A Surface potential of the CcpB dimer from R. cellulolyticum (RcCcpB without the DNA binding domain) and CcpA from B. megaterium (BmCcpA). The RcCcpB dimer was superimposed to the BmCcpA dimer of BmCcpA-HprSer46P complex and the structure of Hpr and Cph was shown in a cyan cartoon representation, with the Ser46P shown in sticks. The BmCcpA residues responsible for interacting with HprSer46P and the equivalent residues in RcCcpB were shown in sticks and labeled. B Multiple sequence alignment showing residues involved in conformational switching are different between BmCcpA and R. cellulolyticum CcpA homologs (RcCcpA and RcCcpB). The sequence logo of the conserved segment (294–310) in CcpA homologs (n = 8651) depicts the usage frequency of amino acids, in reference to the relative position in BmCcpA. C Hydrogen bonding network essential for CcpA conformational switching in B. megaterium apo-CcpA (white), CcpA-HprSer46P complex (yellow) and R. cellulolyticum apo-CcpB (magenta). The residue labels for BmCcpA were shown in parentheses. Effect of site-specific mutations in RcCcpA (D) and RcCcpB (E) on DNA binding (n = 2–3 EMSA gels). The significance was determined by ANOVA. F Distribution of 8651 CcpA orthologs predicted in 14,998 bacterial genomes. The classic HPr-dependent CcpA orthologs (with R/H/K at 303 and T at 306) and RcCcpA/RcCcpB-like orthologs (with Q/N at 303 and V/L at 306) were colored in blue and red, respectively.