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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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Variations in the crystal packing of the MNV NS6pro A and B chains in the asymmetric unit.(A) Crystal packing of A and B chains of MNV NS6pro. The A and B chains of one asymmetric unit are shown along with the neighbouring molecules (labelled A' and B') into which they insert their C-termini. (B) This panel shows the same molecules that are depicted in panel A (with the same colouring) but in this case the A and B chains within the asymmetric unit are superposed; this reveals the very different contacts that they make with their closest neighbour in the crystal. (C) Here the A' and B' chains from panel A are now superposed in order to show the similarity of the conformations of the bound C-termini (shown as sticks) from the A and B chains respectively. Colour-coding is the same as panel A.
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pone-0038723-g002: Variations in the crystal packing of the MNV NS6pro A and B chains in the asymmetric unit.(A) Crystal packing of A and B chains of MNV NS6pro. The A and B chains of one asymmetric unit are shown along with the neighbouring molecules (labelled A' and B') into which they insert their C-termini. (B) This panel shows the same molecules that are depicted in panel A (with the same colouring) but in this case the A and B chains within the asymmetric unit are superposed; this reveals the very different contacts that they make with their closest neighbour in the crystal. (C) Here the A' and B' chains from panel A are now superposed in order to show the similarity of the conformations of the bound C-termini (shown as sticks) from the A and B chains respectively. Colour-coding is the same as panel A.

Mentions: In the norovirus NS6pro crystal structures published to date it has already been observed that, whereas the N-terminal helix found in picornavirus 3C proteases is retained in NS6pro, the C-terminal helix in 3Cpro is not found in the norovirus proteases [19], [22], [23]. Instead the C-terminal polypeptide of NS6pro appears to be largely unstructured; it is either disordered or adopts very different conformations that appear to be determined by crystal contacts (Fig. 1B). Strikingly, in our structure the flexibility of the C-terminal polypeptide has actually allowed it to associate with the peptide-binding sites of a neighbouring molecule in the crystal – a happy accident that has revealed the structure of an MNV NS6pro-peptide complex (Fig. 2A). The interaction occurs for both molecules of the asymmetric unit, although in each case the C-terminus extends from the body of the protease in a different direction towards its neighbour (Fig. 2B). For chain A, the interaction is symmetric: it donates its C-terminus to a neighbouring A-chain that is related by crystallographic two-fold symmetry and binds the C-terminus received from this molecule. This pair of molecules thus forms a closed complex. In contrast, chain B donates its C-terminus to a neighbouring B-chain but binds the C-terminus of a third molecule (also a B-chain). Despite these differences, the interactions made with the neighbouring protease by the residues corresponding to the P4-P1 amino acids of the donated C-terminus are in each case essentially identical (Fig. 2C).


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

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

Variations in the crystal packing of the MNV NS6pro A and B chains in the asymmetric unit.(A) Crystal packing of A and B chains of MNV NS6pro. The A and B chains of one asymmetric unit are shown along with the neighbouring molecules (labelled A' and B') into which they insert their C-termini. (B) This panel shows the same molecules that are depicted in panel A (with the same colouring) but in this case the A and B chains within the asymmetric unit are superposed; this reveals the very different contacts that they make with their closest neighbour in the crystal. (C) Here the A' and B' chains from panel A are now superposed in order to show the similarity of the conformations of the bound C-termini (shown as sticks) from the A and B chains respectively. Colour-coding is the same as panel A.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369882&req=5

pone-0038723-g002: Variations in the crystal packing of the MNV NS6pro A and B chains in the asymmetric unit.(A) Crystal packing of A and B chains of MNV NS6pro. The A and B chains of one asymmetric unit are shown along with the neighbouring molecules (labelled A' and B') into which they insert their C-termini. (B) This panel shows the same molecules that are depicted in panel A (with the same colouring) but in this case the A and B chains within the asymmetric unit are superposed; this reveals the very different contacts that they make with their closest neighbour in the crystal. (C) Here the A' and B' chains from panel A are now superposed in order to show the similarity of the conformations of the bound C-termini (shown as sticks) from the A and B chains respectively. Colour-coding is the same as panel A.
Mentions: In the norovirus NS6pro crystal structures published to date it has already been observed that, whereas the N-terminal helix found in picornavirus 3C proteases is retained in NS6pro, the C-terminal helix in 3Cpro is not found in the norovirus proteases [19], [22], [23]. Instead the C-terminal polypeptide of NS6pro appears to be largely unstructured; it is either disordered or adopts very different conformations that appear to be determined by crystal contacts (Fig. 1B). Strikingly, in our structure the flexibility of the C-terminal polypeptide has actually allowed it to associate with the peptide-binding sites of a neighbouring molecule in the crystal – a happy accident that has revealed the structure of an MNV NS6pro-peptide complex (Fig. 2A). The interaction occurs for both molecules of the asymmetric unit, although in each case the C-terminus extends from the body of the protease in a different direction towards its neighbour (Fig. 2B). For chain A, the interaction is symmetric: it donates its C-terminus to a neighbouring A-chain that is related by crystallographic two-fold symmetry and binds the C-terminus received from this molecule. This pair of molecules thus forms a closed complex. In contrast, chain B donates its C-terminus to a neighbouring B-chain but binds the C-terminus of a third molecule (also a B-chain). Despite these differences, the interactions made with the neighbouring protease by the residues corresponding to the P4-P1 amino acids of the donated C-terminus are in each case essentially identical (Fig. 2C).

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