Structure of an SspH1-PKN1 Complex Reveals the Basis for Host Substrate Recognition and Mechanism of Activation for a Bacterial E3 Ubiquitin Ligase

Abstract

IpaH proteins are bacterium-specific E3 enzymes that function as type three secretion system (T3SS) effectors in Salmonella, Shigella, and other Gram-negative bacteria. IpaH enzymes recruit host substrates for ubiquitination via a leucine-rich repeat (LRR) domain, which can inhibit the catalytic domain in the absence of substrate. The basis for substrate recognition and the alleviation of autoinhibition upon substrate binding is unknown. Here, we report the X-ray structure of Salmonella SspH1 in complex with human PKN1. The LRR domain of SspH1 interacts specifically with the HR1b coiled-coil subdomain of PKN1 in a manner that sterically displaces the catalytic domain from the LRR domain, thereby activating catalytic function. SspH1 catalyzes the ubiquitination and proteasome-dependent degradation of PKN1 in cells, which attenuates androgen receptor responsiveness but not NF-κB activity. These regulatory features are conserved in other IpaH-substrate interactions. Our results explain the mechanism whereby substrate recognition and enzyme autoregulation are coupled in this class of bacterial ubiquitin ligases.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01360-13.

ACKNOWLEDGMENTS

We thank Igor Kurinov at the Argonne National Laboratory for assistance with microdiffraction experiments at the Advanced Photon Source on the Northeastern Collaborative Access Team beamlines (supported by award RR-15301 from the National Center for Research Resources at the National Institutes of Health and contract DE-AC02-06CH11357 from the U.S. Department of Energy) and the CMCF staff for data collection at beamline 08ID-1 at the Canadian Light Source (supported by the Natural Sciences and Engineering Research Council of Canada, the National Research Council Canada, the Canadian Institutes of Health Research, the Province of Saskatchewan, Western Economic Diversification Canada, and the University of Saskatchewan). We also thank members of the Trevor Moraes laboratory at the University of Toronto for crystal data collection.

A.F.A.K. is funded by a Natural Sciences and Engineering Research Council postgraduate scholarship. This work was supported by grants to F.S. and M.T. from the Canadian Institutes of Health Research (MOP-57795 and MOP-126129), by a Genome Quebec International Recruitment Award to M.T., by a Canada Research Chair in Structural Biology to F.S., and by a Canada Research Chair in Systems and Synthetic Biology to M.T.