Structure and function of REP34 implicates carboxypeptidase activity in Francisella tularensis host cell invasion

Geoffrey K. Feld, Sahar El-Etr, Michele H. Corzett, Mark S. Hunter, Kamila Belhocine, Denise M. Monack, Matthias Frank, Brent W. Segelke, Amy Rasley

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

Francisella tularensis is the etiological agent of tularemia, or rabbit fever. Although F. tularensis is a recognized biothreat agent with broad and expanding geographical range, its mechanism of infection and environmental persistence remain poorly understood. Previously, we identified seven F. tularensis proteins that induce a rapid encystment phenotype (REP) in the free-living amoeba, Acanthamoeba castellanii. Encystment is essentialtothe pathogen's long term intracellular survivalinthe amoeba. Here, we characterize the cellular and molecular function of REP34, a REP protein with a mass of 34 kDa. A REP34 knock-out strain of F. tularensis has a reduced ability to both induce encystment in A. castellanii and invade human macrophages. We determined the crystal structure of REP34 to 2.05-Å resolution and demonstrate robust carboxypeptidase B-like activity for the enzyme. REP34 is a zinc-containing monomeric protein with close structural homology to the metallocarboxypeptidase family of peptidases. REP34 possesses a novel topology and substrate binding pocket that deviates from the canonical funnelin structure of carboxypeptidases, putatively resulting in a catalytic role for a conserved tyrosine and distinct S1' recognition site. Taken together, these results identify REP34 as an active carboxypeptidase, implicate the enzyme as a potential key F. tularensis effector protein, and may help elucidate a mechanistic understanding of F. tularensis infection of phagocytic cells.

Original languageEnglish (US)
Pages (from-to)30668-30679
Number of pages12
JournalJournal of Biological Chemistry
Volume289
Issue number44
DOIs
StatePublished - Oct 31 2014
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

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    Feld, G. K., El-Etr, S., Corzett, M. H., Hunter, M. S., Belhocine, K., Monack, D. M., Frank, M., Segelke, B. W., & Rasley, A. (2014). Structure and function of REP34 implicates carboxypeptidase activity in Francisella tularensis host cell invasion. Journal of Biological Chemistry, 289(44), 30668-30679. https://doi.org/10.1074/jbc.M114.599381