The structure of MxiC
Reductive methylation of MxiCNΔ73 yielded crystals that diffracted to higher resolution: 2.85 Å and 2.5 Å in space groups P212121 and P222, respectively (Table 1). MxiC is an elongated rod-shaped molecule with a long axis of 86 Å (Fig. 2a). It is composed of three domains, each possessing a four-helix X-bundle fold (Fig. 2b).19 The first and last domains consist only of the X-bundle motif while the central domain also possesses a bent helix (α5) that is packed against domain 1. It is the first two domains of MxiC that are equivalent to YopN while the third domain of MxiC, equivalent to TyeA, is connected to domain 2 via a ten-residue linker. This linker acts only to tether the domains and does not have any major structural role, allowing the equivalent regions of MxiC and YopN–TyeA to adopt similar folds. There are a total of seven independent MxiC molecules in the crystallographic asymmetric units of the three refined crystal forms (see Table 1). These structures reveal that, although the fold of each domain is maintained in all structures (rmsd over Cα atoms of domains 1, 2 and 3 are 0.5 Å, 0.9 Å and 0.8 Å, respectively), there is some flexibility at the interfaces between domains resulting in a “wobble” of the terminal domains about the central domain (rmsd over all Cα atoms of 1.4 Å; Fig. 2c). The elongated shape of MxiC means that the most distal regions undergo the greatest displacement while the more central interdomain interfaces undergo minimal change.
The structural similarity between the domains of MxiC suggests that they might be the product of gene duplication events. Such internal repetition arising via intragenic duplication and recombination events has been a successful stratagem throughout evolution for enlargement of the available surface area.20 This, combined with the elongated shape of MxiC, provides the maximal exposure of surface area and considerable binding interfaces suitable for large substrates, linear peptides or multiple partners. This feature is typical of scaffolding proteins that act to recruit multiple proteins and enhance signaling.21,22
Pallen et al. used multiple sequence alignments to identify members of the MxiC/YopN–TyeA family from divergent Gram-negative species;7 a structure-based sequence alignment of MxiC and YopN–TyeA (Fig. 3a) differs in the N-terminal region from the alignment obtained in that work, and highlights the need to be cautious when interpreting conservation based on sequence alignments alone. The combination of structural and sequence alignment information identifies that most of the highly conserved residues are involved in maintaining the correct fold of these proteins. In support of this, mutations of YopN that constitutively block secretion replace highly conserved residues that are buried in the interfaces between domains of YopN.23 Ferracci et al. suggested that these mutations are likely to cause part of YopN to become unfolded or less well-ordered.23 Interestingly, mapping of sequence conservation onto the structure across the more closely related Salmonella, Burkholderia and Shigella species reveals a hydrophobic patch on the surface of the central domain consisting of residues Leu222, Met226, Gly239, Leu242 and Leu245 (Fig. 3b, red circle). This region is buried by crystal contacts in all seven independent molecules of MxiC in the P212121, P222 and P43212 crystal forms, suggesting that this region may be a “hot spot” for protein–protein interactions.
A structural homology search using the DALI algorithm,24 and the Secondary Structure Matching algorithm of MSDfold,25 was unable to identify any protein, other than YopN, possessing multiple X-bundle motifs. However, two classes of proteins were identified that possess X-bundle motifs similar to the individual domains of MxiC. The structural homologues belonged to the Bcl-2 family of apoptosis regulators, specifically the Caenorhabditis elegans homologue Ced9 (DALI Z-score 4.3, 1ohu)26 and the programmed cell death proteins involved in inhibition of protein synthesis, specifically the MA3 domain of Pdcd4 (DALI Z-score 3.9, 2nsz).27 Although the significance of these similarities is uncertain, structural mimicry of apoptotic factors is a common theme seen in viral and bacterial pathogens and represents an important defense against the host immune response.28,29 This structural similarity may be of functional relevance, as MxiC has been identified as a protein that is secreted by the T3SS under conditions that mimic those encountered by bacteria during infection of a host.3