A bacterial type III effector family uses the papain-like hydrolytic activity to arrest the host cell cycle

doi:10.1073/pnas.0900212106

A bacterial type III effector family uses the papain-like hydrolytic activity to arrest the host cell cycle

^aNational Institute of Biological Sciences, Beijing 102206, China; and
^bGraduate School of Chinese Academy of Medical Sciences and Beijing Union Medical College, Beijing 100730, China

Communicated by Steven L. McKnight, University of Texas Southwestern Medical School, Dallas, TX, January 9, 2009
↵¹Q.Y. and J.C. contributed equally to this work. (received for review October 24, 2008)

Abstract

Pathogenic bacteria deliver effector proteins into host cells through the type III secretion apparatus to modulate the host function. We identify a family of proteins, homologous to the type III effector Cif from enteropathogenic Escherichia coli, in pathogens including Yersinia, Photorhabdus, and Burkholderia that contain functional type III secretion systems. Like Cif, this family of proteins is capable of arresting the host cell cycle at G₂/M. Structure of one of the family members, Cif homolog in Burkholderia pseudomallei (CHBP), reveals a papain-like fold and a conserved Cys-His-Gln catalytic triad despite the lack of primary sequence identity. For CHBP and Cif, only the putative catalytic Cys is susceptible to covalent modification by E-64, a specific inhibitor of papain-like cysteine proteases. Unlike papain-like enzymes where the S2 site is the major determinant of cleavage-site specificity, CHBP has a characteristic negatively charged pocket occupying surface areas corresponding to the S1/S1′ site in papain-like proteases. The negative charge is provided by a conserved aspartate, and the pocket best fits an arginine, as revealed by molecular docking analysis. Mutation analysis establishes the essential role of the catalytic triad and the negatively charged pocket in inducing cell cycle arrest in host cells. Our results demonstrate that bacterial pathogens have evolved a unique papain-like hydrolytic activity to block the normal host cell cycle progression.

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Footnotes

³To whom correspondence should be addressed at:
National Institute of Biological Sciences, 7 Science Park Road, Zhongguancun Life Science Park, Beijing 102206, China.
E-mail: shaofeng{at}nibs.ac.cn
Author contributions: J.C. and F.S. designed research; Q.Y., J.C., Y.Z., L.H., C.L., R.C., X.L., and N.H. performed research; G.W., S.C., and L.L. contributed new reagents/analytic tools; Q.Y., J.C., and F.S. analyzed data; and Q.Y., J.C., and F.S. wrote the paper.
↵²Present address: College of Life Science, Nankai University, Tianjin 300071, China.
The authors declare no conflict of interest.
Data deposition: The atomic coordinates and structure factors for CHBP have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 3EIR and 3EIT).
This article contains supporting information online at www.pnas.org/cgi/content/full/0900212106/DCSupplemental.