Combined NMR and molecular dynamics conformational filter identifies unambiguously dynamic ensembles of Dengue protease NS2B/NS3pro

The dengue protease NS2B/NS3pro has been reported to adopt either an ‘open’ or a ‘closed’ conformation. We have developed a conformational filter that combines NMR with MD simulations to identify conformational ensembles that dominate in solution. Experimental values derived from relaxation parameters for the backbone and methyl side chains were compared with the corresponding back-calculated relaxation parameters of different conformational ensembles obtained from free MD simulations. Our results demonstrate a high prevalence for the ‘closed’ conformational ensemble while the ‘open’ conformation is absent, indicating that the latter conformation is most probably due to crystal contacts. Conversely, conformational ensembles in which the positioning of the co-factor NS2B results in a ‘partially’ open conformation, previously described in both MD simulations and X-ray studies, were identified by our conformational filter. Altogether, we believe that our approach allows for unambiguous identification of true conformational ensembles, an essential step for reliable drug discovery

Probing contacts of inhibitor locked in transition states in the catalytic triad of DENV2 type serine protease and its mutants by 1H, 19F and 15 N NMR spectroscopy

Background: Detailed structural knowledge of enzyme-inhibitor complexes trapped in intermediate state is the key for a fundamental understanding of reaction mechanisms taking place in enzymes and is indispensable as a structure-guided drug design tool. Solution state NMR uniquely allows the study of active sites of enzymes in equilibrium between different tautomeric forms. In this study 1H, 19F and 15 N NMR spectroscopy has been used to probe the interaction contacts of inhibitors locked in transition states of the catalytic triad of a serine protease. It was demonstrated on the serotype II Dengue virus NS2B:NS3pro serine protease and its mutants, H51N and S135A, in complex with high-affinity ligands containing trifluoromethyl ketone (tfk) and boronic groups in the C-terminal of tetra-peptides. Results: Monitoring 19F resonances, shows that only one of the two isomers of the tfk tetra-peptide binds with NS2B:NS3pro and that access to the bulk of the active site is limited. Moreover, there were no bound water found in proximity of the active site for any of the ligands manifesting in a favorable condition for formation of low barrier hydrogen bonds (LBHB) in the catalytic triad. Based on this data we were able to identify a locked conformation of the protein active site. The data also indicates that the different parts of the binding site most likely act independently of each other. Conclusions: Our reported findings increases the knowledge of the detailed function of the catalytic triad in serine proteases and could facilitate the development of rational structure based inhibitors that can selectively target the NS3 protease of Dengue type II (DENV2) virus. In addition the results shows the usefulness of probing active sites using F-19 NMR spectroscopy

Novel NMR Assignment Strategy Reveals Structural Heterogeneity in Solution of the nsP3 HVD Domain of Venezuelan Equine Encephalitis Virus

In recent years, intrinsically disordered proteins (IDPs) and disordered domains have attracted great attention. Many of them contain linear motifs that mediate interactions with other factors during formation of multicomponent protein complexes. NMR spectrometry is a valuable tool for characterizing this type of interactions on both amino acid (aa) and atomic levels. Alphaviruses encode a nonstructural protein nsP3, which drives viral replication complex assembly. nsP3 proteins contain over 200-aa-long hypervariable domains (HVDs), which exhibits no homology between different alphavirus species, are predicted to be intrinsically disordered and appear to be critical for alphavirus adaptation to different cells. Previously, we have shown that nsP3 HVD of chikungunya virus (CHIKV) is completely disordered with low tendency to form secondary structures in free form. In this new study, we used novel NMR approaches to assign the spectra for the nsP3 HVD of Venezuelan equine encephalitis virus (VEEV). The HVDs of CHIKV and VEEV have no homology but are both involved in replication complex assembly and function. We have found that VEEV nsP3 HVD is also mostly disordered but contains a short stable alpha-helix in its C-terminal fragment, which mediates interaction with the members of cellular Fragile X syndrome protein family. Our NMR data also suggest that VEEV HVD has several regions with tendency to form secondary structures

H-1, C-13 and N-15 resonance assignment of the SARS-CoV-2 full-length nsp1 protein and its mutants reveals its unique secondary structure features in solution

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 H-1, C-13 and N-15 resonance backbone assignment as well as the C beta 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

All Domains of SARS-CoV-2 nsp1 Determine Translational Shutoff and Cytotoxicity of the Protein

The nsp1 of SARS-CoV-2 is a multifunctional protein that modifies the intracellular environment for the needs of viral replication. It is responsible for the development of translational shutoff, and its expression alone is sufficient to cause a cytopathic effect (CPE).Replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strongly affects cellular metabolism and results in rapid development of the cytopathic effect (CPE). The hallmarks of virus-induced modifications are inhibition of translation of cellular mRNAs and redirection of the cellular translational machinery to the synthesis of virus-specific proteins. The multifunctional nonstructural protein 1 (nsp1) of SARS-CoV-2 is a major virulence factor and a key contributor to the development of translational shutoff. In this study, we applied a wide range of virological and structural approaches to further analyze nsp1 functions. The expression of this protein alone was found to be sufficient to cause CPE. However, we selected several nsp1 mutants exhibiting noncytopathic phenotypes. The attenuating mutations were detected in three clusters, located in the C-terminal helices, in one of the loops of the structured domain and in the junction of the disordered and structured fragment of nsp1. NMR-based analysis of the wild type nsp1 and its mutants did not confirm the existence of a stable beta 5-strand that was proposed by the X-ray structure. In solution, this protein appears to be present in a dynamic conformation, which is required for its functions in CPE development and viral replication. The NMR data also suggest a dynamic interaction between the N-terminal and C-terminal domains. The identified nsp1 mutations make this protein noncytotoxic and incapable of inducing translational shutoff, but they do not result in deleterious effects on viral cytopathogenicity.IMPORTANCE The nsp1 of SARS-CoV-2 is a multifunctional protein that modifies the intracellular environment for the needs of viral replication. It is responsible for the development of translational shutoff, and its expression alone is sufficient to cause a cytopathic effect (CPE). In this study, we selected a wide range of nsp1 mutants exhibiting noncytopathic phenotypes. The attenuating mutations, clustered in three different fragments of nsp1, were extensively characterized via virological and structural methods. Our data strongly suggest interactions between the nsp1 domains, which are required for the protein's functions in CPE development. Most of the mutations made nsp1 noncytotoxic and incapable of inducing translational shutoff. Most of them did not affect the viability of the viruses, but they did decrease the rates of replication in cells competent in type I IFN induction and signaling. These mutations, and their combinations, in particular, can be used for the development of SARS-CoV-2 variants with attenuated phenotypes

Site-specific recognition of SARS-CoV-2 nsp1 protein with a tailored titanium dioxide nanoparticle - elucidation of the complex structure using NMR data and theoretical calculation

The ongoing world-wide Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) pandemic shows the need for new potential sensing and therapeutic means against the CoV viruses. The SARS-CoV-2 nsp1 protein is important, both for replication and pathogenesis, making it an attractive target for intervention. In this study we investigated the interaction of this protein with two types of titania nanoparticles by NMR and discovered that while lactate capped particles essentially did not interact with the protein chain, the aminoalcohol-capped ones showed strong complexation with a distinct part of an ordered alpha-helix fragment. The structure of the forming complex was elucidated based on NMR data and theoretical calculation. To the best of our knowledge, this is the first time that a tailored titanium oxide nanoparticle was shown to interact specifically with a unique site of the full-length SARS-CoV-2 nsp1 protein, possibly interfering with its functionality

NMR spectrum reconstruction as a pattern recognition problem

A new deep neural network based on the WaveNet architecture (WNN) is presented, which is designed to grasp specific patterns in the NMR spectra. When trained at a fixed non-uniform sampling (NUS) schedule, the WNN benefits from pattern recognition of the corresponding point spread function (PSF) pattern produced by each spectral peak resulting in the highest quality and robust reconstruction of the NUS spectra as demonstrated in simulations and exemplified in this work on 2D 1H-15N correlation spectra of three representative globular proteins with different sizes: Ubiquitin (8.6 kDa), Azurin (14 kDa), and Malt1 (44 kDa). The pattern recognition by WNN is also demonstrated for successful virtual homo-decoupling in a 2D methyl 1H-13 HMQC spectrum of MALT1. We demonstrate using WNN that prior knowledge about the NUS schedule, which so far was not fully exploited, can be used for designing new powerful NMR processing techniques that surpass the existing algorithmic methods

H-1, C-13 and N-15 resonance assignment of backbone and IVL-methyl side chain of the S135A mutant NS3pro/NS2B protein of Dengue II virus reveals unique secondary structure features in solution

The serotype II Dengue (DENV 2) virus is the most prevalent of all four known serotypes. Herein, we present nearly complete H-1, N-15, and C-13 backbone and H-1, C-13 isoleucine, valine, and leucine methyl resonance assignment of the apo S135A catalytically inactive variant of the DENV 2 protease enzyme folded as a tandem formed between the serine protease domain NS3pro and the cofactor NS2B, as well as the secondary structure prediction of this complex based on the assigned chemical shifts using the TALOS-N software. Our results provide a solid ground for future elucidation of the structure and dynamic of the apo NS3pro/NS2B complex, key for adequate development of inhibitors, and a thorough molecular understanding of their function(s)

Assigned NMR backbone resonances of the ligand-binding region domain of the pneumococcal serine-rich repeat protein (PsrP-BR) reveal a rigid monomer in solution

The pneumococcal serine rich repeat protein (PsrP) is displayed on the surface of Streptococcus pneumoniae with a suggested role in colonization in the human upper respiratory tract. Full-length PsrP is a 4000 residue-long multi-domain protein comprising a positively charged functional binding region (BR) domain for interaction with keratin and extracellular DNA during pneumococcal adhesion and biofilm formation, respectively. The previously determined crystal structure of the BR domain revealed a flat compressed barrel comprising two sides with an extended beta-sheet on one side, and another beta-sheet that is distorted by loops and beta-turns on the other side. Crystallographic B-factors indicated a relatively high mobility of loop regions that were hypothesized to be important for binding. Furthermore, the crystal structure revealed an inter-molecular beta-sheet formed between edge strands of two symmetry-related molecules, which could promote bacterial aggregation during biofilm formation. Here we report the near complete N-15/C-13/H-1 backbone resonance assignment of the BR domain of PsrP, revealing a secondary structure profile that is almost identical to the X-ray structure. Dynamic N-15-T-1, T-2 and NOE data suggest a monomeric and rigid structure of BR with disordered residues only at the N- and C-termini. The presented peak assignment will allow us to identify BR residues that are crucial for ligand binding

Expanding DP4: application to drug compounds and automation.

The DP4 parameter, which provides a confidence level for NMR assignment, has been widely used to help assign the structures of many stereochemically-rich molecules. We present an improved version of the procedure, which can be downloaded as Python script instead of running within a web-browser, and which analyses output from open-source molecular modelling programs (TINKER and NWChem) in addition to being able to use output from commercial packages (Schrodinger's Macromodel and Jaguar; Gaussian). The new open-source workflow incorporates a method for the automatic generation of diastereomers using InChI strings and has been tested on a range of new structures. This improved workflow permits the rapid and convenient computational elucidation of structure and relative stereochemistry.The authors wish to thank Medivir for the generous financial support.This is the final version of the article. It first appeared from the Royal Society of Chemistry via https://doi.org/10.1039/C6OB00015