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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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Sequence conservation of polyprotein junction in MNV that are cleaved by NS6pro.(A) The five cleavage junctions of MNV CW1 polyprotein (NCBI accession number YP_720001) [14]. (B) Weblogo of polyprotein cleavage junctions cleaved by MNV NS6pro. This Weblogo was generated using 39 MNV polyprotein sequences and the Weblogo sequence logo generator [44]. The height of the letter in each case is indicative of the degree of conservation. The Genbank accession numbers of the other sequences used to prepare the alignment are ABU55618, ABU55627, ABU55615, ABU55621, ABU55612, ABU55624, AEE10026, ABU55600, AEY83582, AEE10023, ABU55606, AEE10020, ABU55609, AEE10017, ABB02416, AEE10002, ACJ72215, AEE09999, ABU55591, AEE10005, ABU55570, AEE10008, ABU55585, AEE10014, ABU55579, AEE10011, ABU55576, ABU55597, ABU55573, ABU55603, ABU55582, ABU55594, ABU55588, ABU55567, ACS70958, ACJ72218, ABS29272, ABS29274.
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pone-0038723-g004: Sequence conservation of polyprotein junction in MNV that are cleaved by NS6pro.(A) The five cleavage junctions of MNV CW1 polyprotein (NCBI accession number YP_720001) [14]. (B) Weblogo of polyprotein cleavage junctions cleaved by MNV NS6pro. This Weblogo was generated using 39 MNV polyprotein sequences and the Weblogo sequence logo generator [44]. The height of the letter in each case is indicative of the degree of conservation. The Genbank accession numbers of the other sequences used to prepare the alignment are ABU55618, ABU55627, ABU55615, ABU55621, ABU55612, ABU55624, AEE10026, ABU55600, AEY83582, AEE10023, ABU55606, AEE10020, ABU55609, AEE10017, ABB02416, AEE10002, ACJ72215, AEE09999, ABU55591, AEE10005, ABU55570, AEE10008, ABU55585, AEE10014, ABU55579, AEE10011, ABU55576, ABU55597, ABU55573, ABU55603, ABU55582, ABU55594, ABU55588, ABU55567, ACS70958, ACJ72218, ABS29272, ABS29274.

Mentions: The side-chain of F182 at the P2 in the MNV NS6pro structure is sandwiched between H30 and I109 at the entrance to the hydrophobic S2 pocket (Fig 3A, B) but also contacts V114, V158, A159 and D54. The largely apolar nature of the S2 pocket accounts for the general selectivity for hydrophobic amino acids at the P2 amino acid in norovirus cleavage junctions [14] (Fig. 4). An exception to this is the NS4/NS5 junction of the MNV polyprotein, which has a P2-Ser. It seems unlikely that at the side-chain hydroxyl of this amino acid could interact specifically with the S2 pocket, suggesting that the NS4/NS5 junction may be a sub-optimal substrate for the protease. Intriguingly, the S2 pocket is more enclosed in human norovirus NS6pro structures because the Q110-G111-R112 sequence at the top of the loop that forms one flank of the pocket is replaced in MNV NS6pro by the G110-S111-A112 sequence, which has much smaller amino acids (Fig. 3A–D).


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

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

Sequence conservation of polyprotein junction in MNV that are cleaved by NS6pro.(A) The five cleavage junctions of MNV CW1 polyprotein (NCBI accession number YP_720001) [14]. (B) Weblogo of polyprotein cleavage junctions cleaved by MNV NS6pro. This Weblogo was generated using 39 MNV polyprotein sequences and the Weblogo sequence logo generator [44]. The height of the letter in each case is indicative of the degree of conservation. The Genbank accession numbers of the other sequences used to prepare the alignment are ABU55618, ABU55627, ABU55615, ABU55621, ABU55612, ABU55624, AEE10026, ABU55600, AEY83582, AEE10023, ABU55606, AEE10020, ABU55609, AEE10017, ABB02416, AEE10002, ACJ72215, AEE09999, ABU55591, AEE10005, ABU55570, AEE10008, ABU55585, AEE10014, ABU55579, AEE10011, ABU55576, ABU55597, ABU55573, ABU55603, ABU55582, ABU55594, ABU55588, ABU55567, ACS70958, ACJ72218, ABS29272, ABS29274.
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Related In: Results  -  Collection

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pone-0038723-g004: Sequence conservation of polyprotein junction in MNV that are cleaved by NS6pro.(A) The five cleavage junctions of MNV CW1 polyprotein (NCBI accession number YP_720001) [14]. (B) Weblogo of polyprotein cleavage junctions cleaved by MNV NS6pro. This Weblogo was generated using 39 MNV polyprotein sequences and the Weblogo sequence logo generator [44]. The height of the letter in each case is indicative of the degree of conservation. The Genbank accession numbers of the other sequences used to prepare the alignment are ABU55618, ABU55627, ABU55615, ABU55621, ABU55612, ABU55624, AEE10026, ABU55600, AEY83582, AEE10023, ABU55606, AEE10020, ABU55609, AEE10017, ABB02416, AEE10002, ACJ72215, AEE09999, ABU55591, AEE10005, ABU55570, AEE10008, ABU55585, AEE10014, ABU55579, AEE10011, ABU55576, ABU55597, ABU55573, ABU55603, ABU55582, ABU55594, ABU55588, ABU55567, ACS70958, ACJ72218, ABS29272, ABS29274.
Mentions: The side-chain of F182 at the P2 in the MNV NS6pro structure is sandwiched between H30 and I109 at the entrance to the hydrophobic S2 pocket (Fig 3A, B) but also contacts V114, V158, A159 and D54. The largely apolar nature of the S2 pocket accounts for the general selectivity for hydrophobic amino acids at the P2 amino acid in norovirus cleavage junctions [14] (Fig. 4). An exception to this is the NS4/NS5 junction of the MNV polyprotein, which has a P2-Ser. It seems unlikely that at the side-chain hydroxyl of this amino acid could interact specifically with the S2 pocket, suggesting that the NS4/NS5 junction may be a sub-optimal substrate for the protease. Intriguingly, the S2 pocket is more enclosed in human norovirus NS6pro structures because the Q110-G111-R112 sequence at the top of the loop that forms one flank of the pocket is replaced in MNV NS6pro by the G110-S111-A112 sequence, which has much smaller amino acids (Fig. 3A–D).

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