The cAMP receptor-like protein CLP is a novel c-di-GMP receptor linking cell-cell signaling to virulence gene expression in Xanthomonas campestris.
Cyclic-di-GMP [bis-(3'-5')-cyclic diguanosine monophosphate] controls a wide range of functions in eubacteria, yet little is known about the underlying regulatory mechanisms. In the plant pathogen Xanthomonas campestris, expression of a subset of virulence genes is regulated by c-di-GMP and also by the CAP (catabolite activation protein)-like protein XcCLP, a global regulator in the CRP/FNR superfamily. Here, we report structural and functional insights into the interplay between XcCLP and c-di-GMP in regulation of gene expression. XcCLP bound target promoter DNA with submicromolar affinity in the absence of any ligand. This DNA-binding capability was abrogated by c-di-GMP, which bound to XcCLP with micromolar affinity. The crystal structure of XcCLP showed that the protein adopted an intrinsically active conformation for DNA binding. Alteration of residues of XcCLP implicated in c-di-GMP binding through modeling studies caused a substantial reduction in binding affinity for the nucleotide and rendered DNA binding by these variant proteins insensitive to inhibition by c-di-GMP. Together, these findings reveal the structural mechanism behind a novel class of c-di-GMP effector proteins in the CRP/FNR superfamily and indicate that XcCLP regulates bacterial virulence gene expression in a manner negatively controlled by the c-di-GMP concentrations.
Chin
Ko-Hsin
KH
Institute of Biochemistry, National Chung-Hsing University, Taichung 40227, Taiwan, ROC.
Lee
Yen-Chung
YC
Tu
Zhi-Le
ZL
Chen
Chih-Hua
CH
Tseng
Yi-Hsiung
YH
Yang
Jinn-Moon
JM
Ryan
Robert P
RP
McCarthy
Yvonne
Y
Dow
J Maxwell
JM
Wang
Andrew H-J
AH
Chou
Shan-Ho
SH
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D015398:Signal Transduction; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D014774:Virulence; D016959:Xanthomonas campestris / Q000472:pathogenicity / Q000502:physiology
D001426:Bacterial Proteins; C436751:Clp protein, Xanthomonas campestris; D004269:DNA, Bacterial; D050505:Mutant Proteins; D014157:Transcription Factors; C062025:bis(3',5')-cyclic diguanylic acid; D006152:Cyclic GMP