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1.
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
2.
Biomol NMR Assign ; 15(1): 219-227, 2021 04.
Article in English | MEDLINE | ID: covidwho-1384623

ABSTRACT

The nucleocapsid protein N from SARS-CoV-2 is one of the most highly expressed proteins by the virus and plays a number of important roles in the transcription and assembly of the virion within the infected host cell. It is expected to be characterized by a highly dynamic and heterogeneous structure as can be inferred by bioinformatics analyses as well as from the data available for the homologous protein from SARS-CoV. The two globular domains of the protein (NTD and CTD) have been investigated while no high-resolution information is available yet for the flexible regions of the protein. We focus here on the 1-248 construct which comprises two disordered fragments (IDR1 and IDR2) in addition to the N-terminal globular domain (NTD) and report the sequence-specific assignment of the two disordered regions, a step forward towards the complete characterization of the whole protein.


Subject(s)
Coronavirus Nucleocapsid Proteins/chemistry , Magnetic Resonance Spectroscopy , SARS-CoV-2/chemistry , Carbon Isotopes , Computational Biology , Hydrogen , Nitrogen Isotopes , Phosphoproteins/chemistry , Protein Binding , Protein Domains , Protein Structure, Secondary
3.
Biomol NMR Assign ; 15(1): 165-171, 2021 04.
Article in English | MEDLINE | ID: covidwho-1384622

ABSTRACT

SARS-CoV-2 RNA, nsP3c (non-structural Protein3c) spans the sequence of the so-called SARS Unique Domains (SUDs), first observed in SARS-CoV. Although the function of this viral protein is not fully elucidated, it is believed that it is crucial for the formation of the replication/transcription viral complex (RTC) and of the interaction of various viral "components" with the host cell; thus, it is essential for the entire viral life cycle. The first two SUDs, the so-called SUD-N (the N-terminal domain) and SUD-M (domain following SUD-N) domains, exhibit topological and conformational features that resemble the nsP3b macro (or "X") domain. Indeed, they are all folded in a three-layer α/ß/α sandwich structure, as revealed through crystallographic structural investigation of SARS-CoV SUDs, and they have been attributed to different substrate selectivity as they selectively bind to oligonucleotides. On the other hand, the C-terminal SUD (SUD-C) exhibit much lower sequence similarities compared to the SUD-N & SUD-M, as reported in previous crystallographic and NMR studies of SARS-CoV. In the absence of the 3D structures of SARS-CoV-2, we report herein the almost complete NMR backbone and side-chain resonance assignment (1H,13C,15N) of SARS-CoV-2 SUD-M and SUD-C proteins, and the NMR chemical shift-based prediction of their secondary structure elements. These NMR data will set the base for further understanding at the atomic-level conformational dynamics of these proteins and will allow the effective screening of a large number of small molecules as binders with potential biological impact on their function.


Subject(s)
Coronavirus Papain-Like Proteases/chemistry , Magnetic Resonance Spectroscopy , SARS-CoV-2/chemistry , Carbon Isotopes , Hydrogen , Nitrogen Isotopes , Protein Binding , Protein Domains , Protein Structure, Secondary
4.
Biomol NMR Assign ; 15(1): 85-89, 2021 04.
Article in English | MEDLINE | ID: covidwho-1384621

ABSTRACT

Among the proteins encoded by the SARS-CoV-2 RNA, nsP3 (non-structural Protein3) is the largest multi-domain protein. Its role is multifaceted and important for the viral life cycle. Nonetheless, regarding the specific role of each domain there are many aspects of their function that have to be investigated. SARS Unique Domains (SUDs), constitute the nsP3c region of the nsP3, and were observed for the first time in SARS-CoV. Two of them, namely SUD-N (the first SUD) and the SUD-M (sequential to SUD-N), exhibit structural homology with nsP3b ("X" or macro domain); indeed all of them are folded in a three-layer α/ß/α sandwich. On the contrary, they do not exhibit functional similarities, like ADP-ribose binding properties and ADP-ribose hydrolase activity. There are reports that suggest that these two SUDs may exhibit a binding selectivity towards G-oligonucleotides, a feature which may contribute to the characterization of their role in the formation of the replication/transcription viral complex (RTC) and of the interaction of various viral "components" with the host cell. While the structures of these domains of SARS-CoV-2 have not been determined yet, SUDs interaction with oligonucleotides and/or RNA molecules may provide a platform for drug discovery. Here, we report the almost complete NMR backbone and side-chain resonance assignment (1H,13C,15N) of SARS-CoV-2 SUD-N protein, and the NMR chemical shift-based prediction of the secondary structure elements. These data may be exploited for its 3D structure determination and the screening of chemical compounds libraries, which may alter SUD-N function.


Subject(s)
Coronavirus Papain-Like Proteases/chemistry , Magnetic Resonance Spectroscopy , SARS-CoV-2/chemistry , Carbon Isotopes , Drug Design , Hydrogen , Nitrogen Isotopes , Oligonucleotides/chemistry , Protein Domains , Protein Structure, Secondary , Virus Replication
5.
Biomol NMR Assign ; 15(1): 65-71, 2021 04.
Article in English | MEDLINE | ID: covidwho-1184741

ABSTRACT

The international Covid19-NMR consortium aims at the comprehensive spectroscopic characterization of SARS-CoV-2 RNA elements and proteins and will provide NMR chemical shift assignments of the molecular components of this virus. The SARS-CoV-2 genome encodes approximately 30 different proteins. Four of these proteins are involved in forming the viral envelope or in the packaging of the RNA genome and are therefore called structural proteins. The other proteins fulfill a variety of functions during the viral life cycle and comprise the so-called non-structural proteins (nsps). Here, we report the near-complete NMR resonance assignment for the backbone chemical shifts of the non-structural protein 10 (nsp10). Nsp10 is part of the viral replication-transcription complex (RTC). It aids in synthesizing and modifying the genomic and subgenomic RNAs. Via its interaction with nsp14, it ensures transcriptional fidelity of the RNA-dependent RNA polymerase, and through its stimulation of the methyltransferase activity of nsp16, it aids in synthesizing the RNA cap structures which protect the viral RNAs from being recognized by the innate immune system. Both of these functions can be potentially targeted by drugs. Our data will aid in performing additional NMR-based characterizations, and provide a basis for the identification of possible small molecule ligands interfering with nsp10 exerting its essential role in viral replication.


Subject(s)
Magnetic Resonance Spectroscopy , SARS-CoV-2/chemistry , Viral Regulatory and Accessory Proteins/chemistry , Amino Acid Motifs , Carbon Isotopes , Exoribonucleases/chemistry , Hydrogen , Hydrogen Bonding , Ligands , Methyltransferases , Nitrogen Isotopes , Protein Structure, Secondary , RNA, Viral , Viral Envelope , Viral Nonstructural Proteins/chemistry , Virus Replication , Zinc Fingers
6.
Biomol NMR Assign ; 15(1): 153-157, 2021 04.
Article in English | MEDLINE | ID: covidwho-1141506

ABSTRACT

Coronaviruses have become of great medical and scientific interest because of the Covid-19 pandemic. The hCoV-HKU1 is an endemic betacoronavirus that causes mild respiratory symptoms, although the infection can progress to severe lung disease and death. During viral replication, a discontinuous transcription of the genome takes place, producing the subgenomic messenger RNAs. The nucleocapsid protein (N) plays a pivotal role in the regulation of this process, acting as an RNA chaperone and participating in the nucleocapsid assembly. The isolated N-terminal domain of protein N (N-NTD) specifically binds to the transcriptional regulatory sequences and control the melting of the double-stranded RNA. Here, we report the resonance assignments of the N-NTD of HKU1-CoV.


Subject(s)
Betacoronavirus/chemistry , Coronavirus Nucleocapsid Proteins/chemistry , Magnetic Resonance Spectroscopy , Carbon Isotopes , Escherichia coli/metabolism , Hydrogen , Nitrogen Isotopes , Protein Binding , Protein Domains , Protein Structure, Secondary , Software
7.
Biomol NMR Assign ; 15(1): 129-135, 2021 04.
Article in English | MEDLINE | ID: covidwho-1141504

ABSTRACT

The current outbreak of the highly infectious COVID-19 respiratory disease is caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). To fight the pandemic, the search for promising viral drug targets has become a cross-border common goal of the international biomedical research community. Within the international Covid19-NMR consortium, scientists support drug development against SARS-CoV-2 by providing publicly available NMR data on viral proteins and RNAs. The coronavirus nucleocapsid protein (N protein) is an RNA-binding protein involved in viral transcription and replication. Its primary function is the packaging of the viral RNA genome. The highly conserved architecture of the coronavirus N protein consists of an N-terminal RNA-binding domain (NTD), followed by an intrinsically disordered Serine/Arginine (SR)-rich linker and a C-terminal dimerization domain (CTD). Besides its involvement in oligomerization, the CTD of the N protein (N-CTD) is also able to bind to nucleic acids by itself, independent of the NTD. Here, we report the near-complete NMR backbone chemical shift assignments of the SARS-CoV-2 N-CTD to provide the basis for downstream applications, in particular site-resolved drug binding studies.


Subject(s)
Coronavirus Nucleocapsid Proteins/chemistry , Magnetic Resonance Spectroscopy , SARS-CoV-2/chemistry , Carbon Isotopes , Crystallography, X-Ray , Dimerization , Drug Design , Hydrogen , Hydrogen-Ion Concentration , Nitrogen Isotopes , Phosphoproteins/chemistry , Protein Binding , Protein Domains , Protein Interaction Mapping , Protein Structure, Secondary
8.
Biomol NMR Assign ; 15(1): 73-77, 2021 04.
Article in English | MEDLINE | ID: covidwho-1141500

ABSTRACT

The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project covid19-nmr, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein nsp7. The 83 amino acid nsp7 protein is an essential cofactor in the RNA-dependent RNA polymerase. The polymerase activity and processivity of nsp12 are greatly enhanced by binding 1 copy of nsp7 and 2 copies of nsp8 to form a 160 kD complex. A separate hexadecameric complex of nsp7 and nsp8 (8 copies of each) forms a large ring-like structure. Thus, nsp7 is an important component of several large protein complexes that are required for replication of the large and complex coronavirus genome. We here report the near-complete NMR backbone and sidechain resonance assignment (1H,13C,15N) of isolated nsp7 from SARS-CoV-2 in solution. Further, we derive the secondary structure and compare it to the previously reported assignments and structure of the SARS-CoV nsp7.


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase/chemistry , Magnetic Resonance Spectroscopy , SARS-CoV-2/chemistry , Carbon Isotopes , Genome, Viral , Hydrogen , Hydrogen-Ion Concentration , Nitrogen Isotopes , Protein Binding , Protein Domains , Protein Structure, Secondary
9.
Biomol NMR Assign ; 15(1): 219-227, 2021 04.
Article in English | MEDLINE | ID: covidwho-1116314

ABSTRACT

The nucleocapsid protein N from SARS-CoV-2 is one of the most highly expressed proteins by the virus and plays a number of important roles in the transcription and assembly of the virion within the infected host cell. It is expected to be characterized by a highly dynamic and heterogeneous structure as can be inferred by bioinformatics analyses as well as from the data available for the homologous protein from SARS-CoV. The two globular domains of the protein (NTD and CTD) have been investigated while no high-resolution information is available yet for the flexible regions of the protein. We focus here on the 1-248 construct which comprises two disordered fragments (IDR1 and IDR2) in addition to the N-terminal globular domain (NTD) and report the sequence-specific assignment of the two disordered regions, a step forward towards the complete characterization of the whole protein.


Subject(s)
Coronavirus Nucleocapsid Proteins/chemistry , Magnetic Resonance Spectroscopy , SARS-CoV-2/chemistry , Carbon Isotopes , Computational Biology , Hydrogen , Nitrogen Isotopes , Phosphoproteins/chemistry , Protein Binding , Protein Domains , Protein Structure, Secondary
10.
Biomol NMR Assign ; 15(1): 203-211, 2021 04.
Article in English | MEDLINE | ID: covidwho-1046684

ABSTRACT

The SARS-CoV-2 (SCoV-2) virus is the causative agent of the ongoing COVID-19 pandemic. It contains a positive sense single-stranded RNA genome and belongs to the genus of Betacoronaviruses. The 5'- and 3'-genomic ends of the 30 kb SCoV-2 genome are potential antiviral drug targets. Major parts of these sequences are highly conserved among Betacoronaviruses and contain cis-acting RNA elements that affect RNA translation and replication. The 31 nucleotide (nt) long highly conserved stem-loop 5a (SL5a) is located within the 5'-untranslated region (5'-UTR) important for viral replication. SL5a features a U-rich asymmetric bulge and is capped with a 5'-UUUCGU-3' hexaloop, which is also found in stem-loop 5b (SL5b). We herein report the extensive 1H, 13C and 15N resonance assignment of SL5a as basis for in-depth structural studies by solution NMR spectroscopy.


Subject(s)
5' Untranslated Regions , Coronavirus Papain-Like Proteases/chemistry , Magnetic Resonance Spectroscopy , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Carbon Isotopes , Genes, Viral , Hydrogen , Nitrogen Isotopes , Protein Binding , Protein Domains , Protein Structure, Secondary
11.
Biomol NMR Assign ; 15(1): 173-176, 2021 04.
Article in English | MEDLINE | ID: covidwho-1043969

ABSTRACT

The non-structural protein nsp3 from SARS-CoV-2 plays an essential role in the viral replication transcription complex. Nsp3a constitutes the N-terminal domain of nsp3, comprising a ubiquitin-like folded domain and a disordered acidic chain. This region of nsp3a has been linked to interactions with the viral nucleoprotein and the structure of double membrane vesicles. Here, we report the backbone resonance assignment of both domains of nsp3a. The study is carried out in the context of the international covid19-nmr consortium, which aims to characterize SARS-CoV-2 proteins and RNAs, providing for example NMR chemical shift assignments of the different viral components. Our assignment will provide the basis for the identification of inhibitors and further functional and interaction studies of this essential protein.


Subject(s)
Coronavirus Papain-Like Proteases/chemistry , Magnetic Resonance Spectroscopy , SARS-CoV-2/chemistry , Carbon Isotopes , Escherichia coli , Hydrogen , Hydrogen-Ion Concentration , Nitrogen Isotopes , Plasmids/metabolism , Protein Binding , Protein Domains , Protein Structure, Secondary
12.
Biomol NMR Assign ; 15(1): 137-142, 2021 04.
Article in English | MEDLINE | ID: covidwho-1042942

ABSTRACT

SARS-CoV-2 is a novel pathogen causing pneumonia named COVID-19 and leading to a severe pandemic since the end of 2019. The genome of SARS-CoV-2 contains a macro domain that may play an important role in regulating ADP-ribosylation in host cells and initiating viral replication. Here, we report the 1H, 13C, and 15N resonance assignments of the SARS-CoV-2 macro domain. This work provides the ground for further structural deciphering and biophysical investigation in protein function and antiviral agent design.


Subject(s)
Magnetic Resonance Spectroscopy , SARS-CoV-2/chemistry , Viral Nonstructural Proteins/chemistry , Viral Proteins/chemistry , Carbon Isotopes , Genome, Viral , Hydrogen , Hydrogen-Ion Concentration , Nitrogen Isotopes , Protein Binding , Protein Domains , Protein Structure, Secondary , Temperature
13.
Biomol NMR Assign ; 15(1): 153-157, 2021 04.
Article in English | MEDLINE | ID: covidwho-1009212

ABSTRACT

Coronaviruses have become of great medical and scientific interest because of the Covid-19 pandemic. The hCoV-HKU1 is an endemic betacoronavirus that causes mild respiratory symptoms, although the infection can progress to severe lung disease and death. During viral replication, a discontinuous transcription of the genome takes place, producing the subgenomic messenger RNAs. The nucleocapsid protein (N) plays a pivotal role in the regulation of this process, acting as an RNA chaperone and participating in the nucleocapsid assembly. The isolated N-terminal domain of protein N (N-NTD) specifically binds to the transcriptional regulatory sequences and control the melting of the double-stranded RNA. Here, we report the resonance assignments of the N-NTD of HKU1-CoV.


Subject(s)
Betacoronavirus/chemistry , Coronavirus Nucleocapsid Proteins/chemistry , Magnetic Resonance Spectroscopy , Carbon Isotopes , Escherichia coli/metabolism , Hydrogen , Nitrogen Isotopes , Protein Binding , Protein Domains , Protein Structure, Secondary , Software
14.
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
15.
Biomol NMR Assign ; 15(1): 85-89, 2021 04.
Article in English | MEDLINE | ID: covidwho-938616

ABSTRACT

Among the proteins encoded by the SARS-CoV-2 RNA, nsP3 (non-structural Protein3) is the largest multi-domain protein. Its role is multifaceted and important for the viral life cycle. Nonetheless, regarding the specific role of each domain there are many aspects of their function that have to be investigated. SARS Unique Domains (SUDs), constitute the nsP3c region of the nsP3, and were observed for the first time in SARS-CoV. Two of them, namely SUD-N (the first SUD) and the SUD-M (sequential to SUD-N), exhibit structural homology with nsP3b ("X" or macro domain); indeed all of them are folded in a three-layer α/ß/α sandwich. On the contrary, they do not exhibit functional similarities, like ADP-ribose binding properties and ADP-ribose hydrolase activity. There are reports that suggest that these two SUDs may exhibit a binding selectivity towards G-oligonucleotides, a feature which may contribute to the characterization of their role in the formation of the replication/transcription viral complex (RTC) and of the interaction of various viral "components" with the host cell. While the structures of these domains of SARS-CoV-2 have not been determined yet, SUDs interaction with oligonucleotides and/or RNA molecules may provide a platform for drug discovery. Here, we report the almost complete NMR backbone and side-chain resonance assignment (1H,13C,15N) of SARS-CoV-2 SUD-N protein, and the NMR chemical shift-based prediction of the secondary structure elements. These data may be exploited for its 3D structure determination and the screening of chemical compounds libraries, which may alter SUD-N function.


Subject(s)
Coronavirus Papain-Like Proteases/chemistry , Magnetic Resonance Spectroscopy , SARS-CoV-2/chemistry , Carbon Isotopes , Drug Design , Hydrogen , Nitrogen Isotopes , Oligonucleotides/chemistry , Protein Domains , Protein Structure, Secondary , Virus Replication
16.
Biomol NMR Assign ; 15(1): 65-71, 2021 04.
Article in English | MEDLINE | ID: covidwho-915245

ABSTRACT

The international Covid19-NMR consortium aims at the comprehensive spectroscopic characterization of SARS-CoV-2 RNA elements and proteins and will provide NMR chemical shift assignments of the molecular components of this virus. The SARS-CoV-2 genome encodes approximately 30 different proteins. Four of these proteins are involved in forming the viral envelope or in the packaging of the RNA genome and are therefore called structural proteins. The other proteins fulfill a variety of functions during the viral life cycle and comprise the so-called non-structural proteins (nsps). Here, we report the near-complete NMR resonance assignment for the backbone chemical shifts of the non-structural protein 10 (nsp10). Nsp10 is part of the viral replication-transcription complex (RTC). It aids in synthesizing and modifying the genomic and subgenomic RNAs. Via its interaction with nsp14, it ensures transcriptional fidelity of the RNA-dependent RNA polymerase, and through its stimulation of the methyltransferase activity of nsp16, it aids in synthesizing the RNA cap structures which protect the viral RNAs from being recognized by the innate immune system. Both of these functions can be potentially targeted by drugs. Our data will aid in performing additional NMR-based characterizations, and provide a basis for the identification of possible small molecule ligands interfering with nsp10 exerting its essential role in viral replication.


Subject(s)
Magnetic Resonance Spectroscopy , SARS-CoV-2/chemistry , Viral Regulatory and Accessory Proteins/chemistry , Amino Acid Motifs , Carbon Isotopes , Exoribonucleases/chemistry , Hydrogen , Hydrogen Bonding , Ligands , Methyltransferases , Nitrogen Isotopes , Protein Structure, Secondary , RNA, Viral , Viral Envelope , Viral Nonstructural Proteins/chemistry , Virus Replication , Zinc Fingers
17.
Biomol NMR Assign ; 14(2): 329-333, 2020 10.
Article in English | MEDLINE | ID: covidwho-774089

ABSTRACT

The ongoing pandemic caused by the Betacoronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) demonstrates the urgent need of coordinated and rapid research towards inhibitors of the COVID-19 lung disease. The covid19-nmr consortium seeks to support drug development by providing publicly accessible NMR data on the viral RNA elements and proteins. The SARS-CoV-2 genome encodes for approximately 30 proteins, among them are the 16 so-called non-structural proteins (Nsps) of the replication/transcription complex. The 217-kDa large Nsp3 spans one polypeptide chain, but comprises multiple independent, yet functionally related domains including the viral papain-like protease. The Nsp3e sub-moiety contains a putative nucleic acid-binding domain (NAB) with so far unknown function and consensus target sequences, which are conceived to be both viral and host RNAs and DNAs, as well as protein-protein interactions. Its NMR-suitable size renders it an attractive object to study, both for understanding the SARS-CoV-2 architecture and drugability besides the classical virus' proteases. We here report the near-complete NMR backbone chemical shifts of the putative Nsp3e NAB that reveal the secondary structure and compactness of the domain, and provide a basis for NMR-based investigations towards understanding and interfering with RNA- and small-molecule-binding by Nsp3e.


Subject(s)
Betacoronavirus/metabolism , Carbon-13 Magnetic Resonance Spectroscopy , Nitrogen Isotopes/chemistry , Nucleic Acids/metabolism , Proton Magnetic Resonance Spectroscopy , Viral Nonstructural Proteins/chemistry , Protein Binding , Protein Domains , SARS-CoV-2
18.
Int J Mol Sci ; 21(14)2020 Jul 11.
Article in English | MEDLINE | ID: covidwho-646270

ABSTRACT

A dodecadepsipeptide valinomycin (VLM) has been most recently reported to be a potential anti-coronavirus drug that could be efficiently produced on a large scale. It is thus of importance to study solid-phase forms of VLM in order to be able to ensure its polymorphic purity in drug formulations. The previously available solid-state NMR (SSNMR) data are combined with the plane-wave DFT computations in the NMR crystallography framework. Structural/spectroscopical predictions (the PBE functional/GIPAW method) are obtained to characterize four polymorphs of VLM. Interactions which confer a conformational stability to VLM molecules in these crystalline forms are described in detail. The way how various structural factors affect the values of SSNMR parameters is thoroughly analyzed, and several SSNMR markers of the respective VLM polymorphs are identified. The markers are connected to hydrogen bonding effects upon the corresponding (13C/15N/1H) isotropic chemical shifts of (CO, Namid, Hamid, Hα) VLM backbone nuclei. These results are expected to be crucial for polymorph control of VLM and in probing its interactions in dosage forms.


Subject(s)
Magnetic Resonance Spectroscopy/methods , Valinomycin/chemistry , Betacoronavirus/chemistry , Betacoronavirus/isolation & purification , Betacoronavirus/metabolism , COVID-19 , Carbon Isotopes/chemistry , Coronavirus Infections/pathology , Coronavirus Infections/virology , Crystallography , Hydrogen Bonding , Nitrogen Isotopes/chemistry , Pandemics , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , SARS-CoV-2 , Valinomycin/metabolism
19.
Biomol NMR Assign ; 14(2): 339-346, 2020 10.
Article in English | MEDLINE | ID: covidwho-716391

ABSTRACT

The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project COVID19-NMR, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein Nsp3b, a domain of Nsp3. The 217-kDa large Nsp3 protein contains multiple structurally independent, yet functionally related domains including the viral papain-like protease and Nsp3b, a macrodomain (MD). In general, the MDs of SARS-CoV and MERS-CoV were suggested to play a key role in viral replication by modulating the immune response of the host. The MDs are structurally conserved. They most likely remove ADP-ribose, a common posttranslational modification, from protein side chains. This de-ADP ribosylating function has potentially evolved to protect the virus from the anti-viral ADP-ribosylation catalyzed by poly-ADP-ribose polymerases (PARPs), which in turn are triggered by pathogen-associated sensing of the host immune system. This renders the SARS-CoV-2 Nsp3b a highly relevant drug target in the viral replication process. We here report the near-complete NMR backbone resonance assignment (1H, 13C, 15N) of the putative Nsp3b MD in its apo form and in complex with ADP-ribose. Furthermore, we derive the secondary structure of Nsp3b in solution. In addition, 15N-relaxation data suggest an ordered, rigid core of the MD structure. These data will provide a basis for NMR investigations targeted at obtaining small-molecule inhibitors interfering with the catalytic activity of Nsp3b.


Subject(s)
Adenosine Diphosphate Ribose/metabolism , Apoproteins/chemistry , Betacoronavirus/metabolism , Carbon-13 Magnetic Resonance Spectroscopy , Nitrogen Isotopes/chemistry , Proton Magnetic Resonance Spectroscopy , Viral Nonstructural Proteins/chemistry , Amino Acid Sequence , Apoproteins/metabolism , Protein Domains , Protein Structure, Secondary , SARS-CoV-2 , Viral Nonstructural Proteins/metabolism
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