Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 12 de 12
Filter
1.
J Mol Biol ; : 167696, 2022 Jul 08.
Article in English | MEDLINE | ID: covidwho-1926683

ABSTRACT

The family of coarse-grained models for protein dynamics known as Elastic Network Models (ENMs) require careful choice of parameters to represent well experimental measurements or fully-atomistic simulations. The most basic ENM that represents each protein residue by a node at the position of its C-alpha atom, all connected by springs of equal stiffness, up to a cut-off in distance. Even at this level a choice is required of the optimum cut-off distance and the upper limit of elastic normal modes taken in any sum for physical properties, such as dynamic correlation or allosteric effects on binding. Additionally, backbone-enhanced ENM (BENM) may improve the model by allocating a higher stiffness to springs that connect along the protein backbone. This work reports on the effect of varying these three parameters (distance and mode cutoffs, backbone stiffness) on the dynamical structure of three proteins, Catabolite Activator Protein (CAP), Glutathione S-transferase (GST), and the SARS-CoV-2 Main Protease (M pro ). Our main results are: (1) balancing B-factor and dispersion-relation predictions, a near-universal optimal value of 8.5 Å is advisable for ENMs; (2) inhomogeneity in elasticity brings the first mode containing spatial structure not well-resolved by the ENM typically within the first 20; (3) the BENM only affects modes in the upper third of the distribution, and, additionally to the ENM, is only able to model the dispersion curve better in this vicinity; (4) BENM does not typically affect fluctuation-allostery, which also requires careful treatment of the effector binding to the host protein to capture.

2.
Molecules ; 27(6)2022 Mar 11.
Article in English | MEDLINE | ID: covidwho-1765795

ABSTRACT

Protein-protein assemblies act as a key component in numerous cellular processes. Their accurate modeling at the atomic level remains a challenge for structural biology. To address this challenge, several docking and a handful of deep learning methodologies focus on modeling protein-protein interfaces. Although the outcome of these methods has been assessed using static reference structures, more and more data point to the fact that the interaction stability and specificity is encoded in the dynamics of these interfaces. Therefore, this dynamics information must be taken into account when modeling and assessing protein interactions at the atomistic scale. Expanding on this, our review initially focuses on the recent computational strategies aiming at investigating protein-protein interfaces in a dynamic fashion using enhanced sampling, multi-scale modeling, and experimental data integration. Then, we discuss how interface dynamics report on the function of protein assemblies in globular complexes, in fuzzy complexes containing intrinsically disordered proteins, as well as in active complexes, where chemical reactions take place across the protein-protein interface.


Subject(s)
Intrinsically Disordered Proteins , Intrinsically Disordered Proteins/chemistry
3.
Elife ; 112022 03 24.
Article in English | MEDLINE | ID: covidwho-1761116

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells through binding to angiotensin-converting enzyme 2 (ACE2). This interaction is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural and dynamic data have shown that S can adopt multiple conformations, which controls the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Förster resonance energy transfer (smFRET) imaging, we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and in the presence of the D614G mutation. We find that D614G modulates the energetics of the RBD position in a manner similar to ACE2 binding. We also find that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicate antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.


Subject(s)
SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Angiotensin-Converting Enzyme 2/chemistry , COVID-19 , Humans , Protein Domains , Spike Glycoprotein, Coronavirus/chemistry
4.
Arch Microbiol ; 203(9): 5463-5473, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1363730

ABSTRACT

The causative agent of COVID-19 is a novel betacoronavirus or severe acute respiratory syndrome coronavirus (SARS-CoV-2), which has emerged as a pandemic of global concern. Considering its rapid transmission, WHO has declared public health emergency on 11th March 2020 worldwide. SARS-CoV-2 is a genetically diverse positive sense RNA virus that typically exhibit high rates of mutation than DNA viruses. Higher rates of mutation bring higher genomic variability which may lead to viral evolution and enabling viruses to evade the pre-existing immunity of host and quickly acquire drug resistance properties. The objective of our study was to compare the SARS-CoV-2 RdRp sequences of Indian SARS-CoV-2 isolates with those of Wuhan type virus. A total of 384 point mutations were detected from 488 sequence of the RdRp protein of Indian SARS-CoV-2 genome, out of which seven were used for subsequent study. Furthermore, prediction of secondary structure, protein modeling and its dynamics were performed which revealed that seven mutations (R118C, T148I, Y149C, E802A, Q822H, V880I and D893Y) significantly altered the stability and flexibility of RdRp protein. Present study was therefore, undertaken to analyze the variations occurring in RdRp due to multiple mutations leading to the alterations in the structure and function of RNA-dependent RNA polymerase which is essential for the replication /transcription of this virus and hence can be utilized as a promising therapeutic target to curb SARS-CoV-2 infections.


Subject(s)
COVID-19 , RNA-Dependent RNA Polymerase , Antiviral Agents/pharmacology , Humans , Mutation , RNA, Viral/genetics , RNA-Dependent RNA Polymerase/genetics , SARS-CoV-2
5.
Proteins ; 90(5): 1044-1053, 2022 May.
Article in English | MEDLINE | ID: covidwho-1347428

ABSTRACT

Since the identification of the SARS-CoV-2 virus as the causative agent of the current COVID-19 pandemic, considerable effort has been spent characterizing the interaction between the Spike protein receptor-binding domain (RBD) and the human angiotensin converting enzyme 2 (ACE2) receptor. This has provided a detailed picture of the end point structure of the RBD-ACE2 binding event, but what remains to be elucidated is the conformation and dynamics of the RBD prior to its interaction with ACE2. In this work, we utilize molecular dynamics simulations to probe the flexibility and conformational ensemble of the unbound state of the receptor-binding domain from SARS-CoV-2 and SARS-CoV. We have found that the unbound RBD has a localized region of dynamic flexibility in Loop 3 and that mutations identified during the COVID-19 pandemic in Loop 3 do not affect this flexibility. We use a loop-modeling protocol to generate and simulate novel conformations of the CoV2-RBD Loop 3 region that sample conformational space beyond the ACE2 bound crystal structure. This has allowed for the identification of interesting substates of the unbound RBD that are lower energy than the ACE2-bound conformation, and that block key residues along the ACE2 binding interface. These novel unbound substates may represent new targets for therapeutic design.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Angiotensin-Converting Enzyme 2 , Binding Sites , Humans , Molecular Dynamics Simulation , Pandemics , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry
6.
Proc Natl Acad Sci U S A ; 118(26)2021 06 29.
Article in English | MEDLINE | ID: covidwho-1284759

ABSTRACT

The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), invades a human cell via human angiotensin-converting enzyme 2 (hACE2) as the entry, causing the severe coronavirus disease (COVID-19). The interactions between hACE2 and the spike glycoprotein (S protein) of SARS-CoV-2 hold the key to understanding the molecular mechanism to develop treatment and vaccines, yet the dynamic nature of these interactions in fluctuating surroundings is very challenging to probe by those structure determination techniques requiring the structures of samples to be fixed. Here we demonstrate, by a proof-of-concept simulation of infrared (IR) spectra of S protein and hACE2, that time-resolved spectroscopy may monitor the real-time structural information of the protein-protein complexes of interest, with the help of machine learning. Our machine learning protocol is able to identify fine changes in IR spectra associated with variation of the secondary structures of S protein of the coronavirus. Further, it is three to four orders of magnitude faster than conventional quantum chemistry calculations. We expect our machine learning protocol would accelerate the development of real-time spectroscopy study of protein dynamics.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Machine Learning , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Humans , Kinetics , Protein Binding , Protein Structure, Secondary , Spectrophotometry, Infrared , Spike Glycoprotein, Coronavirus/chemistry
7.
Biology (Basel) ; 10(6)2021 May 21.
Article in English | MEDLINE | ID: covidwho-1243947

ABSTRACT

The latest coronavirus SARS-CoV-2, which causes coronavirus disease 2019 (COVID-19) pneumonia leading to the pandemic, contains 29 proteins. Among them, nucleocapsid protein (NCoV2) is one of the abundant proteins and shows multiple functions including packaging the RNA genome during the infection cycle. It has also emerged as a potential drug target. In this review, the current status of the research of NCoV2 is described in terms of molecular structure and dynamics. NCoV2 consists of two domains, i.e., the N-terminal domain (NTD) and the C-terminal domain (CTD) with a disordered region between them. Recent simulation studies have identified several potential drugs that can bind to NTD or CTD with high affinity. Moreover, it was shown that the degree of flexibility in the disordered region has a large effect on drug binding rate, suggesting the importance of molecular flexibility for the NCoV2 function. Molecular flexibility has also been shown to be integral to the formation of droplets, where NCoV2, RNA and/or other viral proteins gather through liquid-liquid phase separation and considered important for viral replication. Finally, as one of the future research directions, a strategy for obtaining the structural and dynamical information on the proteins contained in droplets is presented.

8.
Comput Struct Biotechnol J ; 19: 3187-3197, 2021.
Article in English | MEDLINE | ID: covidwho-1242911

ABSTRACT

The new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes pathological pulmonary symptoms. Most efforts to develop vaccines and drugs against this virus target the spike glycoprotein, particularly its S1 subunit, which is recognised by angiotensin-converting enzyme 2. Here we use the in-house developed tool CaverDock to perform virtual screening against spike glycoprotein using a cryogenic electron microscopy structure (PDB-ID: 6VXX) and the representative structures of five most populated clusters from a previously published molecular dynamics simulation. The dataset of ligands was obtained from the ZINC database and consists of drugs approved for clinical use worldwide. Trajectories for the passage of individual drugs through the tunnel of the spike glycoprotein homotrimer, their binding energies within the tunnel, and the duration of their contacts with the trimer's three subunits were computed for the full dataset. Multivariate statistical methods were then used to establish structure-activity relationships and select top candidate for movement inhibition. This new protocol for the rapid screening of globally approved drugs (4359 ligands) in a multi-state protein structure (6 states) showed high robustness in the rate of finished calculations. The protocol is universal and can be applied to any target protein with an experimental tertiary structure containing protein tunnels or channels. The protocol will be implemented in the next version of CaverWeb (https://loschmidt.chemi.muni.cz/caverweb/) to make it accessible to the wider scientific community.

9.
Proteins ; 89(9): 1134-1144, 2021 09.
Article in English | MEDLINE | ID: covidwho-1188037

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused substantially more infections, deaths, and economic disruptions than the 2002-2003 SARS-CoV. The key to understanding SARS-CoV-2's higher infectivity lies partly in its host receptor recognition mechanism. Experiments show that the human angiotensin converting enzyme 2 (ACE2) protein, which serves as the primary receptor for both CoVs, binds to the receptor binding domain (RBD) of CoV-2's spike protein stronger than SARS-CoV's spike RBD. The molecular basis for this difference in binding affinity, however, remains unexplained from X-ray structures. To go beyond insights gained from X-ray structures and investigate the role of thermal fluctuations in structure, we employ all-atom molecular dynamics simulations. Microseconds-long simulations reveal that while CoV and CoV-2 spike-ACE2 interfaces have similar conformational binding modes, CoV-2 spike interacts with ACE2 via a larger combinatorics of polar contacts, and on average, makes 45% more polar contacts. Correlation analysis and thermodynamic calculations indicate that these differences in the density and dynamics of polar contacts arise from differences in spatial arrangements of interfacial residues, and dynamical coupling between interfacial and non-interfacial residues. These results recommend that ongoing efforts to design spike-ACE2 peptide blockers will benefit from incorporating dynamical information as well as allosteric coupling effects.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Molecular Dynamics Simulation , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Allosteric Regulation , Humans , Mutation , Protein Binding , Receptors, Virus/chemistry , Receptors, Virus/metabolism , Thermodynamics
10.
J R Soc Interface ; 18(174): 20200591, 2021 01.
Article in English | MEDLINE | ID: covidwho-1010695

ABSTRACT

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has no publicly available vaccine or antiviral drugs at the time of writing. An attractive coronavirus drug target is the main protease (Mpro, also known as 3CLpro) because of its vital role in the viral cycle. A significant body of work has been focused on finding inhibitors which bind and block the active site of the main protease, but little has been done to address potential non-competitive inhibition, targeting regions other than the active site, partly because the fundamental biophysics of such allosteric control is still poorly understood. In this work, we construct an elastic network model (ENM) of the SARS-CoV-2 Mpro homodimer protein and analyse its dynamics and thermodynamics. We found a rich and heterogeneous dynamical structure, including allosterically correlated motions between the homodimeric protease's active sites. Exhaustive 1-point and 2-point mutation scans of the ENM and their effect on fluctuation free energies confirm previously experimentally identified bioactive residues, but also suggest several new candidate regions that are distant from the active site, yet control the protease function. Our results suggest new dynamically driven control regions as possible candidates for non-competitive inhibiting binding sites in the protease, which may assist the development of current fragment-based binding screens. The results also provide new insights into the active biophysical research field of protein fluctuation allostery and its underpinning dynamical structure.


Subject(s)
COVID-19/virology , SARS-CoV-2/metabolism , Viral Proteases/chemistry , Computer Simulation , Crystallization , Humans , Models, Molecular , Protein Conformation , SARS-CoV-2/enzymology , Thermodynamics , Viral Proteases/drug effects , Viral Proteases/metabolism
11.
Biomol NMR Assign ; 15(1): 107-116, 2021 04.
Article in English | MEDLINE | ID: covidwho-1002175

ABSTRACT

The Betacoronavirus SARS-CoV-2 non-structural protein Nsp9 is a 113-residue protein that is essential for viral replication, and consequently, a potential target for the development of therapeutics against COVID19 infections. To capture insights into the dynamics of the protein's backbone in solution and accelerate the identification and mapping of ligand-binding surfaces through chemical shift perturbation studies, the backbone 1H, 13C, and 15N NMR chemical shifts for Nsp9 have been extensively assigned. These assignments were assisted by the preparation of an ~ 70% deuterated sample and residue-specific, 15N-labelled samples (V, L, M, F, and K). A major feature of the assignments was the "missing" amide resonances for N96-L106 in the 1H-15N HSQC spectrum, a region that comprises almost the complete C-terminal α-helix that forms a major part of the homodimer interface in the crystal structure of SARS-CoV-2 Nsp9, suggesting this region either undergoes intermediate motion in the ms to µs timescale and/or is heterogenous. These "missing" amide resonances do not unambiguously appear in the 1H-15N HSQC spectrum of SARS-CoV-2 Nsp9 collected at a concentration of 0.0007 mM. At this concentration, at the detection limit, native mass spectrometry indicates the protein is exclusively in the monomeric state, suggesting the intermediate motion in the C-terminal of Nsp9 may be due to intramolecular dynamics. Perhaps this intermediate ms to µs timescale dynamics is the physical basis for a previously suggested "fluidity" of the C-terminal helix that may be responsible for homophilic (Nsp9-Nsp9) and postulated heterophilic (Nsp9-Unknown) protein-protein interactions.


Subject(s)
Magnetic Resonance Spectroscopy , RNA-Binding Proteins/chemistry , SARS-CoV-2/chemistry , Viral Nonstructural Proteins/chemistry , Binding Sites , Carbon Isotopes , Codon , Crystallography, X-Ray , Dimerization , Disulfides , Hydrogen , Hydrogen-Ion Concentration , Kinetics , Ligands , Nitrogen Isotopes , Protein Binding , Protein Domains , Protein Structure, Secondary
12.
ChemMedChem ; 15(18): 1682-1690, 2020 09 16.
Article in English | MEDLINE | ID: covidwho-641524

ABSTRACT

Angiotensin converting enzyme 2 (ACE2) is the human receptor that interacts with the spike protein of coronaviruses, including the one that produced the 2020 coronavirus pandemic (COVID-19). Thus, ACE2 is a potential target for drugs that disrupt the interaction of human cells with SARS-CoV-2 to abolish infection. There is also interest in drugs that inhibit or activate ACE2, that is, for cardiovascular disorders or colitis. Compounds binding at alternative sites could allosterically affect the interaction with the spike protein. Herein, we review biochemical, chemical biology, and structural information on ACE2, including the recent cryoEM structures of full-length ACE2. We conclude that ACE2 is very dynamic and that allosteric drugs could be developed to target ACE2. At the time of the 2020 pandemic, we suggest that available ACE2 inhibitors or activators in advanced development should be tested for their ability to allosterically displace the interaction between ACE2 and the spike protein.


Subject(s)
Angiotensin-Converting Enzyme Inhibitors/metabolism , Betacoronavirus/chemistry , Peptidyl-Dipeptidase A/metabolism , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Allosteric Regulation , Angiotensin-Converting Enzyme 2 , Angiotensin-Converting Enzyme Inhibitors/chemistry , Catalytic Domain , Humans , Peptidyl-Dipeptidase A/chemistry , Protein Binding , Protein Domains , Receptors, Virus/antagonists & inhibitors , Receptors, Virus/chemistry , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry
SELECTION OF CITATIONS
SEARCH DETAIL