NMR identification of the binding surfaces involved in the Salmonella and Shigella Type III secretion tip‐translocon protein–protein interactions

The type III secretion system (T3SS) is essential for the pathogenesis of many bacteria including Salmonella and Shigella, which together are responsible for millions of deaths worldwide each year. The structural component of the T3SS consists of the needle apparatus, which is assembled in part by the protein–protein interaction between the tip and the translocon. The atomic detail of the interaction between the tip and the translocon proteins is currently unknown. Here, we used NMR methods to identify that the N‐terminal domain of the Salmonella SipB translocon protein interacts with the SipD tip protein at a surface at the distal region of the tip formed by the mixed α/β domain and a portion of its coiled‐coil domain. Likewise, the Shigella IpaB translocon protein and the IpaD tip protein interact with each other using similar surfaces identified for the Salmonella homologs. Furthermore, removal of the extreme N‐terminal residues of the translocon protein, previously thought to be important for the interaction, had little change on the binding surface. Finally, mutations at the binding surface of SipD reduced invasion of Salmonella into human intestinal epithelial cells. Together, these results reveal the binding surfaces involved in the tip‐translocon protein–protein interaction and advance our understanding of the assembly of the T3SS needle apparatus. Proteins 2016; 84:1097–1107. © 2016 Wiley Periodicals, Inc.

[1]  Andrew J. Olive,et al.  Bile Salts Stimulate Recruitment of IpaB to the Shigella flexneri Surface, Where It Colocalizes with IpaD at the Tip of the Type III Secretion Needle , 2007, Infection and Immunity.

[2]  Nicholas Sawyer,et al.  Protein–protein interactions: General trends in the relationship between binding affinity and interfacial buried surface area , 2013, Protein science : a publication of the Protein Society.

[3]  D. Baker,et al.  High-resolution structure of a Shigella type III secretion needle by solid-state NMR and cryo-electron microscopy , 2014, Nature Communications.

[4]  D. Baker,et al.  Atomic model of the type III secretion system needle , 2012, Nature.

[5]  E. McGhie,et al.  The purified Shigella IpaB and Salmonella SipB translocators share biochemical properties and membrane topology , 2003, Molecular microbiology.

[6]  D. Swerdlow,et al.  Global burden of Shigella infections: implications for vaccine development and implementation of control strategies. , 1999, Bulletin of the World Health Organization.

[7]  Gerald J. Wyckoff,et al.  The structures of coiled-coil domains from type III secretion system translocators reveal homology to pore-forming toxins. , 2012, Journal of molecular biology.

[8]  Natalie G Ahn,et al.  Phosphorylation releases constraints to domain motion in ERK2 , 2014, Proceedings of the National Academy of Sciences.

[9]  Andrew J. Olive,et al.  Deoxycholate Interacts with IpaD of Shigella flexneri in Inducing the Recruitment of IpaB to the Type III Secretion Apparatus Needle Tip* , 2008, Journal of Biological Chemistry.

[10]  M. Kolbe,et al.  Crystal Structure of PrgI-SipD: Insight into a Secretion Competent State of the Type Three Secretion System Needle Tip and its Interaction with Host Ligands , 2011, PLoS pathogens.

[11]  B. Geisbrecht,et al.  Identification of the bile salt binding site on ipad from Shigella flexneri and the influence of ligand binding on IpaD structure , 2012, Proteins.

[12]  Samuel Wagner,et al.  Bacterial type III secretion systems: specialized nanomachines for protein delivery into target cells. , 2014, Annual review of microbiology.

[13]  J. Galán,et al.  Salmonella enterica Serovar Typhimurium Pathogenicity Island 1-Encoded Type III Secretion System Translocases Mediate Intimate Attachment to Nonphagocytic Cells , 2009, Infection and Immunity.

[14]  M. Pop,et al.  An optimized system for expression and purification of secreted bacterial proteins. , 2006, Protein expression and purification.

[15]  E. McGhie,et al.  Membrane fusion activity of purified SipB, a Salmonella surface protein essential for mammalian cell invasion , 2000, Molecular microbiology.

[16]  Sebenzile K. Myeni,et al.  SipB-SipC Complex Is Essential for Translocon Formation , 2013, PloS one.

[17]  R. D. De Guzman,et al.  NMR characterization of the interaction of the Salmonella type III secretion system protein SipD and bile salts. , 2010, Biochemistry.

[18]  A. Blocker,et al.  Shigella IpaD has a dual role: signal transduction from the type III secretion system needle tip and intracellular secretion regulation , 2013, Molecular microbiology.

[19]  J. Torres,et al.  Topology of the Salmonella invasion protein SipB in a model bilayer , 2002, Molecular microbiology.

[20]  K. Kaur,et al.  Characterization of the Shigella and Salmonella Type III Secretion System Tip–Translocon Protein–Protein Interaction by Paramagnetic Relaxation Enhancement , 2016, Chembiochem : a European journal of chemical biology.

[21]  S Falkow,et al.  The Salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Aamir Fazil,et al.  The global burden of nontyphoidal Salmonella gastroenteritis. , 2010, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[23]  G. Cornelis,et al.  The type III secretion system tip complex and translocon , 2008, Molecular microbiology.

[24]  L. Kay,et al.  New developments in isotope labeling strategies for protein solution NMR spectroscopy. , 2000, Current opinion in structural biology.

[25]  R. Hayward,et al.  Direct nucleation and bundling of actin by the SipC protein of invasive Salmonella , 1999, The EMBO journal.

[26]  K. Namba,et al.  Three-dimensional electron microscopy reconstruction and cysteine-mediated crosslinking provide a model of the type III secretion system needle tip complex , 2014, Molecular microbiology.

[27]  K. Battaile,et al.  The crystal structures of the Salmonella type III secretion system tip protein SipD in complex with deoxycholate and chenodeoxycholate , 2011, Protein science : a publication of the Protein Society.

[28]  J. Gunn,et al.  Salmonella enterica Serovar Typhimurium Invasion Is Repressed in the Presence of Bile , 2000, Infection and Immunity.

[29]  T. Rathinavelan,et al.  Characterization of the Interaction between the Salmonella Type III Secretion System Tip Protein SipD and the Needle Protein PrgI by Paramagnetic Relaxation Enhancement* , 2010, The Journal of Biological Chemistry.

[30]  Y. Park,et al.  Analysis of functional domains present in the N-terminus of the SipB protein. , 2007, Microbiology.

[31]  M. Kok,et al.  Human Alveolar Macrophages Infected by Virulent Bacteria Expressing SipB Are a Major Source of Active Interleukin-18 , 2003, Infection and Immunity.

[32]  W. Picking,et al.  Conformational changes in IpaD from Shigella flexneri upon binding bile salts provide insight into the second step of type III secretion. , 2011, Biochemistry.

[33]  W. Picking,et al.  N-terminus of IpaB provides a potential anchor to the Shigella type III secretion system tip complex protein IpaD. , 2013, Biochemistry.

[34]  Mok Yu-Keung.  Structure of AcrH-AopB chaperone-translocator complex reveals a role for membrane hairpins in type III secretion system translocon assembly , 2017 .

[35]  A. Vaheri,et al.  Interaction between hantaviral nucleocapsid protein and the cytoplasmic tail of surface glycoprotein Gn. , 2010, Virus research.

[36]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[37]  Andrew J. Olive,et al.  IpaD Localizes to the Tip of the Type III Secretion System Needle of Shigella flexneri , 2006, Infection and Immunity.

[38]  S. Falkow,et al.  Transcriptional regulation of Salmonella enterica serovar Typhimurium genes by bile. , 2004, FEMS immunology and medical microbiology.

[39]  Chun Tang,et al.  NMR model of PrgI-SipD interaction and its implications in the needle-tip assembly of the Salmonella type III secretion system. , 2014, Journal of molecular biology.

[40]  G. Cornelis,et al.  Insertion of a Yop translocation pore into the macrophage plasma membrane by Yersinia enterocolitica: requirement for translocators YopB and YopD, but not LcrG , 1999, Molecular microbiology.

[41]  K. Kaur,et al.  Structure and biophysics of type III secretion in bacteria. , 2013, Biochemistry.

[42]  Bruce A Johnson,et al.  Using NMRView to visualize and analyze the NMR spectra of macromolecules. , 2004, Methods in molecular biology.

[43]  Andrew J. Olive,et al.  Self-chaperoning of the Type III Secretion System Needle Tip Proteins IpaD and BipD* , 2006, Journal of Biological Chemistry.