Poster abstracts
Poster number 14 submitted by Lucas Runyan
Unconventional binding of Salmonella effector SipA to F-actin mimics the structural effects of inorganic phosphate and phalloidin
Lucas A. Runyan (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH), Ewa Niedzialkowska (Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA), Elena Kudryashova (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH), Edward Egelman (Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA), Dmitri S. Kudryashov (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH)
Abstract:
Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen whose entry to host enterocytes strictly relies on the Salmonella pathogenicity island 1 effector SipA, injected via type III secretion system 1. The C-terminal half of SipA binds and stabilizes the filamentous form of actin (F-actin) by a poorly understood mechanism. We found that SipA binds to actin with exceptionally high (in the picomolar range) affinity achieved through a unique binding mode. A 3.2 Å resolution cryo-EM reconstruction showed that the globular core domain of SipA binds at the inter-strand groove of actin, connecting two pairs of adjacent subunits in both strands. Most remarkably, the extended C-terminal arm of SipA is deeply inserted in the inter-strand space, stabilizing F-actin from within the filament core and excluding the globular core from binding to the same region resulting in a 1:2 binding stoichiometry to actin. Kinetics experiments revealed that the globular domain bestows fast docking allowing the arm to explore the otherwise hidden intra-filament surfaces to anchor the toxin to actin, slowing its dissociation by ~100-fold, inhibiting inorganic phosphate (Pi) release, and increasing filament rigidity. The increased rigidity mimics that caused by Pi/BeF3 and small-molecule toxins and correlates with potent cooperative inhibition of actin severing and depolymerization by ADF/cofilin. This work revealed a new mode of actin interaction with its partners and demonstrated how disordered peptides can penetrate to narrow clefts occupying a niche otherwise accessible only to small-molecule drugs and toxins.
Keywords: Salmonella, Actin, TIRF