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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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Comparative analysis of protease-peptide interactions for the P6–P1 residues in MNV and SV NS6pro and CAV 3Cpro.The N-terminal and C-terminal β-barrel domains of each protease are coloured green and orange respectively. (A) Binding of residues P5–P1 (C-terminus of NS6pro), shown as sticks colour-coded by atom type (Carbon – light-blue; Oxygen – red; Nitrogen – blue), within the peptide binding grove of MNV NS6pro. Selected side-chains from the protease are also shown as sticks. Hydrogen bonds and salt-bridges mentioned in the text are indicated by black dashed lines; all such bonds shown are ≤3.1 Å. (B) Same view as in A but showing the surface of MNV NS6pro. (C) Binding of residues P5–P1 from a peptide-like inhibitor to SV (a human norovirus) [23]. Water molecules involved in the protease-peptide interaction are shown as red spheres. (D) Same view as in B but showing the surface of SV NS6pro. (E) The refined σ-weighted 2Fo-Fc map electron density (where Fo and Fc are the observed and calculated structure factors respectively) for an A-chain C-terminal peptide, shown at 1.5 σ. (F) The interaction between residues P6–P1 of a peptide ‘product’ and CAV 3Cpro[30].
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pone-0038723-g003: Comparative analysis of protease-peptide interactions for the P6–P1 residues in MNV and SV NS6pro and CAV 3Cpro.The N-terminal and C-terminal β-barrel domains of each protease are coloured green and orange respectively. (A) Binding of residues P5–P1 (C-terminus of NS6pro), shown as sticks colour-coded by atom type (Carbon – light-blue; Oxygen – red; Nitrogen – blue), within the peptide binding grove of MNV NS6pro. Selected side-chains from the protease are also shown as sticks. Hydrogen bonds and salt-bridges mentioned in the text are indicated by black dashed lines; all such bonds shown are ≤3.1 Å. (B) Same view as in A but showing the surface of MNV NS6pro. (C) Binding of residues P5–P1 from a peptide-like inhibitor to SV (a human norovirus) [23]. Water molecules involved in the protease-peptide interaction are shown as red spheres. (D) Same view as in B but showing the surface of SV NS6pro. (E) The refined σ-weighted 2Fo-Fc map electron density (where Fo and Fc are the observed and calculated structure factors respectively) for an A-chain C-terminal peptide, shown at 1.5 σ. (F) The interaction between residues P6–P1 of a peptide ‘product’ and CAV 3Cpro[30].

Mentions: Superposition of the crystal structure of Southampton Virus NS6pro with a covalently-attached peptide-like inhibitor shows that the positions of the P4-P1 amino acids are very similar to our MNV NS6pro ‘co-crystal’ structure [23] (Fig. 3A, C). The positions of the P4-P1 amino acids observed in the peptide-binding site of MNV NS6pro are also very similar to the equivalent residues in the co-crystal structure of FMDV 3Cpro:peptide complex [29] (not shown). This indicates that, although the formation of an MNV protease-peptide complex is an accident of crystallisation, crystal-packing constraints do not prevent the C-terminal residues from adopting a physiologically-relevant conformation in the binding site of a neighbouring molecule.


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

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

Comparative analysis of protease-peptide interactions for the P6–P1 residues in MNV and SV NS6pro and CAV 3Cpro.The N-terminal and C-terminal β-barrel domains of each protease are coloured green and orange respectively. (A) Binding of residues P5–P1 (C-terminus of NS6pro), shown as sticks colour-coded by atom type (Carbon – light-blue; Oxygen – red; Nitrogen – blue), within the peptide binding grove of MNV NS6pro. Selected side-chains from the protease are also shown as sticks. Hydrogen bonds and salt-bridges mentioned in the text are indicated by black dashed lines; all such bonds shown are ≤3.1 Å. (B) Same view as in A but showing the surface of MNV NS6pro. (C) Binding of residues P5–P1 from a peptide-like inhibitor to SV (a human norovirus) [23]. Water molecules involved in the protease-peptide interaction are shown as red spheres. (D) Same view as in B but showing the surface of SV NS6pro. (E) The refined σ-weighted 2Fo-Fc map electron density (where Fo and Fc are the observed and calculated structure factors respectively) for an A-chain C-terminal peptide, shown at 1.5 σ. (F) The interaction between residues P6–P1 of a peptide ‘product’ and CAV 3Cpro[30].
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038723-g003: Comparative analysis of protease-peptide interactions for the P6–P1 residues in MNV and SV NS6pro and CAV 3Cpro.The N-terminal and C-terminal β-barrel domains of each protease are coloured green and orange respectively. (A) Binding of residues P5–P1 (C-terminus of NS6pro), shown as sticks colour-coded by atom type (Carbon – light-blue; Oxygen – red; Nitrogen – blue), within the peptide binding grove of MNV NS6pro. Selected side-chains from the protease are also shown as sticks. Hydrogen bonds and salt-bridges mentioned in the text are indicated by black dashed lines; all such bonds shown are ≤3.1 Å. (B) Same view as in A but showing the surface of MNV NS6pro. (C) Binding of residues P5–P1 from a peptide-like inhibitor to SV (a human norovirus) [23]. Water molecules involved in the protease-peptide interaction are shown as red spheres. (D) Same view as in B but showing the surface of SV NS6pro. (E) The refined σ-weighted 2Fo-Fc map electron density (where Fo and Fc are the observed and calculated structure factors respectively) for an A-chain C-terminal peptide, shown at 1.5 σ. (F) The interaction between residues P6–P1 of a peptide ‘product’ and CAV 3Cpro[30].
Mentions: Superposition of the crystal structure of Southampton Virus NS6pro with a covalently-attached peptide-like inhibitor shows that the positions of the P4-P1 amino acids are very similar to our MNV NS6pro ‘co-crystal’ structure [23] (Fig. 3A, C). The positions of the P4-P1 amino acids observed in the peptide-binding site of MNV NS6pro are also very similar to the equivalent residues in the co-crystal structure of FMDV 3Cpro:peptide complex [29] (not shown). This indicates that, although the formation of an MNV protease-peptide complex is an accident of crystallisation, crystal-packing constraints do not prevent the C-terminal residues from adopting a physiologically-relevant conformation in the binding site of a neighbouring molecule.

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