Binding Affects the Tertiary and Quaternary Structures of the Shigella Translocator Protein IpaB and Its Chaperone IpgC

Abstract

Shigella flexneri uses its type III secretion system (T3SS) to promote invasion of human intestinal epithelial cells as the first step in causing shigellosis, a life threatening form of dysentery. The Shigella type III secretion apparatus (T3SA) consists of a basal body that spans the bacterial envelope and an exposed needle that injects effector proteins into target cells. The nascent Shigella T3SA needle is topped with a pentamer of the needle tip protein invasion plasmid antigen D (IpaD). Bile salts trigger recruitment of the first hydrophobic translocator protein, IpaB, to the tip complex where it senses contact with a host membrane. In the bacterial cytoplasm, IpaB exists in a complex with its chaperone IpgC. Several structures of IpgC have been solved and we recently reported the 2.1-Å crystal structure of the N-terminal domain (IpaB74.224) of IpaB. Like IpgC, the IpaB N-terminal domain exists as a homodimer in solution. We now report that when the two are mixed, these homodimers dissociate and form heterodimers having a nanomolar dissociation constant. This is consistent with the equivalent complexes co-purified after being co-expressed in E. coli. Fluorescence data presented here also indicate that the N-terminal domain of IpaB possesses two regions that appear to contribute additively to chaperone binding. It is also likely that the IpaB N terminus adopts an alternative conformation as a result of chaperone binding. The importance of these findings within the functional context of these proteins is discussed.

LcrH, a Class II Chaperone from the Type Three Secretion System, Has a Highly Flexible Native Structure

“…The peptides each bind to a similar region of the concave groove formed by all three TPR motifs. Additionally, a recent study shows that the translocator chaperones may also bind supplementary regions on the translocator proteins as well (7,13,14). However, the mechanism by which these small chaperones find their cognate peptide sequences within the structure of the large membrane translocator proteins remains unclear.…”

“…(i) Although biochemical studies have implicated more than one interaction between chaperone and translocator (7,13,14), structures of IpgC with IpaB(51-72), PcrH(21-160) with PopD(47-56), and SycD(21-163) with YopD(56 -65) show a common main interaction site where an extended peptide from each translocator binds on the concave face of its cognate TPR domain (1:1 ratio) (Fig. 1).…”

YspC: A Unique Translocator Exhibits Structural Alteration in the Complex form with Chaperone SycB

“…These stable fragments obtained after digestion of proteins have significant value for crystallization. Proteolysis of IpaB-IpgC complex has allowed identification of IpaB fragments, which proved useful for further crystallization purpose [1,25].…”

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

“…Using this structure, we investigated the interaction between the soluble Nterminal domain of IpaB and IpgC. 19 In this work we identified two distinct chaperone binding domains near the extreme N-terminus of IpaB. These sites promote the formation of a heterodimer of IpaB:IpgC.…”