3/4A protease has been well characterized, the mechanism for resistance of non-active site mutations is unclear. R155K and V36M often co-evolve and while R155K alters the electrostatic network at the binding site, V36M is more than 13 Å away. In this study the mechanism by which V36M confers resistance, in the context of R155K, is elucidated with drug susceptibility assays, crystal structures, and molecular dynamics (MD) simulations for three protease inhibitors: telaprevir, boceprevir and danoprevir. The R155K and R155K/V36M crystal structures differ in the α-2 helix and E2 strand near the active site, with alternative conformations at M36 and side chains of active site residues D168 and R123, revealing an allosteric coupling, which persists dynamically in MD simulations, between the distal mutation and the active site. This allosteric modulation validates the network hypothesis and elucidates how distal mutations confer resistance through propagation of conformational changes to the active site.

scholarly journals Assessment of the PETase Conformational Changes Induced by Poly(ethylene terephthalate) Binding

2021 ◽  
Author(s):  
Clauber Henrique Costa ◽  
Alberto dos Santos ◽  
Cláudio Nahum Alves ◽  
Sérgio Martí ◽  
Vicente Moliner ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Conformational Changes ◽  
Active Site ◽  
Terephthalic Acid ◽  
Structural Changes ◽  
Ethylene Terephthalate ◽  
Md Simulations ◽  
Poly Ethylene Terephthalate ◽  
Bounded State ◽  
Poly Ethylene

Recently, a bacterium strain of Ideonella sakaiensis was identified with the uncommon ability to degrade the poly(ethylene terephthalate) (PET). The PETase from I. sakaiensis strain 201-F6 catalyzes the hydrolysis of PET converting it to mono(2-hydroxyethyl) terephthalic acid (MHET), bis(2-hydroxyethyl)-TPA (BHET), and terephthalic acid (TPA). Despite the potential of this enzyme for mitigation or elimination of environmental contaminants, one of the limitations of the use of PETase for PET degradation is the fact that it acts only at moderate temperature due to its low thermal stability. Besides, molecular details of the main interaction of PET in the active site of PETase remains unclear. Herein, molecular docking and molecular dynamics (MD) simulations were applied to analyze structural changes of PETase induced by PET binding. Results from the essential dynamics revealed that β1-β2 connecting loop is very flexible. This Loop is located far from the active site of PETase and we suggest that it can be considered for mutagenesis in order to increase the thermal stability of PETase. The free energy landscape (FEL) demonstrates that the main change in the transition between the unbounded to the bounded state is associated with β7-α5 connecting loop, where the catalytic residue Asp206 is located. Overall, the present study provides insights into the molecular binding mechanism of PET into the PETase structure and a computational strategy for mapping flexible regions of this enzyme, which can be useful for the engineering of more efficient enzymes for recycling the plastic polymers using biological systems.

scholarly journals Coupling of HSP72 α-Helix Subdomains by the Unexpected Irreversible Targeting of Lysine-56 over Cysteine-17; Coevolution of Covalent Bonding

Molecules ◽  
2020 ◽  
Vol 25 (18) ◽  
pp. 4239
Author(s):  
Aimen Aljoundi ◽  
Ahmed El Rashedy ◽  
Patrick Appiah-Kubi ◽  
Mahmoud E. S. Soliman
Keyword(s):  
Conformational Changes ◽  
Conformational Dynamics ◽  
Salt Bridge ◽  
Structural Basis ◽  
Irreversible Inhibitor ◽  
Closed Conformation ◽  
Covalent Inhibition ◽  
Covalent Inhibitors ◽  
Biological Target ◽  
Α Helix

Covalent inhibition has recently gained a resurgence of interest in several drug discovery areas. The expansion of this approach is based on evidence elucidating the selectivity and potency of covalent inhibitors when bound to particular amino acids of a biological target. The unexpected covalent inhibition of heat shock protein 72 (HSP72) by covalently targeting Lys-56 instead of Cys-17 was an interesting observation. However, the structural basis and conformational changes associated with this preferential coupling to Lys-56 over Cys-17 remain unclear. To resolve this mystery, we employed structural and dynamic analyses to investigate the structural basis and conformational dynamics associated with the unexpected covalent inhibition. Our analyses reveal that the coupling of the irreversible inhibitor to Lys-56 is intrinsically less dynamic than Cys-17. Conformational dynamics analyses further reveal that the coupling of the inhibitor to Lys-56 induced a closed conformation of the nucleotide-binding subdomain (NBD) α-helices, in contrast, an open conformation was observed in the case of Cys-17. The closed conformation maintained the crucial salt-bridge between Glu-268 and Lys-56 residues, which strengthens the interaction affinity of the inhibitor nearly identical to adenosine triphosphate (ADP/Pi) bound to the HSP72-NBD. The outcome of this report provides a substantial shift in the conventional direction for the design of more potent covalent inhibitors.

Crystallographic binding studies with an engineered monomeric variant of triosephosphate isomerase

2010 ◽  
Vol 66 (8) ◽  
pp. 934-944 ◽  
Author(s):  
Mikko Salin ◽  
Evangelia G. Kapetaniou ◽  
Matti Vaismaa ◽  
Marja Lajunen ◽  
Marco G. Casteleijn ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Active Site ◽  
Triosephosphate Isomerase ◽  
Binding Pocket ◽  
Sugar Phosphate ◽  
Wild Type ◽  
Binding Studies ◽  
Closed Conformation ◽  
Transition State Analogue ◽  
Site Binding

Crystallographic binding studies have been carried out to probe the active-site binding properties of a monomeric variant (A-TIM) of triosephosphate isomerase (TIM). These binding studies are part of a structure-based directed-evolution project aimed towards changing the substrate specificity of monomeric TIM and are therefore aimed at finding binders which are substrate-like molecules. A-TIM has a modified more extended binding pocket between loop-7 and loop-8 compared with wild-type TIM. The A-TIM crystals were grown in the presence of citrate, which is bound in the active site of each of the two molecules in the asymmetric unit. In this complex, the active-site loops loop-6 and loop-7 adopt the closed conformation, similar to that observed in liganded wild-type TIM. Extensive crystal-soaking protocols have been developed to flush the bound citrate out of the active-site pocket of both molecules and the crystal structure shows that the unliganded open conformation of the A-TIM active site is the same as in unliganded wild-type TIM. It is also shown that sulfonate compounds corresponding to the transition-state analogue 2-phosphoglycolate bind in the active site, which has a closed conformation. It is also shown that the new binding pocket of A-TIM can bind 3-phosphoglycerate (3PGA; an analogue of a C4-sugar phosphate) and 4-phospho-D-erythronohydroxamic acid (4PEH; an analogue of a C5-sugar phosphate). Therefore, these studies have provided a rationale for starting directed-evolution experiments aimed at generating the catalytic properties of a C5-sugar phosphate isomerase on the A-TIM framework.

scholarly journals SARS-CoV-2 variants impact RBD conformational dynamics and ACE2 accessibility

2021 ◽  
Author(s):  
Mariana Fidalgo Valerio ◽  
Luis Borges-Araujo ◽  
Manuel N. Melo ◽  
Diana Lousa ◽  
Claudio Soares
Keyword(s):  
Conformational Dynamics ◽  
Md Simulations ◽  
Natural Immunity ◽  
Angiotensin Converting Enzyme 2 ◽  
Closed Conformation ◽  
Fitness Advantage ◽  
Open Conformation ◽  
Binding Surface ◽  
Difficult Challenge ◽  
Host Cell Membranes

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has killed over 5 million people and is causing a devastating social and economic impact all over the world. The rise of new variants of concern (VOCs) represents a difficult challenge due to the loss vaccine and natural immunity, and increased transmissibility. All circulating VOCs contain mutations in the spike glycoprotein, which mediates fusion between the viral and host cell membranes, via its receptor binding domain (RBD) that binds to angiotensin-converting enzyme 2 (ACE2). In an attempt to understand the effect of RBD mutations in circulating VOCs, a lot of attention has been given to the RBD-ACE2 interaction. However, this type of analysis is limited, since it ignores more indirect effects, such as the conformational dynamics of the RBD itself. Observing that some VOCs mutations occur in residues that are not in direct contact with ACE2, we hypothesized that they could affect RBD conformational dynamics. To test this, we performed long atomistic (AA) molecular dynamics (MD) simulations to investigate the structural dynamics of wt RBD, and that of three circulating VOCs (alpha, beta, and delta). Our results show that in solution, wt RBD presents two distinct conformations: an open conformation where it is free to bind ACE2; and a closed conformation, where the RBM ridge blocks the binding surface. The alpha and beta variants significantly impact the open/closed equilibrium, shifting it towards the open conformation by roughly 20%. This shift likely increases ACE2 binding affinity. Simulations of the currently predominant delta variant RBD were extreme in this regard, in that a closed conformation was never observed. Instead, the system alternated between the before mentioned open conformation and an alternative reversed one, with a significantly changed orientation of the RBM ridge flanking the RBD. This alternate conformation could potentially provide a fitness advantage not only due to increased availability for ACE2 binding, but also by aiding antibody escape through epitope occlusion. These results support the hypothesis that VOCs, and particularly the delta variant, impact RBD conformational dynamics in a direction that simultaneously promotes efficient binding to ACE2 and antibody escape.

scholarly journals Conformational Transition Pathways in Major Facilitator Superfamily Transporters

2019 ◽  
Author(s):  
Dylan Ogden ◽  
Kalyan Immadisetty ◽  
Mahmoud Moradi
Keyword(s):  
Conformational Changes ◽  
Glucose Transporter ◽  
Conformational Transition ◽  
Conformational Dynamics ◽  
Salt Bridge ◽  
Md Simulations ◽  
Major Facilitator Superfamily ◽  
Glucose Transporter 1 ◽  
Major Facilitator ◽  
Mfs Transporters

AbstractMajor facilitator superfamily (MFS) of transporters consists of three classes of membrane transporters: symporters, uniporters, and antiporters. Despite such diverse functions, MFS transporters are believed to undergo similar conformational changes within their distinct transport cycles. While the similarities between conformational changes are noteworthy, the differences are also important since they could potentially explain the distinct functions of symporters, uniporters, and antiporters of MFS superfamily. We have performed a variety of equilibrium, non-equilibrium, biased, and unbiased all-atom molecular dynamics (MD) simulations of bacterial proton-coupled oligopeptide transporter GkPOT, glucose transporter 1 (GluT1), and glycerol-3-phosphate transporter (GlpT) to compare the similarities and differences of the conformational dynamics of three different classes of transporters. Here we have simulated the apo protein in an explicit membrane environment. Our results suggest a very similar conformational transition involving interbundle salt-bridge formation/disruption coupled with the orientation changes of transmembrane (TM) helices, specifically H1/H7 and H5/H11, resulting in an alternation in the accessibility of water at the cyto- and periplasmic gates.

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