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 High-speed atomic force microscopy directly visualizes conformational dynamics of the HIV Vif protein in complex with three host proteins

2020 ◽  
Vol 295 (34) ◽  
pp. 11995-12001
Author(s):  
Yangang Pan ◽  
Luda S. Shlyakhtenko ◽  
Yuri L. Lyubchenko
Keyword(s):  
Atomic Force Microscopy ◽  
Conformational Changes ◽  
High Speed ◽  
Conformational Dynamics ◽  
Time Lapse ◽  
Hiv Treatment ◽  
Force Microscopy ◽  
X Ray Crystallography ◽  
Atomic Force ◽  
Dynamics Simulations

Vif (viral infectivity factor) 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 host APOBEC3 (apolipoprotein B mRNA-editing enzyme catalytic subunit 3) proteins, which mutate viral nucleic acids. Inside the cell, Vif binds to the host cell proteins Elongin-C, Elongin-B, and core-binding factor subunit β, forming a four-protein complex called VCBC. The structure of VCBC–Cullin5 has recently been solved by X-ray crystallography, and, using molecular dynamics simulations, the dynamics of VCBC have been characterized. Here, we applied time-lapse high-speed atomic force microscopy to visualize the conformational changes of the VCBC complex. We determined the three most favorable conformations of this complex, which we identified as the triangle, dumbbell, and globular structures. Moreover, we characterized the dynamics of each of these structures. Our data revealed the very dynamic behavior of all of them, with the triangle and dumbbell structures being the most dynamic. These findings provide insight into the structure and dynamics of the VCBC complex and may support efforts to improve HIV treatment, because Vif is essential for virus survival in the cell.

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.

Quantitative studies of ribosome conformational dynamics

2007 ◽  
Vol 40 (2) ◽  
pp. 163-189 ◽  
Author(s):  
Christopher S. Fraser ◽  
Jennifer A. Doudna
Keyword(s):  
Protein Synthesis ◽  
Single Molecule ◽  
Conformational Changes ◽  
Conformational Dynamics ◽  
Start Codon ◽  
X Ray Crystallography ◽  
Initiation Factors ◽  
Quantitative Studies ◽  
Order Of Magnitude ◽  
Protein Synthesis Initiation

AbstractThe ribosome is a dynamic machine that undergoes many conformational rearrangements during the initiation of protein synthesis. Significant differences exist between the process of protein synthesis initiation in eubacteria and eukaryotes. In particular, the initiation of eukaryotic protein synthesis requires roughly an order of magnitude more initiation factors to promote efficient mRNA recruitment and ribosomal recognition of the start codon than are needed for eubacterial initiation. The mechanisms by which these initiation factors promote ribosome conformational changes during stages of initiation have been studied using cross-linking, footprinting, site-directed probing, cryo-electron microscopy, X-ray crystallography, fluorescence spectroscopy and single-molecule techniques. Here, we review how the results of these different approaches have begun to converge to yield a detailed molecular understanding of the dynamic motions that the eukaryotic ribosome cycles through during the initiation of protein synthesis.

scholarly journals A chimeric prokaryotic pentameric ligand–gated channel reveals distinct pathways of activation

2015 ◽  
Vol 146 (4) ◽  
pp. 323-340 ◽  
Author(s):  
Nicolaus Schmandt ◽  
Phanindra Velisetty ◽  
Sreevatsa V. Chalamalasetti ◽  
Richard A. Stein ◽  
Ross Bonner ◽  
...  
Keyword(s):  
Ion Channel ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Conformational Dynamics ◽  
Primary Amines ◽  
Molecular Architecture ◽  
X Ray Crystallography ◽  
Gated Channel ◽  
Activation Mechanisms ◽  
Double Electron Electron Resonance

Recent high resolution structures of several pentameric ligand–gated ion channels have provided unprecedented details of their molecular architecture. However, the conformational dynamics and structural rearrangements that underlie gating and allosteric modulation remain poorly understood. We used a combination of electrophysiology, double electron–electron resonance (DEER) spectroscopy, and x-ray crystallography to investigate activation mechanisms in a novel functional chimera with the extracellular domain (ECD) of amine-gated Erwinia chrysanthemi ligand–gated ion channel, which is activated by primary amines, and the transmembrane domain of Gloeobacter violaceus ligand–gated ion channel, which is activated by protons. We found that the chimera was independently gated by primary amines and by protons. The crystal structure of the chimera in its resting state, at pH 7.0 and in the absence of primary amines, revealed a closed-pore conformation and an ECD that is twisted with respect to the transmembrane region. Amine- and pH-induced conformational changes measured by DEER spectroscopy showed that the chimera exhibits a dual mode of gating that preserves the distinct conformational changes of the parent channels. Collectively, our findings shed light on both conserved and divergent features of gating mechanisms in this class of channels, and will facilitate the design of better allosteric modulators.

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.

scholarly journals Pyrococcus furiosus Prolyl Oligopeptidase: A Dynamic Supramolecular Host for Peptidase and Dirhodium Catalysis

2018 ◽  
Author(s):  
Ken Ellis-Guardiola ◽  
Huan Rui ◽  
Ryan Beckner ◽  
Poonam Srivastava ◽  
Narayanasami Sukumar ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Pyrococcus Furiosus ◽  
Conformational Dynamics ◽  
Domain Architecture ◽  
Md Simulations ◽  
Peptidase Activity ◽  
Prolyl Oligopeptidase ◽  
Supramolecular Catalysis ◽  
Wide Range ◽  
Synthetic Macromolecules

Supramolecular catalysis involves the design and characterization of synthetic macromolecules that catalyze chemical reactions. While enzymes are often cited as the inspiration for such catalysts, enzymes can also serve as hosts for non-native catalytic components. Protein-based hosts can be readily produced in E. coli and rapidly evolved for particular applications. Moreover, inherent properties of these systems, including their conformational dynamics, can be exploited for non-native transformations that occur within their interior. Studies on the peptidase activity of a prolyl oligopeptidase from Pyrococcus furiosus (Pfu POP) suggest that its unique two-domain architecture regulates substrate access and specificity. We have established that Pfu POP also serves as an efficient host for asymmetric cyclopropanation upon active-site modification with a dirhodium cofactor. To understand how Pfu POP controls both peptidase and dirhodium catalysis, we determined the crystal structures of this enzyme and its S477C mutant and used these structures as starting points for MD simulations of both the apo structures and systems containing a covalently linked peptidase inhibitor or a dirhodium catalyst. Pfu POP was crystalized in an open conformation, and MD simulations reveal spontaneous transitions between open and closed states, in addition to a number of smaller scale conformational changes, suggesting facile inter-domain movement. Importantly, key aspects of previously reported peptidase kinetics and cyclopropanation selectivity can be rationalized in the context of this inter-domain opening and closing. This finding constitutes a remarkable example in which the conformational dynamics of a supramolecular host affect two different catalytic activities and suggests that Pfu POP could serve as a host for a wide range of non-native catalysts.

scholarly journals Molecular dynamics simulations disclose early stages of the photo-activation of cryptochrome 4

2018 ◽  
Author(s):  
D. R. Kattnig ◽  
C. Nielsen ◽  
I. A. Solov’yov
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Large Scale ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
Quantum Processes ◽  
Helical Segment ◽  
High Betweenness ◽  
Dynamics Simulations ◽  
Fad Binding

AbstractBirds appear to be equipped with a light-dependent, radical-pair-based magnetic compass that relies on truly quantum processes. While the identity of the sensory protein has remained speculative, cryptochrome 4 has recently been identified as the most auspicious candidate. Here, we report on allatom molecular dynamics (MD) simulations addressing the structural reorganisations that accompany the photoreduction of the flavin cofactor in a model of the European robin cryptochrome 4 (ErCry4). Extensive MD simulations reveal that the photo-activation of ErCry4 induces large-scale conformational changes on short (hundreds of nanoseconds) timescales. Specifically, the photo-reduction is accompanied with the release of the C-terminal tail, structural rearrangements in the vicinity of the FAD-binding site, and the noteworthy formation of an α-helical segment at the N-terminal part. Some of these rearrangements appear to expose potential phosphorylation sites. We describe the conformational dynamics of the protein using a graph-based approach that is informed by the adjacency of residues and the correlation of their local motions. This approach reveals densely coupled reorganisation communities, which facilitate an efficient signal transduction due to a high density of hubs. These communities are interconnected by a small number of highly important residues characterized by high betweenness centrality. The network approach clearly identifies the sites restructuring upon photoactivation, which appear as protrusions or delicate bridges in the reorganisation network. We also find that, unlike in the homologous cryptochrome from D. melanogaster, the release of the C-terminal domain does not appear to be correlated with the transposition of a histidine residue close to the FAD cofactor.

scholarly journals Structural titration of receptor ion channel GLIC gating by HS-AFM

2018 ◽  
Vol 115 (41) ◽  
pp. 10333-10338 ◽  
Author(s):  
Yi Ruan ◽  
Kevin Kao ◽  
Solène Lefebvre ◽  
Arin Marchesi ◽  
Pierre-Jean Corringer ◽  
...  
Keyword(s):  
Ion Channel ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Interactions ◽  
High Speed ◽  
Conformational Dynamics ◽  
Protein Protein Interactions ◽  
Ion Conductance ◽  
Single Molecule Level ◽  
Selective Channel

Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated, cation-selective channel, is a prokaryotic homolog of the pentameric Cys-loop receptor ligand-gated ion channel family. Despite large changes in ion conductance, small conformational changes were detected in X-ray structures of detergent-solubilized GLIC at pH 4 (active/desensitized state) and pH 7 (closed state). Here, we used high-speed atomic force microscopy (HS-AFM) combined with a buffer exchange system to perform structural titration experiments to visualize GLIC gating at the single-molecule level under native conditions. Reference-free 2D classification revealed channels in multiple conformational states during pH gating. We find changes of protein–protein interactions so far elusive and conformational dynamics much larger than previously assumed. Asymmetric pentamers populate early stages of activation, which provides evidence for an intermediate preactivated state.

scholarly journals Shining light on cysteine modification: connecting protein conformational dynamics to catalysis and regulation

2019 ◽  
Vol 26 (4) ◽  
pp. 958-966 ◽  
Author(s):  
Henry van den Bedem ◽  
Mark A Wilson
Keyword(s):  
Conformational Changes ◽  
Conformational Dynamics ◽  
Covalent Bond ◽  
Covalent Modification ◽  
Cysteine Residues ◽  
Cysteine Oxidation ◽  
X Ray ◽  
Photo Oxidation ◽  
X Ray Crystallography ◽  
Important Amino Acid

Cysteine is a rare but functionally important amino acid that is often subject to covalent modification. Cysteine oxidation plays an important role in many human disease processes, and basal levels of cysteine oxidation are required for proper cellular function. Because reactive cysteine residues are typically ionized to the thiolate anion (Cys-S−), their formation of a covalent bond alters the electrostatic and steric environment of the active site. X-ray-induced photo-oxidation to sulfenic acids (Cys-SOH) can recapitulate some aspects of the changes that occur under physiological conditions. Here we propose how site-specific cysteine photo-oxidation can be used to interrogate ensuing changes in protein structure and dynamics at atomic resolution. Although this powerful approach can connect cysteine covalent modification to global protein conformational changes and function, careful biochemical validation must accompany all such studies to exclude misleading artifacts. New types of X-ray crystallography experiments and powerful computational methods are creating new opportunities to connect conformational dynamics to catalysis for the large class of systems that use covalently modified cysteine residues for catalysis or regulation.

scholarly journals Ligand-Induced Conformational Dynamics of A Tyramine Receptor from Sitophilus oryzae

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mac Kevin E. Braza ◽  
Jerrica Dominique N. Gazmen ◽  
Eizadora T. Yu ◽  
Ricky B. Nellas
Keyword(s):  
Conformational Changes ◽  
Cellular Response ◽  
Conformational Dynamics ◽  
Sitophilus Oryzae ◽  
Md Simulations ◽  
Homology Model ◽  
Activation Process ◽  
Intracellular Loop ◽  
Binding Effect ◽  
Natural Ligand

Abstract Tyramine receptor (TyrR) is a biogenic amine G protein-coupled receptor (GPCR) associated with many important physiological functions in insect locomotion, reproduction, and pheromone response. Binding of specific ligands to the TyrR triggers conformational changes, relays the signal to G proteins, and initiates an appropriate cellular response. Here, we monitor the binding effect of agonist compounds, tyramine and amitraz, to a Sitophilus oryzae tyramine receptor (SoTyrR) homology model and their elicited conformational changes. All-atom molecular dynamics (MD) simulations of SoTyrR-ligand complexes have shown varying dynamic behavior, especially at the intracellular loop 3 (IL3) region. Moreover, in contrast to SoTyrR-tyramine, SoTyrR-amitraz and non-liganded SoTyrR shows greater flexibility at IL3 residues and were found to be coupled to the most dominant motion in the receptor. Our results suggest that the conformational changes induced by amitraz are different from the natural ligand tyramine, albeit being both agonists of SoTyrR. This is the first attempt to understand the biophysical implication of amitraz and tyramine binding to the intracellular domains of TyrR. Our data may provide insights into the early effects of ligand binding to the activation process of SoTyrR.

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