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1.
Int J Biol Macromol ; 244: 125096, 2023 Jul 31.
Article in English | MEDLINE | ID: covidwho-20231041

ABSTRACT

Baricitinib is a Janus Kinase (JAK) inhibitor that is primarily used to treat moderately to severely active rheumatoid arthritis in adults and has recently been reported for the treatment of patients with severe COVID-19. This paper describes the investigation of the binding behavior of baricitinib to human α1-acid glycoprotein (HAG) employing a variety of spectroscopic techniques, molecular docking and dynamics simulations. Baricitinib can quench the fluorescence from amino acids in HAG through a mix of dynamic and static quenching, according to steady-state fluorescence and UV spectra observations, but it is mainly static quenching at low concentration. The binding constant (Kb) of baricitinib to HAG at 298 K was at the level of 104 M-1, indicating a moderate affinity of baricitinib to HAG. Hydrogen bonding and hydrophobic interactions conducted the main effect, according to thermodynamic characteristics, competition studies between ANS and sucrose, and molecular dynamics simulations. For the change in HAG conformation, the results of multiple spectra showed that baricitinib was able to alter the secondary structure of HAG as well as increase the polarity of the microenvironment around the Trp amino acid. Furthermore, the binding behavior of baricitinib to HAG was investigated by molecular docking and molecular dynamics simulations, which validated experimental results. Also explored is the influence of K+, Co2+, Ni2+, Ca2+, Fe3+, Zn2+, Mg2+ and Cu2+plasma on binding affinity.


Subject(s)
COVID-19 , Janus Kinase Inhibitors , Humans , Molecular Docking Simulation , Protein Binding , Orosomucoid/chemistry , COVID-19 Drug Treatment , Molecular Dynamics Simulation , Protein Structure, Secondary , Thermodynamics , Binding Sites , Spectrometry, Fluorescence
2.
Comput Biol Med ; 158: 106814, 2023 05.
Article in English | MEDLINE | ID: covidwho-2273828

ABSTRACT

This paper presents a novel framework, called PSAC-PDB, for analyzing and classifying protein structures from the Protein Data Bank (PDB). PSAC-PDB first finds, analyze and identifies protein structures in PDB that are similar to a protein structure of interest using a protein structure comparison tool. Second, the amino acids (AA) sequences of identified protein structures (obtained from PDB), their aligned amino acids (AAA) and aligned secondary structure elements (ASSE) (obtained by structural alignment), and frequent AA (FAA) patterns (discovered by sequential pattern mining), are used for the reliable detection/classification of protein structures. Eleven classifiers are used and their performance is compared using six evaluation metrics. Results show that three classifiers perform well on overall, and that FAA patterns can be used to efficiently classify protein structures in place of providing the whole AA sequences, AAA or ASSE. Furthermore, better classification results are obtained using AAA of protein structures rather than AA sequences. PSAC-PDB also performed better than state-of-the-art approaches for SARS-CoV-2 genome sequences classification.


Subject(s)
COVID-19 , Humans , SARS-CoV-2 , Protein Structure, Secondary , Amino Acids , Databases, Protein , Protein Conformation
3.
Proc Natl Acad Sci U S A ; 120(13): e2300360120, 2023 03 28.
Article in English | MEDLINE | ID: covidwho-2287540

ABSTRACT

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) introduced a relatively large number of mutations, including three mutations in the highly conserved heptad repeat 1 (HR1) region of the spike glycoprotein (S) critical for its membrane fusion activity. We show that one of these mutations, N969K induces a substantial displacement in the structure of the heptad repeat 2 (HR2) backbone in the HR1HR2 postfusion bundle. Due to this mutation, fusion-entry peptide inhibitors based on the Wuhan strain sequence are less efficacious. Here, we report an Omicron-specific peptide inhibitor designed based on the structure of the Omicron HR1HR2 postfusion bundle. Specifically, we inserted an additional residue in HR2 near the Omicron HR1 K969 residue to better accommodate the N969K mutation and relieve the distortion in the structure of the HR1HR2 postfusion bundle it introduced. The designed inhibitor recovers the loss of inhibition activity of the original longHR2_42 peptide with the Wuhan strain sequence against the Omicron variant in both a cell-cell fusion assay and a vesicular stomatitis virus (VSV)-SARS-CoV-2 chimera infection assay, suggesting that a similar approach could be used to combat future variants. From a mechanistic perspective, our work suggests the interactions in the extended region of HR2 may mediate the initial landing of HR2 onto HR1 during the transition of the S protein from the prehairpin intermediate to the postfusion state.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Viral Envelope Proteins/genetics , Amino Acid Sequence , Protein Structure, Secondary , Spike Glycoprotein, Coronavirus/metabolism , Peptides/genetics , Peptides/pharmacology , Peptides/chemistry , Anti-Retroviral Agents
4.
Phys Chem Chem Phys ; 24(41): 25391-25402, 2022 Oct 27.
Article in English | MEDLINE | ID: covidwho-2077132

ABSTRACT

Here, we have carried out a proof-of-concept molecular dynamics (MD) simulation with adaptive tempering in a membrane mimetic environment to study the folding of single-pass membrane peptides. We tested the influenza A M2 viroporin, influenza B M2 viroporin, and protein E from coronaviruses MERS-Cov-2 and SARS-CoV-2 peptides with known experimental secondary structures in membrane bilayers. The two influenza-derived peptides are significantly different in the peptide sequence and secondary structure and more polar than the two coronavirus-derived peptides. Through a total of more than 50 µs of simulation time that could be accomplished in trifluoroethanol (TFE), as a membrane model, we characterized comparatively the folding behavior, helical stability, and helical propensity of these transmembrane peptides that match perfectly their experimental secondary structures, and we identified common motifs that reflect their quaternary organization and known (or not) biochemical function. We showed that BM2 is organized into two structurally distinct parts: a significantly more stable N-terminal half, and a fast-converting C-terminal half that continuously folds and unfolds between α-helical structures and non-canonical structures, which are mostly turns. In AM2, both the N-terminal half and C-terminal half are very flexible. In contrast, the two coronavirus-derived transmembrane peptides are much more stable and fast helix-formers when compared with the influenza ones. In particular, the SARS-derived peptide E appears to be the fastest and most stable helix-former of all the four viral peptides studied, with a helical structure that persists almost without disruption for the whole of its 10 µs simulation. By comparing the results with experimental observations, we benchmarked TFE in studying the conformation of membrane and hydrophobic peptides. This work provided accurate results suggesting a methodology to run long MD simulations and predict structural properties of biologically important membrane peptides.


Subject(s)
COVID-19 , Influenza, Human , Humans , Molecular Dynamics Simulation , Peptides/chemistry , Polytetrafluoroethylene , Protein Folding , Protein Structure, Secondary , SARS-CoV-2 , Solvents , Trifluoroethanol/chemistry , Viroporin Proteins , Influenzavirus B , Middle East Respiratory Syndrome Coronavirus
5.
Science ; 369(6511): 1586-1592, 2020 09 25.
Article in English | MEDLINE | ID: covidwho-2038226

ABSTRACT

Intervention strategies are urgently needed to control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. The trimeric viral spike (S) protein catalyzes fusion between viral and target cell membranes to initiate infection. Here, we report two cryo-electron microscopy structures derived from a preparation of the full-length S protein, representing its prefusion (2.9-angstrom resolution) and postfusion (3.0-angstrom resolution) conformations, respectively. The spontaneous transition to the postfusion state is independent of target cells. The prefusion trimer has three receptor-binding domains clamped down by a segment adjacent to the fusion peptide. The postfusion structure is strategically decorated by N-linked glycans, suggesting possible protective roles against host immune responses and harsh external conditions. These findings advance our understanding of SARS-CoV-2 entry and may guide the development of vaccines and therapeutics.


Subject(s)
Host-Pathogen Interactions/immunology , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2 , Cryoelectron Microscopy , HEK293 Cells , Humans , Peptidyl-Dipeptidase A/chemistry , Protein Domains , Protein Multimerization , Protein Structure, Secondary , Receptors, Virus/chemistry , Virus Internalization
6.
Int J Mol Sci ; 21(16)2020 Aug 06.
Article in English | MEDLINE | ID: covidwho-1934101

ABSTRACT

The recently discovered 340-cavity in influenza neuraminidase (NA) N6 and N7 subtypes has introduced new possibilities for rational structure-based drug design. However, the plasticity of the 340-loop (residues 342-347) and the role of the 340-loop in NA activity and substrate binding have not been deeply exploited. Here, we investigate the mechanism of 340-cavity formation and demonstrate for the first time that seven of nine NA subtypes are able to adopt an open 340-cavity over 1.8 µs total molecular dynamics simulation time. The finding that the 340-loop plays a role in the sialic acid binding pathway suggests that the 340-cavity can function as a druggable pocket. Comparing the open and closed conformations of the 340-loop, the side chain orientation of residue 344 was found to govern the formation of the 340-cavity. Additionally, the conserved calcium ion was found to substantially influence the stability of the 340-loop. Our study provides dynamical evidence supporting the 340-cavity as a druggable hotspot at the atomic level and offers new structural insight in designing antiviral drugs.


Subject(s)
Antiviral Agents/pharmacology , Drug Development , Neuraminidase/chemistry , Orthomyxoviridae/enzymology , Binding Sites , Calcium/chemistry , Ions , Models, Molecular , Molecular Dynamics Simulation , N-Acetylneuraminic Acid/chemistry , Principal Component Analysis , Protein Structure, Secondary , Thermodynamics
7.
J Virol ; 96(13): e0068522, 2022 07 13.
Article in English | MEDLINE | ID: covidwho-1891738

ABSTRACT

Since its outbreak in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread with high transmission efficiency across the world, putting health care as well as economic systems under pressure. During the course of the pandemic, the originally identified SARS-CoV-2 variant has been multiple times replaced by various mutant versions, which showed enhanced fitness due to increased infection and transmission rates. In order to find an explanation for why SARS-CoV-2 and its emerging mutated versions showed enhanced transmission efficiency compared with SARS-CoV (2002), an enhanced binding affinity of the spike protein to human angiotensin converting enzyme 2 (hACE2) has been proposed by crystal structure analysis and was identified in cell culture models. Kinetic analysis of the interaction of various spike protein constructs with hACE2 was considered to be best described by a Langmuir-based 1:1 stoichiometric interaction. However, we demonstrate in this report that the SARS-CoV-2 spike protein interaction with hACE2 is best described by a two-step interaction, which is defined by an initial binding event followed by a slower secondary rate transition that enhances the stability of the complex by a factor of ~190 (primary versus secondary state) with an overall equilibrium dissociation constant (KD) of 0.20 nM. In addition, we show that the secondary rate transition is not only present in SARS-CoV-2 wild type ("wt"; Wuhan strain) but also found in the B.1.1.7 variant, where its transition rate is 5-fold increased. IMPORTANCE The current SARS-CoV-2 pandemic is characterized by the high infectivity of SARS-CoV-2 and its derived variants of concern (VOCs). It has been widely assumed that the reason for its increased cell entry compared with SARS-CoV (2002) is due to alterations in the viral spike protein, where single amino acid residue substitutions can increase affinity for hACE2. So far, the interaction of a single unit of the CoV-2 spike protein has been described using the 1:1 Langmuir interaction kinetic. However, we demonstrate here that there is a secondary state binding step that may be essential for novel VOCs in order to further increase their infectivity. These findings are important for quantitatively understanding the infection process of SARS-CoV-2 and characterization of emerging SARS-CoV-2 variants of spike proteins. Thus, they provide a tool for predicting the potential infectivity of the respective viral variants based on secondary rate transition and secondary complex stability.


Subject(s)
Angiotensin-Converting Enzyme 2 , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , Humans , Kinetics , Peptidyl-Dipeptidase A/metabolism , Protein Binding , Protein Structure, Secondary , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
8.
Biomed Res Int ; 2022: 2273648, 2022.
Article in English | MEDLINE | ID: covidwho-1832664

ABSTRACT

Protein is the material foundation of living things, and it directly takes part in and runs the process of living things itself. Predicting protein complexes helps us understand the structure and function of complexes, and it is an important foundation for studying how cells work. Genome-wide protein interaction (PPI) data is growing as high-throughput experiments become more common. The aim of this research is that it provides a dual-tree complex wavelet transform which is used to find out about the structure of proteins. It also identifies the secondary structure of protein network. Many computer-based methods for predicting protein complexes have also been developed in the field. Identifying the secondary structure of a protein is very important when you are studying protein characteristics and properties. This is how the protein sequence is added to the distance matrix. The scope of this research is that it can confidently predict certain protein complexes rapidly, which compensates for shortcomings in biological research. The three-dimensional coordinates of C atom are used to do this. According to the texture information in the distance matrix, the matrix is broken down into four levels by the double-tree complex wavelet transform because it has four levels. The subband energy and standard deviation in different directions are taken, and then, the two-dimensional feature vector is used to show the secondary structure features of the protein in a way that is easy to understand. Then, the KNN and SVM classifiers are used to classify the features that were found. Experiments show that a new feature called a dual-tree complex wavelet can improve the texture granularity and directionality of the traditional feature extraction method, which is called secondary structure.


Subject(s)
Computational Biology , Support Vector Machine , Protein Structure, Secondary , Proteins/chemistry , Wavelet Analysis
9.
Adv Exp Med Biol ; 1366: 87-100, 2022.
Article in English | MEDLINE | ID: covidwho-1782742

ABSTRACT

The continued HIV/AIDS epidemic worldwide and the battle against emerging infectious diseases caused by coronaviruses underscore the need for the development of an ever-expanding repertoire of antiviral drugs. Entry inhibitors are of particular interest because of their potential to be used as therapeutic or prophylactic treatments for blocking viral invasion. HIV and coronaviruses utilize class I fusion proteins to facilitate their entry and membrane fusion. Discovery of a common hexameric coiled-coil fusion complex resulting from the packing of three C-terminal heptad repeat region from the fusion-mediating subunit of viral fusion proteins against trimeric coiled-coil made up by their N-terminal heptad repeat prompted the search for peptides mimicking the heptad repeat regions that could potentially inhibit viral entry. This has led to the development of effective peptides that are specific to the virus that is developed for. In this review, we focus on peptide-based entry dual inhibitors that block fusion process not only of HIV but also coronaviruses through interrupting their fusogenic six-helical bundle core and which hopefully will help to gain insight into the α-helical secondary structure- and coiled-coil superstructure-based strategies to design entry inhibitors with broad-spectrum antiviral activity against enveloped viruses with class I fusion proteins.


Subject(s)
Antiviral Agents , Coronavirus Infections , Coronavirus , HIV Fusion Inhibitors , HIV Infections , Peptides , Amino Acid Sequence , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Coronavirus Infections/drug therapy , HIV Envelope Protein gp41/metabolism , HIV Envelope Protein gp41/pharmacology , HIV Fusion Inhibitors/pharmacology , HIV Fusion Inhibitors/therapeutic use , Humans , Peptides/pharmacology , Protein Structure, Secondary
10.
J Med Virol ; 94(4): 1641-1649, 2022 04.
Article in English | MEDLINE | ID: covidwho-1718410

ABSTRACT

Emerging severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) variants, especially those of concern, may have an impact on the virus's transmissibility and pathogenicity, as well as diagnostic equipment performance and vaccine effectiveness. Even though the SARS-CoV-2 Delta variant (B.1.617.2) emerged during India's second wave of infections, Delta variants have grown dominant internationally and are still evolving. On November 26, 2021, World Health Organization identified the variant B.1.1.529 as a variant of concern, naming it Omicron, based on evidence that Omicron contains numerous mutations that may influence its behavior. However, the mode of transmission and severity of the Omicron variant remains unknown. We used computational studies to examine the Delta and Omicron variants in this study and found that the Omicron variant had a higher affinity for human angiotensin-converting enzyme 2 (ACE2) than the Delta variant due to a significant number of mutations in the SARS-CoV-2 receptor-binding domain (RBD), indicating a higher potential for transmission. Based on docking studies, the Q493R, N501Y, S371L, S373P, S375F, Q498R, and T478K mutations contribute significantly to high binding affinity with human ACE2. In comparison to the Delta variant, both the entire spike protein and the RBD in Omicron include a high proportion of hydrophobic amino acids such as leucine and phenylalanine. These amino acids are located within the protein's core and are required for structural stability. We observed a disorder-order transition in the Omicron variant between spike protein RBD regions 468-473, and it may be significant in the influence of disordered residues/regions on spike protein stability and binding to ACE2. A future study might investigate the epidemiological and biological consequences of the Omicron variant.


Subject(s)
SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/chemistry , Binding Sites , COVID-19/virology , Humans , Hydrophobic and Hydrophilic Interactions , Molecular Docking Simulation , Mutation , Protein Binding , Protein Stability , Protein Structure, Secondary , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
11.
FASEB J ; 36(3): e22199, 2022 03.
Article in English | MEDLINE | ID: covidwho-1684809

ABSTRACT

Spike trimer plays a key role in SARS-CoV-2 infection and vaccine development. It consists of a globular head and a flexible stalk domain that anchors the protein into the viral membrane. While the head domain has been extensively studied, the properties of the adjoining stalk are poorly understood. Here, we characterize the coiled-coil formation and thermodynamic stability of the stalk domain and its segments. We find that the N-terminal segment of the stalk does not form coiled-coils and remains disordered in solution. The C-terminal stalk segment forms a trimeric coiled-coil in solution, which becomes significantly stabilized in the context of the full-length stalk. Its crystal structure reveals a novel antiparallel tetramer coiled-coil with an unusual combination of a-d and e-a-d hydrophobic core packing. Structural analysis shows that a subset of hydrophobic residues stabilizes different coiled-coil structures: trimer, tetramer, and heterohexamer, underscoring a highly polymorphic nature of the SARS-CoV-2 stalk sequence.


Subject(s)
COVID-19/virology , Models, Molecular , Protein Domains , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Sequence , Crystallization , Crystallography, X-Ray , Humans , Hydrophobic and Hydrophilic Interactions , Protein Stability , Protein Structure, Secondary , Scattering, Small Angle , Temperature , X-Ray Diffraction
12.
Int J Mol Sci ; 23(2)2022 Jan 13.
Article in English | MEDLINE | ID: covidwho-1625319

ABSTRACT

A rational therapeutic strategy is urgently needed for combating SARS-CoV-2 infection. Viral infection initiates when the SARS-CoV-2 receptor-binding domain (RBD) binds to the ACE2 receptor, and thus, inhibiting RBD is a promising therapeutic for blocking viral entry. In this study, the structure of lead antiviral candidate binder (LCB1), which has three alpha-helices (H1, H2, and H3), is used as a template to design and simulate several miniprotein RBD inhibitors. LCB1 undergoes two modifications: structural modification by truncation of the H3 to reduce its size, followed by single and double amino acid substitutions to enhance its binding with RBD. We use molecular dynamics (MD) simulations supported by ab initio density functional theory (DFT) calculations. Complete binding profiles of all miniproteins with RBD have been determined. The MD investigations reveal that the H3 truncation results in a small inhibitor with a -1.5 kcal/mol tighter binding to RBD than original LCB1, while the best miniprotein with higher binding affinity involves D17R or E11V + D17R mutation. DFT calculations provide atomic-scale details on the role of hydrogen bonding and partial charge distribution in stabilizing the minibinder:RBD complex. This study provides insights into general principles for designing potential therapeutics for SARS-CoV-2.


Subject(s)
COVID-19 Drug Treatment , SARS-CoV-2/chemistry , Small Molecule Libraries/chemistry , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Substitution , Antiviral Agents/chemistry , Computational Biology , Molecular Dynamics Simulation , Protein Binding , Protein Domains , Protein Structure, Secondary , Virus Internalization
13.
Int J Mol Sci ; 23(3)2022 Jan 18.
Article in English | MEDLINE | ID: covidwho-1625435

ABSTRACT

Spike protein of SARS-CoV-2 contains a single-span transmembrane (TM) domain and plays roles in receptor binding, viral attachment and viral entry to the host cells. The TM domain of spike protein is critical for viral infectivity. Herein, the TM domain of spike protein of SARS-CoV-2 was reconstituted in detergent micelles and subjected to structural analysis using solution NMR spectroscopy. The results demonstrate that the TM domain of the protein forms a helical structure in detergent micelles. An unstructured linker is identified between the TM helix and heptapeptide repeat 2 region. The linker is due to the proline residue at position 1213. Side chains of the three tryptophan residues preceding to and within the TM helix important for the function of S-protein might adopt multiple conformations which may be critical for their function. The side chain of W1212 was shown to be exposed to solvent and the side chains of residues W1214 and W1217 are buried in micelles. Relaxation study shows that the TM helix is rigid in solution while several residues have exchanges. The secondary structure and dynamics of the TM domain in this study provide insights into the function of the TM domain of spike protein.


Subject(s)
Detergents/pharmacology , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Sequence , COVID-19/virology , Cell Membrane/metabolism , Cross-Linking Reagents/pharmacology , Detergents/chemistry , Humans , Magnetic Resonance Spectroscopy , Micelles , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Domains/drug effects , Protein Structure, Secondary/drug effects , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/drug effects , Spike Glycoprotein, Coronavirus/metabolism
14.
J Cell Mol Med ; 26(1): 25-34, 2022 01.
Article in English | MEDLINE | ID: covidwho-1570773

ABSTRACT

Transmission electron microscopy has historically been indispensable for virology research, as it offers unique insight into virus function. In the past decade, as cryo-electron microscopy (cryo-EM) has matured and become more accessible, we have been able to peer into the structure of viruses at the atomic level and understand how they interact with the host cell, with drugs or with antibodies. Perhaps, there was no time in recent history where cryo-EM was more needed, as SARS-CoV-2 has spread around the globe, causing millions of deaths and almost unquantifiable economic devastation. In this concise review, we aim to mark the most important contributions of cryo-EM to understanding the structure and function of SARS-CoV-2 proteins, from surface spikes to the virus core and from virus-receptor interactions to antibody binding.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Viral/chemistry , COVID-19 Vaccines/chemistry , COVID-19/prevention & control , Receptors, Virus/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/biosynthesis , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/biosynthesis , Cryoelectron Microscopy , Epitopes/chemistry , Epitopes/immunology , Epitopes/metabolism , Humans , Models, Molecular , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Receptors, Virus/immunology , Receptors, Virus/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , SARS-CoV-2/ultrastructure , Serine Endopeptidases/chemistry , Serine Endopeptidases/immunology , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Virion/drug effects , Virion/pathogenicity , Virion/ultrastructure
15.
PLoS One ; 16(12): e0251834, 2021.
Article in English | MEDLINE | ID: covidwho-1556859

ABSTRACT

Structural characterization of the SARS-CoV-2 full length nsp1 protein will be an essential tool for developing new target-directed antiviral drugs against SARS-CoV-2 and for further understanding of intra- and intermolecular interactions of this protein. As a first step in the NMR studies of the protein, we report the 1H, 13C and 15N resonance backbone assignment as well as the Cß of the apo form of the full-lengthSARS-CoV-2 nsp1 including the folded domain together with the flaking N- and C- terminal intrinsically disordered fragments. The 19.8 kD protein was characterized by high-resolution NMR. Validation of assignment have been done by using two different mutants, H81P and K129E/D48E as well as by amino acid specific experiments. According to the obtained assignment, the secondary structure of the folded domain in solution was almost identical to its previously published X-ray structure as well as another published secondary structure obtained by NMR, but some discrepancies have been detected. In the solution SARS-CoV-2 nsp1 exhibited disordered, flexible N- and C-termini with different dynamic characteristics. The short peptide in the beginning of the disordered C-terminal domain adopted two different conformations distinguishable on the NMR time scale. We propose that the disordered and folded nsp1 domains are not fully independent units but are rather involved in intramolecular interactions. Studies of the structure and dynamics of the SARS-CoV-2 mutant in solution are on-going and will provide important insights into the molecular mechanisms underlying these interactions.


Subject(s)
Magnetic Resonance Spectroscopy/methods , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/chemistry , Amino Acid Sequence , COVID-19/pathology , COVID-19/virology , Carbon-13 Magnetic Resonance Spectroscopy , Humans , Mutation , Nitrogen Isotopes/chemistry , Protein Structure, Secondary , Proton Magnetic Resonance Spectroscopy , SARS-CoV-2/isolation & purification , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
16.
Commun Biol ; 4(1): 1240, 2021 10 29.
Article in English | MEDLINE | ID: covidwho-1493232

ABSTRACT

Circular tandem repeat proteins ('cTRPs') are de novo designed protein scaffolds (in this and prior studies, based on antiparallel two-helix bundles) that contain repeated protein sequences and structural motifs and form closed circular structures. They can display significant stability and solubility, a wide range of sizes, and are useful as protein display particles for biotechnology applications. However, cTRPs also demonstrate inefficient self-assembly from smaller subunits. In this study, we describe a new generation of cTRPs, with longer repeats and increased interaction surfaces, which enhanced the self-assembly of two significantly different sizes of homotrimeric constructs. Finally, we demonstrated functionalization of these constructs with (1) a hexameric array of peptide-binding SH2 domains, and (2) a trimeric array of anti-SARS CoV-2 VHH domains. The latter proved capable of sub-nanomolar binding affinities towards the viral receptor binding domain and potent viral neutralization function.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , Protein Engineering/methods , Proteins/chemistry , Proteins/metabolism , SARS-CoV-2/metabolism , Tandem Repeat Sequences , Amino Acid Sequence , COVID-19/virology , Computer Simulation , Crystallization , HEK293 Cells , Humans , Models, Molecular , Neutralization Tests , Protein Binding , Protein Domains , Protein Folding , Protein Structure, Secondary , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
17.
Soft Matter ; 17(42): 9772-9785, 2021 Nov 03.
Article in English | MEDLINE | ID: covidwho-1469997

ABSTRACT

A coarse-grained force field for molecular dynamics simulations of the native structures of proteins in a dissipative particle dynamics (DPD) framework is developed. The parameters for bonded interactions are derived by mapping the bonds and angles for 20 amino acids onto target distributions obtained from fully atomistic simulations in explicit solvent. A dual-basin potential is introduced for stabilizing backbone angles, to cover a wide spectrum of protein secondary structures. The backbone dihedral potential enables folding of the protein from an unfolded initial state to the folded native structure. The proposed force field is validated by evaluating the structural properties of several model peptides and proteins including the SARS-CoV-2 fusion peptide, consisting of α-helices, ß-sheets, loops and turns. Detailed comparisons with fully atomistic simulations are carried out to assess the ability of the proposed force field to stabilize the different secondary structures present in proteins. The compact conformations of the native states were evident from the radius of gyration and the high intensity peaks of the root mean square deviation histograms, which were found to be within 0.4 nm. The Ramachandran-like energy landscape on the phase space of backbone angles (θ) and dihedrals (ϕ) effectively captured the conformational phase space of α-helices at ∼(ϕ = 50°,θ = 90°) and ß-strands at ∼(ϕ = ±180°,θ = 90-120°). Furthermore, the residue-residue native contacts were also well reproduced by the proposed DPD model. The applicability of the model to multidomain complexes was assessed using lysozyme and a large α-helical bacterial pore-forming toxin, cytolysin A. Our study illustrates that the proposed force field is generic, and can potentially be extended for efficient in silico investigations of membrane bound polypeptides and proteins using DPD simulations.


Subject(s)
COVID-19 , Humans , Molecular Dynamics Simulation , Protein Structure, Secondary , Proteins , SARS-CoV-2
18.
Biomol NMR Assign ; 15(2): 255-260, 2021 10.
Article in English | MEDLINE | ID: covidwho-1453890

ABSTRACT

The nucleoprotein (N) from SARS-CoV-2 is an essential cofactor of the viral replication transcription complex and as such represents an important target for viral inhibition. It has also been shown to colocalize to the transcriptase-replicase complex, where many copies of N decorate the viral genome, thereby protecting it from the host immune system. N has also been shown to phase separate upon interaction with viral RNA. N is a 419 amino acid multidomain protein, comprising two folded, RNA-binding and dimerization domains spanning residues 45-175 and 264-365 respectively. The remaining 164 amino acids are predicted to be intrinsically disordered, but there is currently no atomic resolution information describing their behaviour. Here we assign the backbone resonances of the first two intrinsically disordered domains (N1, spanning residues 1-44 and N3, spanning residues 176-263). Our assignment provides the basis for the identification of inhibitors and functional and interaction studies of this essential protein.


Subject(s)
Nuclear Magnetic Resonance, Biomolecular , Nucleoproteins/chemistry , SARS-CoV-2 , Viral Proteins/chemistry , Models, Molecular , Protein Domains , Protein Structure, Secondary
19.
Int J Biol Macromol ; 147: 513-520, 2020 Mar 15.
Article in English | MEDLINE | ID: covidwho-1454163

ABSTRACT

The alternative splicing is a mechanism increasing the number of expressed proteins and a variety of these functions. We uncovered the protein domains most frequently lacked or occurred in the splice variants. Proteins presented by several isoforms participate in such processes as transcription regulation, immune response, etc. Our results displayed the association of alternative splicing with branched regulatory pathways. By considering the published data on the protein proteins encoded by the 18th human chromosome, we noted that alternative products display the differences in several functional features, such as phosphorylation, subcellular location, ligand specificity, protein-protein interactions, etc. The investigation of alternative variants referred to the protein kinase domain was performed by comparing the alternative sequences with 3D structures. It was shown that large enough insertions/deletions could be compatible with the kinase fold if they match between the conserved secondary structures. Using the 3D data on human proteins, we showed that conformational flexibility could accommodate fold alterations in splice variants. The investigations of structural and functional differences in splice isoforms are required to understand how to distinguish the isoforms expressed as functioning proteins from the non-realized transcripts. These studies allow filling the gap between genomic and proteomic data.


Subject(s)
Alternative Splicing , Chromosomes, Human, Pair 18 , Databases, Protein , RNA-Binding Proteins , Chromosomes, Human, Pair 18/genetics , Chromosomes, Human, Pair 18/metabolism , Humans , Protein Structure, Secondary , Proteomics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism
20.
Mol Cell ; 81(22): 4605-4621.e11, 2021 11 18.
Article in English | MEDLINE | ID: covidwho-1440263

ABSTRACT

G-protein-coupled receptors (GPCRs), also known as seven transmembrane receptors (7TMRs), typically interact with two distinct signal-transducers, i.e., G proteins and ß-arrestins (ßarrs). Interestingly, there are some non-canonical 7TMRs that lack G protein coupling but interact with ßarrs, although an understanding of their transducer coupling preference, downstream signaling, and structural mechanism remains elusive. Here, we characterize two such non-canonical 7TMRs, namely, the decoy D6 receptor (D6R) and the complement C5a receptor subtype 2 (C5aR2), in parallel with their canonical GPCR counterparts. We discover that D6R and C5aR2 efficiently couple to ßarrs, exhibit distinct engagement of GPCR kinases (GRKs), and activate non-canonical downstream signaling pathways. We also observe that ßarrs adopt distinct conformations for D6R and C5aR2, compared to their canonical GPCR counterparts, in response to common natural agonists. Our study establishes D6R and C5aR2 as ßarr-coupled 7TMRs and provides key insights into their regulation and signaling with direct implication for biased agonism.


Subject(s)
Cell Membrane/metabolism , Protein Conformation , Signal Transduction , beta-Arrestins/chemistry , Animals , GTP-Binding Proteins/chemistry , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , Protein Binding , Protein Domains , Protein Structure, Secondary , Protein Transport , Receptor, Anaphylatoxin C5a/metabolism
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