research papers Acta Cryst. (2011). D67, 997–1008 doi:10.1107/S090744491104039X 997 Acta Crystallographica Section D Biological Crystallography ISSN 0907-4449 The structure and inhibition of a GGDEF diguanylate cyclase complexed with (c-di-GMP) 2 at the active site Chao-Yu Yang, a Ko-Hsin Chin, b Mary Lay-Cheng Chuah, c Zhao-Xun Liang, c Andrew H.-J. Wang d and Shan-Ho Chou a,b,e * a Institute of Biochemistry, National Chung Hsing University, Taichung 40227, Taiwan, b Agricultural Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan, c School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore, d Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei, Taiwan, and e Graduate Institute of Basic Medical Science, China Medical University, Taichung 40402, Taiwan Correspondence e-mail: shchou@nchu.edu.tw # 2011 International Union of Crystallography Printed in Singapore – all rights reserved Cyclic diguanosine monophosphate (c-di-GMP) is a key signalling molecule involved in regulating many important biological functions in bacteria. The synthesis of c-di-GMP is catalyzed by the GGDEF-domain-containing diguanylate cyclase (DGC), the activity of which is regulated by the binding of product at the allosteric inhibitory (I) site. However, a significant number of GGDEF domains lack the RxxD motif characteristic of the allosteric I site. Here, the structure of XCC4471 GGDEF , the GGDEF domain of a DGC from Xanthomonas campestris, in complex with c-di-GMP has been solved. Unexpectedly, the structure of the complex revealed a GGDEF-domain dimer cross-linked by two molecules of c-di-GMP at the strongly conserved active sites. In the complex (c-di-GMP) 2 adopts a novel partially inter- calated form, with the peripheral guanine bases bound to the guanine-binding pockets and the two central bases stacked upon each other. Alteration of the residues involved in specific binding to c-di-GMP led to dramatically reduced K d values between XCC4471 GGDEF and c-di-GMP. In addition, these key residues are strongly conserved among the many thousands of GGDEF-domain sequences identified to date. These results indicate a new product-bound form for GGDEF-domain- containing proteins obtained via (c-di-GMP) 2 binding at the active site. This novel XCC4471 GGDEF –c-di-GMP complex structure may serve as a general model for the design of lead compounds to block the DGC activity of GGDEF-domain- containing proteins in X. campestris or other microorganisms that contain multiple GGDEF-domain proteins. Received 26 July 2011 Accepted 30 September 2011 PDB Reference: XCC4471 GGDEF –c-di-GMP complex, 3qyy. 1. Introduction Cyclic diguanosine monophosphate (c-di-GMP) is a key bacterial secondary messenger that was first described as an allosteric activator of cellulose synthase in Gluconacetobacter xylinus (Ross et al. , 1987), but is now known to regulate a diverse range of important cellular functions, such as viru- lence, biofilm formation, cellular morphology and motility, in most eubacteria (Paul et al., 2004; Ro ¨ mling et al., 2005; Jenal & Malone, 2006). c-di-GMP is synthesized from two molecules of GTP by diguanylate cyclase (DGC) containing the GGDEF domain (Chan et al., 2004; Wassmann et al., 2007; De et al. , 2008) and is degraded by phosphodiesterase (PDE) containing the EAL domain (Christen et al., 2005; Schmidt et al., 2005) or the HD-GYP domain (Ryan et al., 2006). c-di-GMP has been found to bind to a plethora of effector proteins, including Clp (Leduc & Roberts, 2009; Chin et al., 2010; Tao et al. , 2010), PelD (Lee et al. , 2007), PleD (Chan et al., 2004; Wassmann et al., 2007), WspR (De et al. , 2008), FleQ (Hickman & Harwood, 2008) and PilZ domain-containing proteins (Benach et al.,