Crystal structure of the Yersinia enterocolitica type III secretion chaperone SycD in complex with a peptide of the minor translocator YopD

Abstract

BackgroundType III secretion systems are used by Gram-negative bacteria as “macromolecular syringes” to inject effector proteins into eukaryotic cells. Two hydrophobic proteins called translocators form the necessary pore in the host cell membrane. Both translocators depend on binding to a single chaperone in the bacterial cytoplasm to ensure their stability and efficient transport through the secretion needle. It was suggested that the conserved chaperones bind the more divergent translocators via a hexapeptide motif that is found in both translocators and conserved between species.ResultsWe crystallized a synthetic decapeptide from the Yersinia enterocolitica minor type III secretion translocator YopD bound to its cognate chaperone SycD and determined the complex structure at 2.5 Å resolution. The structure of peptide-bound SycD is almost identical to that of apo SycD with an all helical fold consisting of three tetratricopeptide repeats (TPRs) and an additional C-terminal helix. Peptide-bound SycD formed a kinked head-to-head dimer that had previously been observed for the apo form of SycD. The homodimer interface comprises both helices of the first tetratricopeptide repeat. The YopD peptide bound in extended conformation into a mainly hydrophobic groove on the concave side of SycD. TPRs 1 and 2 of SycD form three hydrophobic pockets that accommodated the conserved hydrophobic residues at position 1, 3 and 6 of the translocator hexapeptide sequence. Two tyrosines that are highly conserved among translocator chaperones contribute to the hydrophobic patches but also form hydrogen bonds to the peptide backbone.ConclusionsThe interaction between SycD and YopD is very similar to the binding of the Pseudomonas minor translocator PopD to its chaperone PcrH and the Shigella major translocator IpaB to its chaperone IpgC. This confirms the prediction made by Kolbe and co-workers that a hexapeptide with hydrophobic residues at three positions is a conserved chaperone binding motif. Because the hydrophobic groove on the concave side of translocator chaperones is involved in binding of the major and the minor translocator, simultaneous binding of both translocators to a single type III secretion class II chaperone appears unlikely.

Structure of AcrH–AopB Chaperone-Translocator Complex Reveals a Role for Membrane Hairpins in Type III Secretion System Translocon Assembly

“…Based on our structure, this assumption is incorrect. Although previous structures for IpaB-IpgC (Lunelli et al, 2009), SycD-YopD peptide (Schreiner and Niemann, 2012), and PcrH-PopD 48-55 (Job et al, 2010) concluded that the hydrophobic anchor of the translocator was bound to the concave surface of the chaperone, our structure shows that binding at the concave region is only part of the overall mechanism for the interaction. The N-terminal arm as well as the convex and top surfaces of the TPR are also equally, if not more, important for this interaction.…”

“…The N-terminal molecular anchor is conserved in both the major and minor translocators of the Ysc and Inv-Mxi-Spa families with the consensus sequence of P/VXLXXP (Discola et al, 2014;Job et al, 2010;Lunelli et al, 2009;Schreiner and Niemann, 2012) ( Figure 3D). In the structure of AcrH-AopB 40-264 , we further confirmed that this consensus sequence (VVLPQP; residues Val48 0 -Pro53 0 ) in AopB interacts in an extended conformation with the hydrophobic concave surface of AcrH ( Figure 3B).…”

“…Until now, the structures for class II chaperones in complex with their specific translocators have been solved with limited short peptides of 9-13 residues that are selected from the N-terminal region of the translocator, hereafter referred to as the ''molecular anchor'' and bear a ''P/VXLXXP'' consensus sequence (Discola et al, 2014;Job et al, 2010;Lunelli et al, 2009;Schreiner and Niemann, 2012). Both major and minor translocators use this conserved molecular anchor in their discrete binding to the concave surface of the chaperone.…”

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

“…LcrH protein and buffer were added to the differing denaturant concentrations to give identical protein (from 3 to 80 M) and buffer concentrations (LcrH buffer). The length of time the experiments were left to equilibrate was determined by repeating a denaturation experiment after differing equilibration times (4,8,12, and 24 h). These all gave superimposable curves, showing that equilibrium was reached after 4 h (supplemental Fig.…”

Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa

“…By solving the high resolution crystal structure of PcrH, we had also shown that it folds into seven TPR motifs that generate a concave region that is capable of binding PopD residues 49 -56 (8). Notably, IpgC, the type II chaperone of the type III secretion system of Shigella, also recognizes a short peptide of the N terminus of the major translocator molecule (50,51), and SycD from Yersinia enterocolitica was also structurally characterized in complex with a peptide from minor translocator YopD (52).…”