Insights into cleavage specificity from the crystal structure of foot-and-mouth disease virus 3C protease complexed with a peptide substrate

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Standard

Insights into cleavage specificity from the crystal structure of foot-and-mouth disease virus 3C protease complexed with a peptide substrate. / Zunszain, Patricia A; Knox, Stephen R; Sweeney, Trevor R; Yang, Jingjie; Roqué-Rosell, Núria; Belsham, Graham J; Leatherbarrow, Robin J; Curry, Stephen.

In: Journal of Molecular Biology, Vol. 395, No. 2, 15.01.2010, p. 375-89.

Research output: Contribution to journal › Journal article › Research › peer-review

Harvard

Zunszain, PA, Knox, SR, Sweeney, TR, Yang, J, Roqué-Rosell, N, Belsham, GJ, Leatherbarrow, RJ & Curry, S 2010, 'Insights into cleavage specificity from the crystal structure of foot-and-mouth disease virus 3C protease complexed with a peptide substrate', Journal of Molecular Biology, vol. 395, no. 2, pp. 375-89. https://doi.org/10.1016/j.jmb.2009.10.048

APA

Zunszain, P. A., Knox, S. R., Sweeney, T. R., Yang, J., Roqué-Rosell, N., Belsham, G. J., Leatherbarrow, R. J., & Curry, S. (2010). Insights into cleavage specificity from the crystal structure of foot-and-mouth disease virus 3C protease complexed with a peptide substrate. Journal of Molecular Biology, 395(2), 375-89. https://doi.org/10.1016/j.jmb.2009.10.048

Vancouver

Zunszain PA, Knox SR, Sweeney TR, Yang J, Roqué-Rosell N, Belsham GJ et al. Insights into cleavage specificity from the crystal structure of foot-and-mouth disease virus 3C protease complexed with a peptide substrate. Journal of Molecular Biology. 2010 Jan 15;395(2):375-89. https://doi.org/10.1016/j.jmb.2009.10.048

Author

Zunszain, Patricia A ; Knox, Stephen R ; Sweeney, Trevor R ; Yang, Jingjie ; Roqué-Rosell, Núria ; Belsham, Graham J ; Leatherbarrow, Robin J ; Curry, Stephen. / Insights into cleavage specificity from the crystal structure of foot-and-mouth disease virus 3C protease complexed with a peptide substrate. In: Journal of Molecular Biology. 2010 ; Vol. 395, No. 2. pp. 375-89.

Bibtex

@article{6138b82d2f2445979e1e59be504bd428,

title = "Insights into cleavage specificity from the crystal structure of foot-and-mouth disease virus 3C protease complexed with a peptide substrate",

abstract = "Picornavirus replication is critically dependent on the correct processing of a polyprotein precursor by 3C protease(s) (3C(pro)) at multiple specific sites with related but non-identical sequences. To investigate the structural basis of its cleavage specificity, we performed the first crystallographic structural analysis of non-covalent complexes of a picornavirus 3C(pro) with peptide substrates. The X-ray crystal structure of the foot-and-mouth disease virus 3C(pro), mutated to replace the catalytic Cys by Ala and bound to a peptide (APAKQ|LLNFD) corresponding to the P5-P5' region of the VP1-2A cleavage junction in the viral polyprotein, was determined up to 2.5 A resolution. Comparison with free enzyme reveals significant conformational changes in 3C(pro) on substrate binding that lead to the formation of an extended interface of contact primarily involving the P4-P2' positions of the peptide. Strikingly, the deep S1' specificity pocket needed to accommodate P1'-Leu only forms when the peptide binds. Substrate specificity was investigated using peptide cleavage assays to show the impact of amino acid substitutions within the P5-P4' region of synthetic substrates. The structure of the enzyme-peptide complex explains the marked substrate preferences for particular P4, P2 and P1 residue types, as well as the relative promiscuity at P3 and on the P' side of the scissile bond. Furthermore, crystallographic analysis of the complex with a modified VP1-2A peptide (APAKE|LLNFD) containing a Gln-to-Glu substitution reveals an identical mode of peptide binding and explains the ability of foot-and-mouth disease virus 3C(pro) to cleave sequences containing either P1-Gln or P1-Glu. Structure-based mutagenesis was used to probe interactions within the S1' specificity pocket and to provide direct evidence of the important contribution made by Asp84 of the Cys-His-Asp catalytic triad to proteolytic activity. Our results provide a new level of detail in our understanding of the structural basis of polyprotein cleavage by 3C(pro).",

keywords = "Amino Acid Sequence, Amino Acid Substitution, Base Sequence, Catalytic Domain/genetics, Crystallography, X-Ray, Cysteine Endopeptidases/chemistry, DNA, Viral/genetics, Foot-and-Mouth Disease Virus/enzymology, Models, Molecular, Mutagenesis, Site-Directed, Peptides/chemistry, Protein Binding, Protein Conformation, Protein Structure, Secondary, Recombinant Proteins/chemistry, Substrate Specificity, Viral Proteins/chemistry, Viral Structural Proteins/chemistry",

author = "Zunszain, {Patricia A} and Knox, {Stephen R} and Sweeney, {Trevor R} and Jingjie Yang and N{\'u}ria Roqu{\'e}-Rosell and Belsham, {Graham J} and Leatherbarrow, {Robin J} and Stephen Curry",

year = "2010",

month = jan,

day = "15",

doi = "10.1016/j.jmb.2009.10.048",

language = "English",

volume = "395",

pages = "375--89",

journal = "Journal of Molecular Biology",

issn = "0022-2836",

publisher = "Academic Press",

number = "2",

}

RIS

TY - JOUR

T1 - Insights into cleavage specificity from the crystal structure of foot-and-mouth disease virus 3C protease complexed with a peptide substrate

AU - Zunszain, Patricia A

AU - Knox, Stephen R

AU - Sweeney, Trevor R

AU - Yang, Jingjie

AU - Roqué-Rosell, Núria

AU - Belsham, Graham J

AU - Leatherbarrow, Robin J

AU - Curry, Stephen

PY - 2010/1/15

Y1 - 2010/1/15

N2 - Picornavirus replication is critically dependent on the correct processing of a polyprotein precursor by 3C protease(s) (3C(pro)) at multiple specific sites with related but non-identical sequences. To investigate the structural basis of its cleavage specificity, we performed the first crystallographic structural analysis of non-covalent complexes of a picornavirus 3C(pro) with peptide substrates. The X-ray crystal structure of the foot-and-mouth disease virus 3C(pro), mutated to replace the catalytic Cys by Ala and bound to a peptide (APAKQ|LLNFD) corresponding to the P5-P5' region of the VP1-2A cleavage junction in the viral polyprotein, was determined up to 2.5 A resolution. Comparison with free enzyme reveals significant conformational changes in 3C(pro) on substrate binding that lead to the formation of an extended interface of contact primarily involving the P4-P2' positions of the peptide. Strikingly, the deep S1' specificity pocket needed to accommodate P1'-Leu only forms when the peptide binds. Substrate specificity was investigated using peptide cleavage assays to show the impact of amino acid substitutions within the P5-P4' region of synthetic substrates. The structure of the enzyme-peptide complex explains the marked substrate preferences for particular P4, P2 and P1 residue types, as well as the relative promiscuity at P3 and on the P' side of the scissile bond. Furthermore, crystallographic analysis of the complex with a modified VP1-2A peptide (APAKE|LLNFD) containing a Gln-to-Glu substitution reveals an identical mode of peptide binding and explains the ability of foot-and-mouth disease virus 3C(pro) to cleave sequences containing either P1-Gln or P1-Glu. Structure-based mutagenesis was used to probe interactions within the S1' specificity pocket and to provide direct evidence of the important contribution made by Asp84 of the Cys-His-Asp catalytic triad to proteolytic activity. Our results provide a new level of detail in our understanding of the structural basis of polyprotein cleavage by 3C(pro).

AB - Picornavirus replication is critically dependent on the correct processing of a polyprotein precursor by 3C protease(s) (3C(pro)) at multiple specific sites with related but non-identical sequences. To investigate the structural basis of its cleavage specificity, we performed the first crystallographic structural analysis of non-covalent complexes of a picornavirus 3C(pro) with peptide substrates. The X-ray crystal structure of the foot-and-mouth disease virus 3C(pro), mutated to replace the catalytic Cys by Ala and bound to a peptide (APAKQ|LLNFD) corresponding to the P5-P5' region of the VP1-2A cleavage junction in the viral polyprotein, was determined up to 2.5 A resolution. Comparison with free enzyme reveals significant conformational changes in 3C(pro) on substrate binding that lead to the formation of an extended interface of contact primarily involving the P4-P2' positions of the peptide. Strikingly, the deep S1' specificity pocket needed to accommodate P1'-Leu only forms when the peptide binds. Substrate specificity was investigated using peptide cleavage assays to show the impact of amino acid substitutions within the P5-P4' region of synthetic substrates. The structure of the enzyme-peptide complex explains the marked substrate preferences for particular P4, P2 and P1 residue types, as well as the relative promiscuity at P3 and on the P' side of the scissile bond. Furthermore, crystallographic analysis of the complex with a modified VP1-2A peptide (APAKE|LLNFD) containing a Gln-to-Glu substitution reveals an identical mode of peptide binding and explains the ability of foot-and-mouth disease virus 3C(pro) to cleave sequences containing either P1-Gln or P1-Glu. Structure-based mutagenesis was used to probe interactions within the S1' specificity pocket and to provide direct evidence of the important contribution made by Asp84 of the Cys-His-Asp catalytic triad to proteolytic activity. Our results provide a new level of detail in our understanding of the structural basis of polyprotein cleavage by 3C(pro).

KW - Amino Acid Sequence

KW - Amino Acid Substitution

KW - Base Sequence

KW - Catalytic Domain/genetics

KW - Crystallography, X-Ray

KW - Cysteine Endopeptidases/chemistry

KW - DNA, Viral/genetics

KW - Foot-and-Mouth Disease Virus/enzymology

KW - Models, Molecular

KW - Mutagenesis, Site-Directed

KW - Peptides/chemistry

KW - Protein Binding

KW - Protein Conformation

KW - Protein Structure, Secondary

KW - Recombinant Proteins/chemistry

KW - Substrate Specificity

KW - Viral Proteins/chemistry

KW - Viral Structural Proteins/chemistry

U2 - 10.1016/j.jmb.2009.10.048

DO - 10.1016/j.jmb.2009.10.048

M3 - Journal article

C2 - 19883658

VL - 395

SP - 375

EP - 389

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 2

ER -

ID: 257918152