X-Ray Crystallographic Structure of the Norwalk Virus Protease at 1.5-Å Resolution (original) (raw)
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Structural and antiviral studies of the human norovirus GII.4 protease
Biochemistry, 2019
Noroviruses are single-stranded RNA viruses. They encode a protease that cleaves a viral polyprotein at specific sites to produce mature viral proteins. In addition, the protease also binds to viral RNA, and thus is thought to regulate viral replication. However, to date no structural information is available for protease-substrate complexes that might explain the interactions made by peptide residues P'-side of cleavage junctions or RNA. Here I report the work carried out to characterize these interactions in human norovirus protease using X-ray crystallography. The protease was successfully expressed, purified and the crystallization conditions were optimized to grow crystals for structure determination. Unfortunately, RNA and peptide electron density were not observed in co-crystal structures. The packing of protease molecules in one of the crystal forms shows the interaction of protease C-terminal residues with the peptide-binding groove of a neighboring molecule in the crystal, thereby providing the view of a protease-product complex. iii Acknowledgments It would not have been possible to work on this project without the support and help of people around me. First, I would like to thank my family for their unconditional support. Then there are my friends Saad Rajput, Saad Amjad, Mujeeb Mufti and Alexander Carleton, who made graduate life fun and were there for me whenever I needed them.
Crystal Structure of Inhibitor-Bound GII.4 Sydney 2012 Norovirus 3C-Like Protease
Viruses
Norovirus is the leading cause of viral gastroenteritis worldwide, and there are no approved vaccines or therapeutic treatments for chronic or severe norovirus infections. The structural characterisation of the norovirus protease and drug development has predominantly focused upon GI.1 noroviruses, despite most global outbreaks being caused by GII.4 noroviruses. Here, we determined the crystal structures of the GII.4 Sydney 2012 ligand-free norovirus protease at 2.79 Å and at 1.83 Å with a covalently bound high-affinity (IC50 = 0.37 µM) protease inhibitor (NV-004). We show that the active sites of the ligand-free protease structure are present in both open and closed conformations, as determined by their Arg112 side chain orientation. A comparative analysis of the ligand-free and ligand-bound protease structures reveals significant structural differences in the active site cleft and substrate-binding pockets when an inhibitor is covalently bound. We also report a second molecule of ...
Structural and dynamics characterization of norovirus protease
Protein Science, 2013
Norovirus protease is an essential enzyme for proteolytic maturation of norovirus nonstructural proteins and has been implicated as a potential target for antiviral drug development. Although X-ray structural studies of the protease give us wealth of structural information including interactions of the protease with its substrate and dimeric overall structure, the role of protein dynamics in the substrate recognition and the biological relevance of the protease dimer remain unclear. Here we determined the solution NMR structure of the 3C-like protease from Norwalk virus (NV 3CLpro), a prototype strain of norovirus, and analyzed its backbone dynamics and hydrodynamic behavior in solution. 15 N spin relaxation and analytical ultracentrifugation analyses demonstrate that NV 3CLpro is predominantly a monomer in solution. Solution structure of NV 3CLpro shows significant structural variation in C-terminal domain compared with crystal structures and among lower energy structure ensembles. Also, 15 N spin relaxation and Carr-Purcell-Meiboom-Gill (CPMG)-based relaxation dispersion analyses reveal the dynamic properties of residues in the C-terminal domain over a wide range of timescales. In particular, the long loop spanning residues T123-G133 show fast motion (ps-ns), and the residues in the bII-cII region forming the large hydrophobic pocket (S2 site) undergo conformational exchanges on slower timescales (ls-ms), suggesting their important role in substrate recognition.
In crystallo-screening for discovery of human norovirus 3C-like protease inhibitors
Journal of Structural Biology: X, 2020
Outbreaks of human epidemic nonbacterial gastroenteritis are mainly caused by noroviruses. Viral replication requires a 3C-like cysteine protease (3CL pro) which processes the 200 kDa viral polyprotein into six functional proteins. The 3CL pro has attracted much interest due to its potential as a target for antiviral drugs. A system for growing high-quality crystals of native Southampton norovirus 3CL pro (SV3CP) has been established, allowing the ligand-free crystal structure to be determined to 1.3 Å in a tetrameric state. This also allowed crystal-based fragment screening to be performed with various compound libraries, ultimately to guide drug discovery for SV3CP. A total of 19 fragments were found to bind to the protease out of the 844 which were screened. Two of the hits were located at the active site of SV3CP and showed good inhibitory activity in kinetic assays. Another 5 were found at the enzyme's putative RNA-binding site and a further 11 were located in the symmetric central cavity of the tetramer.
Crystal structure of human enterovirus 71 3C protease
2011
EV71 is the primary pathogenic cause of hand-foot-mouth disease (HFMD), but an effective antiviral drug currently is unavailable. Rupintrivir, an inhibitor against human rhinovirus (HRV), has potent antiviral activities against EV71. We determined the high-resolution crystal structures of the EV71 3C pro /rupintrivir complex, showing that although rupintrivir interacts with EV71 3C pro similarly to HRV 3C pro , the C terminus of the inhibitor cannot accommodate the leaving-group pockets of EV71 3C pro . Our structures reveal that EV71 3C pro possesses a surface-recessive S2 pocket that is not present in HRV 3C pro that contributes to the additional substrate binding affinity. Combined with mutagenic studies, we demonstrated that catalytic Glu71 is irreplaceable for maintaining the overall architecture of the active site and, most importantly, the productive conformation of catalytic His40. We discovered the role of a previously uncharacterized residue, Arg39 of EV71 3C pro , that can neutralize the negative charge of Glu71, which may subsequently assist deprotonation of His40 during proteolysis.
Journal of Virology, 2008
Coronaviruses (CoVs) can infect humans and multiple species of animals, causing a wide spectrum of diseases. The coronavirus main protease (M pro ), which plays a pivotal role in viral gene expression and replication through the proteolytic processing of replicase polyproteins, is an attractive target for anti-CoV drug design. In this study, the crystal structures of infectious bronchitis virus (IBV) M pro and a severe acute respiratory syndrome CoV (SARS-CoV) M pro mutant (H41A), in complex with an N-terminal autocleavage substrate, were individually determined to elucidate the structural flexibility and substrate binding of M pro . A monomeric form of IBV M pro was identified for the first time in CoV M pro structures. A comparison of these two structures to other available M pro structures provides new insights for the design of substrate-based inhibitors targeting CoV M pro s. Furthermore, a Michael acceptor inhibitor (named N3) was cocrystallized with IBV M pro and was found to demonstrate in vitro inactivation of IBV M pro and potent antiviral activity against IBV in chicken embryos. This provides a feasible animal model for designing wide-spectrum inhibitors against CoV-associated diseases. The structure-based optimization of N3 has yielded two more efficacious lead compounds, N27 and H16, with potent inhibition against SARS-CoV M pro .
Structural and functional parameters of the flaviviral protease: a promising antiviral drug target
Future Virology, 2010
Flaviviruses have a single-strand, positive-polarity RNA genome that encodes a single polyprotein. The polyprotein is comprised of seven nonstructural (NS) and three structural proteins. The N-and C-terminal parts of NS3 represent the serine protease and the RNA helicase, respectively. The cleavage of the polyprotein by the protease is required to produce the individual viral proteins, which assemble a new viral progeny. Conversely, inactivation of the protease blocks viral infection. Both the protease and the helicase are conserved among flaviviruses. As a result, NS3 is a promising drug target in flaviviral infections. This article examines the West Nile virus NS3 with an emphasis on the structural and functional parameters of the protease, the helicase and their cofactors.
European Journal of Medicinal Chemistry, 2016
Human noroviruses are the primary cause of epidemic and sporadic acute gastroenteritis. The worldwide high morbidity and mortality associated with norovirus infections, particularly among the elderly, immunocompromised patients and children, constitute a serious public health concern. There are currently no approved human vaccines or norovirus-specific small-molecule therapeutics or prophylactics. Norovirus 3CL protease has recently emerged as a potential therapeutic target for the development of anti-norovirus agents. We hypothesized that the S 4 subsite of the enzyme may provide an effective means of designing potent and cell permeable inhibitors of the enzyme. We report herein the structure-guided exploration and exploitation of the S 4 subsite of norovirus 3CL protease in the design and synthesis of effective inhibitors of the protease.
Structural Basis for Antiviral Inhibition of the Main Protease, 3C, from Human Enterovirus 93
Journal of Virology, 2011
Members of the Enterovirus genus of the Picornaviridae family are abundant, with common human pathogens that belong to the rhinovirus (HRV) and enterovirus (EV) species, including diverse echo-, coxsackie- and polioviruses. They cause a wide spectrum of clinical manifestations ranging from asymptomatic to severe diseases with neurological and/or cardiac manifestations. Pandemic outbreaks of EVs may be accompanied by meningitis and/or paralysis and can be fatal. However, no effective prophylaxis or antiviral treatment against most EVs is available. The EV RNA genome directs the synthesis of a single polyprotein that is autocatalytically processed into mature proteins at Gln↓Gly cleavage sites by the 3C protease (3C pro ), which has narrow, conserved substrate specificity. These cleavages are essential for virus replication, making 3C pro an excellent target for antivirus drug development. In this study, we report the first determination of the crystal structure of 3C pro from an ente...