Tetratricopeptide repeats in the type III secretion chaperone, LcrH: their role in substrate binding and secretion

Abstract

SummaryNon-flagellar type III secretion systems (T3SSs) transport proteins across the bacterial cell and into eukaryotic cells. Targeting of proteins into host cells requires a dedicated translocation apparatus. Efficient secretion of the translocator proteins that make up this apparatus depends on molecular chaperones. Chaperones of the translocators (also called class-II chaperones) are characterized by the possession of three tandem tetratricopeptide repeats (TPRs). We wished to dissect the relations between chaperone structure and function and to validate a structural model using site-directed mutagenesis. Drawing on a number of experimental approaches and focusing on LcrH, a class-II chaperone from the Yersinia Ysc-Yop T3SS, we examined the contributions of different residues, residue classes and regions of the protein to chaperone stability, chaperone-substrate binding, substrate stability and secretion and regulation of Yop protein synthesis. We confirmed the expected role of the conserved canonical residues from the TPRs to chaperone stability and function. Eleven mutations specifically abrogated YopB binding or secretion while three mutations led to a specific loss of YopD secretion. These are the first mutations described for any class-II chaperone that allow interactions with one translocator to be dissociated from interactions with the other. Strikingly, all mutations affecting the interaction with YopB mapped to residues with side chains projecting from the inner, concave surface of the modelled TPR structure, defining a YopB interaction site. Conversely, all mutations preventing YopD secretion affect residues that lie on the outer, convex surface of the triple-TPR cluster in our model, suggesting that this region of the molecule represents a distinct interaction site for YopD. Intriguingly, one of the LcrH double mutants, Y40A/F44A, was able to maintain stable substrates inside bacteria, but unable to secrete them, suggesting that these two residues might influence delivery of substrates to the secretion apparatus.

Yersinia pestis YopD 150–287 fragment is partially unfolded in the native state

“…To the contrary, the C-terminal YopD 278-292 peptide is readily soluble and forms an amphipathic a-helix in solution [18]. The binding site of YopD was postulated to localize to a large surface on the model of SycD, the opposite side of the YopB interactions [19], in agreement with data for a common chaperone of YopD and YopB [16].…”

IpaB–IpgC interaction defines binding motif for type III secretion translocator

“…The spatial distribution of P1, P2, and P3 and the residues forming these pockets are conserved in SycD (Table S3). Indeed, mutations in the predicted pockets of LcrH, IpgC homologue from Yersinia pseudotuberculosis, affect binding and/or secretion of its substrates (26). Together, these observations indicate that the spatial distribution of P1, P2, and P3 is a key feature in TTSS class II chaperones.…”

Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC

“…In this study, escA was demonstrated to function as a specific chaperone for EseC. EscA possesses the (Neyt & Cornelis, 1999;Waterman & Holden, 2003;Zurawski & Stein, 2003;Edqvist et al, 2006). EspD in EPEC requires two chaperones (CesD and CesD2) for complete secretion and accumulation activities (Neves et al, 2003;Waterman & Holden, 2003).…”

“…Two distinct functional classes of chaperones exist for NF-T3SSs: class I chaperones, which bind to effectors, and class II chaperones, which bind to translocons (Page & Parsot, 2002;Parsot et al, 2003). Class I chaperones share a moderate degree of sequence homology, with a common mixed a/b-helical fold (Parsot et al, 2003;Pallen et al, 2005), while some class II chaperones have been recently reported to share TPR-like motifs (Bröms et al, 2006;Edqvist et al, 2006;Büttner et al, 2008). Interactions of class II chaperones and their substrates are necessary for functions involving regulatory, structural and effector mechanisms (Delahay & Frankel, 2002;Olsson et al, 2004), while some class II chaperones are reported to have defined regions that interact with their cognate substrates (Daniell et al, 2003;Edqvist et al, 2006).…”