YfiBNR, a tripartite cyclic-di-GMP (c-di-GMP) signalling system, plays an important role in biofilm formation of the gram-negative bacterium P. aeruginosa, which regulates the cellular processes strongly associated with chronic lung infections and drug resistance. The outer-membrane lipoprotein YfiB can release the inhibition of the inner membrane protein YfiN by sequestering the periplasmatic protein YfiR, resulting in the activation of diguanylate cyclase activity of YfiN and the production of c-di-GMP. In contrast to the extensive studies on c-di-GMP, little is known about how GMP acts in the YfiBNR system. Here, we report the crystal structures of YfiR complexed with GMP and YfiB(L43P)-YfiR complexed with GMP. In the YfiR-GMP complex, GMP is located in a hydrophilic pocket formed by R175/H177/R60, while in the YfiB(L43P)-YfiR-GMP complex, GMP is located in a slightly separated hydrophilic pocket, with GMP forming hydrogen bonds with both YfiB and YfiR. A binding affinity test and biofilm formation assay indicated that GMP may activate diguanylate cyclase activity to promote biofilm production by promoting the affinity of YfiB and YfiR This finding thus provides a new perspective for preventing biofilm-related antibiotic resistance and chronic infections. (C) 2017 Elsevier Inc. All rights reserved.