Comparison with the YopN–TyeA complex
Although the individual domains of MxiC and YopN–TyeA adopt similar folds (rmsd over Cα atoms of the first and second domains of MxiC with the equivalent regions of YopN of 3.9 Å and 3.2 Å, respectively, and the third domain of MxiC with TyeA of 0.8 Å), the arrangement of these domains results in a different overall shape for these molecules (Fig. 3b). This structural rearrangement differs from the “wobble” seen between different MxiC molecules and is particularly noticeable in one orientation (Fig. 3b, top panel). This view highlights the straight conformation adopted by MxiC and the relative curvature of the YopN–TyeA complex. The different position and orientation of the first domain of MxiC may be due, in part, to the missing N-terminal portion, as the YopN–TyeA structure possesses an additional helix at its N terminus.
It seemed likely that the major difference between these structures would be the arrangement of the domain equivalent to TyeA as this is a separate polypeptide in the Yersinia structure. Surprisingly, the most striking difference instead involves the long á-helix (α9) in the central domain of MxiC, which is straight in all of our MxiC structures, but possesses a sharp kink in YopN when complexed with TyeA (Fig. 3b). It is the straightening of this helix that results in a reorientation of the C-terminal domain of MxiC, compared with TyeA, and opens one face of the molecule. As the sequence at the hinge is not conserved, and all MxiC molecules possess the straight helix conformation, these structures may represent genuine differences between species. Alternatively, it may represent a conformational switch that, in this case, was captured due to the very different pH for the YopN–TyeA structure (pH 10.5) compared with the MxiC structures (pH 6.5–7.5).
Despite the lack of sequence conservation on the surface of these structures, an analysis of the surface electrostatics reveals that the face that is curved in YopN–TyeA and open in MxiC possesses a conserved negatively charged patch (Fig. 3c). This patch spreads across one face of the C-terminal half of the molecule (displayed in red on the right-hand side of the surface shown in Fig. 3c). The helices that line this face undergo minor repacking and can accommodate the large movements of the surrounding domains. The conserved patch on this face suggests a role for this region in interactions with partner proteins. If this is a binding face for another component of the T3SS, it is interesting to note that this interface is conserved despite it being intramolecular in one homologue and intermolecular in another.