Vibrio cholerae YaeO is a Structural Homologue of RNA Chaperone Hfq that Inhibits Rho-dependent Transcription Termination by Dissociating its Hexameric State.

Rho-dependent transcription termination is a well-conserved process in bacteria. The Psu and YaeO proteins are the two established inhibitors of the ATP-dependent RNA helicase Rho protein of Escherichia coli. Here, we show a detailed sequence and phylogenetic analysis demonstrating that Vibrio cholerae YaeO (VcYaeO) is significantly distinct from its E. coli counterpart. VcYaeO induces significant growth defect on in vivo expression and inhibits in vitro functions of the V. cholerae Rho on directly binding to the latter. Through various biophysical techniques, we showed that interaction of VcYaeO disrupts the oligomeric state of the VcRho. Structure of VcYaeO solved at 1.75 Å resolution, the first crystal structure of a YaeO protein, demonstrates a beta-sandwich fold distinct from the NMR structure of the EcYaeO. Interestingly, VcYaeO structurally resembles the Hfq protein, and like the latter, it exhibits ssDNA/RNA-binding properties. Docking studies demonstrate probable interactions of VcYaeO with VcRho and mode of inhibition of RNA binding to Rho. We propose that VcYaeO inhibits the function of the Rho protein via disruption of the latter's hexameric assembly and also likely by sequestering the RNA from the Rho primarybinding sites.

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication.

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3'3'-cyclic GMP-AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5'-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5'-pGpG-Ca2+ structure, ß5-α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5'-pGpG-Ca2+ structure quite different from other 5'-pGpG bound structures reported earlier.