Structure of the PilZ-FimXEAL-c-di-GMP and PilZ-PilB complexes responsible for the regulation of bacterial Type IV pilus biogenesis

GUZZO, CRISTIANE; DUNGER, GERMÁN; LLONTOP, EDGAR; KOPKE SALINAS, ROBERTO; FARAH, C.S.

Sao Paulo

Jornada; Alunos na Ciência - Instituto de Química; 2014

Instituto de Química - Universidade de Sao Paulo

Signal transduction pathways mediated by c-diGMP are ubiquitous in bacteria where they control many important and complex behaviors. C-diGMP 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. Several families of c-diGMP receptors have been characterized, the largest of which are PilZ domains and degenerate (enzymatically inactive) EAL domains. Most Gram-negative bacterial genomes code for at least one and often dozens of different proteins with GGDEF, EAL or HD-GYP and PilZ domains. There is mounting evidence that some important c-diGMP-mediated pathways require protein-protein interactions between members of these domain families. For example, in Xanthomonas species, the control of motility via DSF-dependent signaling during quorum sensing is dependent on interactions between the HD-GYP domain from RpfG and at least two GGDEF domaincontaining proteins. Interactions have also been observed between PilZ and the EAL domain from FimX. These latter proteins are involved in the regulation of type IV pilus biogenesis via interactions of PilZ with the hexameric PilB ATPase associated with the bacterial inner membrane. In spite of the importance of protein-protein interactions in c-diGMP-mediated pathways, no high resolution structures of these domains in protein-proteincomplexes are yet available. Here, we present the crystal structure of the ternary complex made up of PilZ, the FimX EAL domain and c-diGMP. PilZ interacts principally with the lobe region and the N-terminal linker helix of the FimX EAL domain. PilZ interactions with the FimXEAL involve both the stitching to together of beta sheets from both proteins as well as a hydrophobic surface made up of amino acids conserved in a non-canonical family of PilZ domains that lack intrinsic c-diGMP binding ability. Interestingly, the c-diGMP binds to isolated FimXEAL and to the PilZ-FimXEAL complex in a novel conformation never before encountered in c-diGMP-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. While direct PilZ-cidiGMP interactions in the complex are very tenuous, the structure points to a means by which both c-diGMP and PilZ binding could work together to modulate both the relative orientation of the EAL domain with respect to the neighboring GGDEF domain in the FimX protein and interactions between the conserved PilZ C-terminus and PilB.