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Structure of a murine norovirus NS6 protease-product complex revealed by adventitious crystallisation.

Leen EN, Baeza G, Curry S - PLoS ONE (2012)

Bottom Line: Murine noroviruses have emerged as a valuable tool for investigating the molecular basis of infection and pathogenesis of the closely related human noroviruses, which are the major cause of non-bacterial gastroenteritis.The replication of noroviruses relies on the proteolytic processing of a large polyprotein precursor into six non-structural proteins (NS1-2, NS3, NS4, NS5, NS6(pro), NS7(pol)) by the virally-encoded NS6 protease.The observed mode of binding of the C-terminal product peptide yields new insights into the structural basis of NS6(pro) specificity.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Imperial College, London, United Kingdom.

ABSTRACT
Murine noroviruses have emerged as a valuable tool for investigating the molecular basis of infection and pathogenesis of the closely related human noroviruses, which are the major cause of non-bacterial gastroenteritis. The replication of noroviruses relies on the proteolytic processing of a large polyprotein precursor into six non-structural proteins (NS1-2, NS3, NS4, NS5, NS6(pro), NS7(pol)) by the virally-encoded NS6 protease. We report here the crystal structure of MNV NS6(pro), which has been determined to a resolution of 1.6 Å. Adventitiously, the crystal contacts are mediated in part by the binding of the C-terminus of NS6(pro) within the peptide-binding cleft of a neighbouring molecule. This insertion occurs for both molecules in the asymmetric unit of the crystal in a manner that is consistent with physiologically-relevant binding, thereby providing two independent views of a protease-peptide complex. Since the NS6(pro) C-terminus is formed in vivo by NS6(pro) processing, these crystal contacts replicate the protease-product complex that is formed immediately following cleavage of the peptide bond at the NS6-NS7 junction. The observed mode of binding of the C-terminal product peptide yields new insights into the structural basis of NS6(pro) specificity.

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Structural comparison of the MNV NS6 protease with human norovirus NS6pro and foot-and-mouth disease virus 3Cpro.(A) Cartoon representation of the MNV NS6pro structure. The N and C-terminal domains are coloured green and orange respectively. The side-chains of the amino acids that make up the catalytic triad, A139 (mutated from Cys), H30 and D54, are shown as sticks. A disordered loop formed by residues 124–130 (residues 124–131 in chain B) is indicated as a dashed line. The peptide bound to NS6pro is not shown in this representation. (B) Overlay of HuNV NS6 protease structures from Chiba (PDB-ID: 1WQS), Norwalk (PDB-ID: 2FYQ) and Southampton (PDB-ID: 2IPH) viruses [19], [22], [23]. Excluding the variable C-termini, the root mean square deviations of the backbone atoms of Chiba, Norwalk and Southampton virus NS6pro from MNV NS6pro are 0.62, 0.43 and 0.41 respectively. The disordered C-terminus of the Chibavirus protease is shown as a dashed line. (C) Structure of FMDV 3Cpro (PDB-ID: 2J92) [26], coloured as in panel (A).
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pone-0038723-g001: Structural comparison of the MNV NS6 protease with human norovirus NS6pro and foot-and-mouth disease virus 3Cpro.(A) Cartoon representation of the MNV NS6pro structure. The N and C-terminal domains are coloured green and orange respectively. The side-chains of the amino acids that make up the catalytic triad, A139 (mutated from Cys), H30 and D54, are shown as sticks. A disordered loop formed by residues 124–130 (residues 124–131 in chain B) is indicated as a dashed line. The peptide bound to NS6pro is not shown in this representation. (B) Overlay of HuNV NS6 protease structures from Chiba (PDB-ID: 1WQS), Norwalk (PDB-ID: 2FYQ) and Southampton (PDB-ID: 2IPH) viruses [19], [22], [23]. Excluding the variable C-termini, the root mean square deviations of the backbone atoms of Chiba, Norwalk and Southampton virus NS6pro from MNV NS6pro are 0.62, 0.43 and 0.41 respectively. The disordered C-terminus of the Chibavirus protease is shown as a dashed line. (C) Structure of FMDV 3Cpro (PDB-ID: 2J92) [26], coloured as in panel (A).

Mentions: The overall structure of the MNV NS6 protease is nearly identical to Norwalk virus, SV and CV NS6pro (Fig. 1), which comes as no surprise given the high degree of amino acid identity (∼60%) between these proteins. As described previously, the structure is an abbreviated form of the chymotrypsin-like 3C proteases from picornaviruses [19], [22], which comprise two β-barrel domains. Specifically, in norovirus NS6 proteases, the β-strands on one side of the N-terminal β-barrel are so much shorter that the domain is better considered as a single β-sheet decorated by loops (Fig. 1A). The active site triad of H30, D54, and C139 (A139 in our mutated protein) is located in the centre of the peptide-binding cleft formed at the interface of these two domains.


Structure of a murine norovirus NS6 protease-product complex revealed by adventitious crystallisation.

Leen EN, Baeza G, Curry S - PLoS ONE (2012)

Structural comparison of the MNV NS6 protease with human norovirus NS6pro and foot-and-mouth disease virus 3Cpro.(A) Cartoon representation of the MNV NS6pro structure. The N and C-terminal domains are coloured green and orange respectively. The side-chains of the amino acids that make up the catalytic triad, A139 (mutated from Cys), H30 and D54, are shown as sticks. A disordered loop formed by residues 124–130 (residues 124–131 in chain B) is indicated as a dashed line. The peptide bound to NS6pro is not shown in this representation. (B) Overlay of HuNV NS6 protease structures from Chiba (PDB-ID: 1WQS), Norwalk (PDB-ID: 2FYQ) and Southampton (PDB-ID: 2IPH) viruses [19], [22], [23]. Excluding the variable C-termini, the root mean square deviations of the backbone atoms of Chiba, Norwalk and Southampton virus NS6pro from MNV NS6pro are 0.62, 0.43 and 0.41 respectively. The disordered C-terminus of the Chibavirus protease is shown as a dashed line. (C) Structure of FMDV 3Cpro (PDB-ID: 2J92) [26], coloured as in panel (A).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369882&req=5

pone-0038723-g001: Structural comparison of the MNV NS6 protease with human norovirus NS6pro and foot-and-mouth disease virus 3Cpro.(A) Cartoon representation of the MNV NS6pro structure. The N and C-terminal domains are coloured green and orange respectively. The side-chains of the amino acids that make up the catalytic triad, A139 (mutated from Cys), H30 and D54, are shown as sticks. A disordered loop formed by residues 124–130 (residues 124–131 in chain B) is indicated as a dashed line. The peptide bound to NS6pro is not shown in this representation. (B) Overlay of HuNV NS6 protease structures from Chiba (PDB-ID: 1WQS), Norwalk (PDB-ID: 2FYQ) and Southampton (PDB-ID: 2IPH) viruses [19], [22], [23]. Excluding the variable C-termini, the root mean square deviations of the backbone atoms of Chiba, Norwalk and Southampton virus NS6pro from MNV NS6pro are 0.62, 0.43 and 0.41 respectively. The disordered C-terminus of the Chibavirus protease is shown as a dashed line. (C) Structure of FMDV 3Cpro (PDB-ID: 2J92) [26], coloured as in panel (A).
Mentions: The overall structure of the MNV NS6 protease is nearly identical to Norwalk virus, SV and CV NS6pro (Fig. 1), which comes as no surprise given the high degree of amino acid identity (∼60%) between these proteins. As described previously, the structure is an abbreviated form of the chymotrypsin-like 3C proteases from picornaviruses [19], [22], which comprise two β-barrel domains. Specifically, in norovirus NS6 proteases, the β-strands on one side of the N-terminal β-barrel are so much shorter that the domain is better considered as a single β-sheet decorated by loops (Fig. 1A). The active site triad of H30, D54, and C139 (A139 in our mutated protein) is located in the centre of the peptide-binding cleft formed at the interface of these two domains.

Bottom Line: Murine noroviruses have emerged as a valuable tool for investigating the molecular basis of infection and pathogenesis of the closely related human noroviruses, which are the major cause of non-bacterial gastroenteritis.The replication of noroviruses relies on the proteolytic processing of a large polyprotein precursor into six non-structural proteins (NS1-2, NS3, NS4, NS5, NS6(pro), NS7(pol)) by the virally-encoded NS6 protease.The observed mode of binding of the C-terminal product peptide yields new insights into the structural basis of NS6(pro) specificity.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Imperial College, London, United Kingdom.

ABSTRACT
Murine noroviruses have emerged as a valuable tool for investigating the molecular basis of infection and pathogenesis of the closely related human noroviruses, which are the major cause of non-bacterial gastroenteritis. The replication of noroviruses relies on the proteolytic processing of a large polyprotein precursor into six non-structural proteins (NS1-2, NS3, NS4, NS5, NS6(pro), NS7(pol)) by the virally-encoded NS6 protease. We report here the crystal structure of MNV NS6(pro), which has been determined to a resolution of 1.6 Å. Adventitiously, the crystal contacts are mediated in part by the binding of the C-terminus of NS6(pro) within the peptide-binding cleft of a neighbouring molecule. This insertion occurs for both molecules in the asymmetric unit of the crystal in a manner that is consistent with physiologically-relevant binding, thereby providing two independent views of a protease-peptide complex. Since the NS6(pro) C-terminus is formed in vivo by NS6(pro) processing, these crystal contacts replicate the protease-product complex that is formed immediately following cleavage of the peptide bond at the NS6-NS7 junction. The observed mode of binding of the C-terminal product peptide yields new insights into the structural basis of NS6(pro) specificity.

Show MeSH
Related in: MedlinePlus