Structure of the PilZ–FimXEAL–c-di-GMP Complex Responsible for the Regulation of Bacterial Type IV Pilus Biogenesis

Abstract

Signal transduction pathways mediated by cyclic-bis(3'→5')-dimeric GMP (c-di-GMP) control many important and complex behaviors in bacteria. C-di-GMP is synthesized through the action of GGDEF domains that possess diguanylate cyclase activity and is degraded by EAL or HD-GYP domains with phosphodiesterase activity. There is mounting evidence that some important c-di-GMP-mediated pathways require protein-protein interactions between members of the GGDEF, EAL, HD-GYP and PilZ protein domain families. For example, interactions have been observed between PilZ and the EAL domain from FimX of Xanthomonas citri (Xac). FimX and PilZ are involved in the regulation of type IV pilus biogenesis via interactions of the latter with the hexameric PilB ATPase associated with the bacterial inner membrane. Here, we present the crystal structure of the ternary complex made up of PilZ, the FimX EAL domain (FimXEAL) and c-di-GMP. PilZ interacts principally with the lobe region and the N-terminal linker helix of the FimXEAL. These interactions involve a hydrophobic surface made up of amino acids conserved in a non-canonical family of PilZ domains that lack intrinsic c-di-GMP binding ability and strand complementation that joins β-sheets from both proteins. Interestingly, the c-di-GMP binds to isolated FimXEAL and to the PilZ-FimXEAL complex in a novel conformation encountered in c-di-GMP-protein complexes in which one of the two glycosidic bonds is in a rare syn conformation while the other adopts the more common anti conformation. The structure points to a means by which c-di-GMP and PilZ binding could be coupled to FimX and PilB conformational states.

Diversity of Cyclic Di-GMP-Binding Proteins and Mechanisms

“…Type I PilZ is a full-length domain containing both RXXXR and (D/N)XSXXG motifs (described in reference 18); it is apparently the only PilZ type capable of directly binding c-di-GMP. Many PilZ domain proteins, including the eponymous PilZ protein from P. aeruginosa, belong to type II, which lacks the Nterminal RXXXR motif and is incapable of binding c-di-GMP (26)(27)(28). PilZ type III is a truncated version that forms a stable tetramer via a helical bundle (29); it does not bind c-di-GMP either.…”

“…While the RXXD motif in the I sites binds the characteristic mutually intercalated dimeric form of c-di-GMP, the EAL-containing PDEs bind and hydrolyze c-di-GMP in its fully extended monomeric form, which is apparently more suitable for cleavage of the central 12-atom ribosephosphate ring (41)(42)(43). The same binding mode of the extended monomeric c-di-GMP is retained in the enzymatically inactive EAL domains that function exclusively as c-di-GMP receptors (28,44,45). Figure 3A shows the structure of the EAL domain of the blue light-regulated PDE BlrP1 in a complex with c-di-GMP (PDB code 3GG1) and a calcium ion, an inhibitor of the c-di-GMP hydrolysis (41).…”

“…The second mechanism of c-di-GMP signaling is through its binding at protein interfaces, where it functions as molecular glue, facilitating protein-protein interactions. A good example is the interaction of the enzymatically inactive EAL domain of FimX with the PilZ protein of X. campestris, which regulates extension and retraction of the pilus in the presence of c-di-GMP (27,28). This XcPilZ protein contains a type II PilZ domain (see above) and cannot bind c-di-GMP on its own.…”

Progress in Understanding the Molecular Basis Underlying Functional Diversification of Cyclic Dinucleotide Turnover Proteins

“…In the conserved Lap system with the GGDEF-EAL receptor LapD, interactive inside-out/outside-in signals mediated by the HAMP domain couple cytoplasmic cyclic di-GMP binding to reinforcement of periplasmic protein-protein interactions controlling, e.g., periplasmic proteolysis of cell surface proteins (46,47). Interestingly, homologous GGDEF-EAL receptors have variations in their cyclic di-GMP binding sites and bind cyclic di-GMP in different conformations, which reflects the structural polymorphism of this second messenger (48,49), as well as binding site flexibility (Fig. 5C) (50).…”

Stand-Alone EAL Domain Proteins Form a Distinct Subclass of EAL Proteins Involved in Regulation of Cell Motility and Biofilm Formation in Enterobacteria

“…Based on the conservation of the active-site motifs and the dimerization loop (11), EAL domains have been categorized into three main classes: (i) bona fide c-di-GMP PDEs, (ii) EAL domains with a degenerate loop 6 that might or might not have PDE activity, and (iii) EAL domains that lack the key catalytic residues and have no enzymatic activity (11,12). The latter class could be further subdivided into those EAL domains that have retained the ability to bind c-di-GMP and serve as c-di-GMP receptors and those domains that do not even bind c-di-GMP (9,10,13,14).…”