Structure and interactions of the cytoplasmic domain of the Yersinia type III secretion protein YscD

Abstract

The virulence of a large number of Gram-negative bacterial pathogens depends on the type III secretion (T3S) system, which transports select bacterial proteins into host cells. An essential component of the Yersinia T3S system is YscD, a single-pass inner membrane protein. We report here the 2.52-Å resolution structure of the cytoplasmic domain of YscD, called YscDc. The structure confirms that YscDc consists of a forkhead-associated (FHA) fold, which in many but not all cases specifies binding to phosphothreonine. YscDc, however, lacks the structural properties associated with phosphothreonine binding and thus most likely interacts with partners in a phosphorylation-independent manner. Structural comparison highlighted two loop regions, L3 and L4, as potential sites of interactions. Alanine substitutions at L3 and L4 had no deleterious effects on protein structure or stability but abrogated T3S in a dominant negative manner. To gain insight into the function of L3 and L4, we identified proteins associated with YscD by affinity purification coupled to mass spectrometry. The lipoprotein YscJ was found associated with wild-type YscD, as was the effector YopH. Notably, the L3 and L4 substitution mutants interacted with more YopH than did wild-type YscD. These substitution mutants also interacted with SycH (the specific chaperone for YopH), the putative C-ring component YscQ, and the ruler component YscP, whereas wild-type YscD did not. These results suggest that substitutions in the L3 and L4 loops of YscD disrupted the dissociation of SycH from YopH, leading to the accumulation of a large protein complex that stalled the T3S apparatus.

Fig 1

YscDc has an FHA fold. (a) Structure of YscDc (residues 1 to 108) in ribbon representation, with β-strands in red, coils in gray, and helices in blue. Loops L3 and L4 are indicated. (b) Structural superposition of YscDc with closely related structurally characterized FHA domains. YscDc is in red, CT664 (RSCB accession code 3GQS) purple, EmbR (2FEZ) cyan, Rv0020c (3PO8) green, KIF13 (3FM8) blue, and Ki67 (2AFF) orange. The proteins are shown as Cα traces. (c) Structure-based sequence alignment of the FHA domains shown in panel b. A red arrow denotes the position of the Ser in loop 4 that is conserved in phosphothreonine-binding FHA domains, and the blue circles denote core residues of YscDc. Secondary structure annotations at the top of the alignment are for YscDc.

Fig 2

YscDc and T3S orthologs. (a) Electrostatic potential of YscDc mapped to its surface. Red is negative (−10 kT), and blue is positive (+10 kT). (b) Structure-based sequence alignments of YscD and Ysc subfamily members AscD, PscD, and SctD. Secondary structure annotations at the top of the alignments are for YscDc. (c) Structure-based sequence alignments of YscD and T3S orthologs PrgH, EscD, and MxiG. Secondary structure annotations at the top of the alignments are for YscDc and those at the bottom for MxiG. Red arrowheads below MxiG denote MxiG R39 and S61. (d) Structural superposition of YscDc and the FHA domain of MxiG. YscDc is in red and MxiG (2XXS) in blue. The proteins are shown as Cα traces.

Fig 3

L3 and L4 mutants. (a) Positions of residues mutated in the L3 loops (blue) and L4 loops (green). YscDc is shown as a bond trace, with β-strands in red and loops in gray. (b) Top panel, type III secretion as detected by SDS-PAGE of culture supernatants. Y. pseudotuberculosis (ΔyscD) was transformed with plasmids expressing wild-type YscD (pHis-YscD) and mutant YscD [pHis-YscD(L3), pHis-YscD(L4), pHis-YscD(S38A), pHis-YscD(S39A), pHis-YscD(S40A)] under the inducible control of arabinose. Yp is wild-type Y. pseudotuberculosis; +Ca²⁺ indicates the presence of high calcium concentration, which suppresses T3S; −arabinose indicates lack of arabinose induction. The sizes of molecular mass standards in kDa are indicated at left. Middle panel, level of intrabacterial YscD for each of the samples, as detected by an anti-YscD Western blot. Lower panel, level of intrabacterial RpoA as a loading control, as detected by anti-RpoA blot. (c) Circular dichroism spectra of wild-type YscDc, YscDc(L3), and YscDc(L4) at 4 and 37°C.

Fig 4

Proteins associated with YscD. Proteins found associated (indicated at top left of each plot) with wild-type YscD, ΔyscD (lacking YscD and thus a specificity control), and the L3 and L4 substitutions mutants of YscD. Peak heights of peptides corresponding to these proteins are plotted relative to an internal standard, as identified by mass spectrometry (see Table S2 in the supplemental material).

Fig 5

Dominant negative effect of YscD L3 and L4. Type III secretion detected by Coomassie-stained SDS-PAGE. The format is as in Fig. 3b, with wild-type Y. pseudotuberculosis being transformed with plasmids expressing wild-type YscD (pHis-YscD) and mutant YscD [pHis-YscD(L3), pHis-YscD(L4)] under the inducible control of arabinose.