Structural basis of ligand binding by a c-di-GMP riboswitch

Abstract

The GEMM riboswitch is conserved in diverse bacteria and recognizes the second messenger c-di-GMP which mediates many processes, such as the transition between sedentary and motile behavior. The structure of the GEMM riboswitch with ligand now elucidates ligand recognition and specificity. The second messenger signaling molecule bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) regulates many processes in bacteria, including motility, pathogenesis and biofilm formation. c-di-GMP–binding riboswitches are important downstream targets in this signaling pathway. Here we report the crystal structure, at 2.7 Å resolution, of a c-di-GMP riboswitch aptamer from Vibrio cholerae bound to c-di-GMP, showing that the ligand binds within a three-helix junction that involves base-pairing and extensive base-stacking. The symmetric c-di-GMP is recognized asymmetrically with respect to both the bases and the backbone. A mutant aptamer was engineered that preferentially binds the candidate signaling molecule c-di-AMP over c-di-GMP. Kinetic and structural data suggest that genetic regulation by the c-di-GMP riboswitch is kinetically controlled and that gene expression is modulated through the stabilization of a previously unidentified P1 helix, illustrating a direct mechanism for c-di-GMP signaling.