Allaire

Belsham

Bergmann

Birtley

Bjorndahl

Blom

Brunger

Brünger

Carrillo

Cordingley

Curry

Delano

Ellard

Frigerio

Fujinaga

Graham J. Belsham

Grubman

Hedstrom

Hodis

Huber

Jaulent

Jewell

Jingjie Yang

Jones

Laskowski

Long

Matthews

McCoy

Mosimann

Nayak

Núria Roqué-Rosell

Pallai

Patricia A. Zunszain

Perona

Phan

Prasad

Reid

Robin J. Leatherbarrow

Schechter

Seipelt

Skern

Stephen Curry

Stephen R. Knox

Sutmoller

Sweeney

Trevor R. Sweeney

Tsunogae

Journal of Molecular Biology

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).</p

Zunszain, Patricia A

Knox, Stephen R

Sweeney, Trevor R

Yang, Jingjie

Roqué-Rosell, Núria

Belsham, Graham J

Leatherbarrow, Robin J

Curry, Stephen

Copenhagen University Research Information System

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

Crossref

Zunszain, PA

Knox, SR

Sweeney, TR

Yang, J

Roqué-Rosell, N

Belsham, GJ

Leatherbarrow, RJ

Curry, S

Spiral - Imperial College Digital Repository

Insights into Cleavage Specificity from the Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease Complexed with a Peptide Substrate.

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 vertical bar LLNFD) corresponding to the P5-P5' region of the VP1-2A cleavage junction in the viral polyprotein, was determined up to 2.5 angstrom 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 vertical bar 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). (C) 2009 Elsevier Ltd. All rights reserved.</p

Zunszain, Patricia

Knox, Stephen R.

Sweeney, Trevor R.

Roque-Rosell, Nuria

Belsham, Graham J.

Leatherbarrow, Robin J.

King's Research Portal

Insights into Cleavage Specificity from the Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease Complexed with a Peptide Substrate

Foot-and-mouth disease (FMD) is a serious, widespread viral disease of cloven-hoofed animals, including important agricultural species such as cattle, sheep, pigs and goats (19, 45). The virus spreads rapidly and, although endemic and epidemic situations can be controlled using vaccines that are based on inactivated virus particles, political and technical difficulties with the maintenance and use of vaccine stocks has stimulated the search for alternative means of tackling the disease, such as anti-viral drugs (16). The development of such treatments will demand a detailed knowledge of the molecular basis of viral replication. In this paper we focus on the structural basis of the cleavage activity of FMDV 3Cpro; as a highly conserved viral enzyme (11), FMDV 3Cpro is a potential drug target

Belsham, Graham

Online Research Database In Technology

http://spiral.imperial.ac.uk/bitstream/10044/1/4403/1/Zunszain_JMB10_OAreprint.pdf

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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Copenhagen University Research Information System

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Spiral - Imperial College Digital Repository

King's Research Portal

Online Research Database In Technology