Browsing by Author "Chou, Shan-Ho"

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Structure and dynamics of polymyxin-resistance-associated response regulator PmrA in complex with promoter DNA
(2015-11-01)
Lou, Yuan-Chao
;
Weng, Tsai-Hsuan
;
Li, Yi-Chuan
;
Kao, Yi-Fen
;
Lin, Wei-Feng
PmrA, an OmpR/PhoB family response regulator, manages genes for antibiotic resistance. Phosphorylation of OmpR/PhoB response regulator induces the formation of a symmetric dimer in the N-terminal receiver domain (REC), promoting two C-terminal DNA-binding domains (DBDs) to recognize promoter DNA to elicit adaptive responses. Recently, determination of the KdpE-DNA complex structure revealed an REC-DBD interface in the upstream protomer that may be necessary for transcription activation. Here, we report the 3.2-angstrom-resolution crystal structure of the PmrA-DNA complex, which reveals a similar yet different REC-DBD interface. However, NMR studies show that in the DNA-bound state, two domains tumble separately and an REC-DBD interaction is transiently populated in solution. Reporter gene analyses of PmrA variants with altered interface residues suggest that the interface is not crucial for supporting gene expression. We propose that REC-DBD interdomain dynamics and the DBD-DBD interface help PmrA interact with RNA polymerase holoenzyme to activate downstream gene transcription.
The cAMP Receptor-Like Protein CLP Is a Novel c-di-GMP Receptor Linking Cell-Cell Signaling to Virulence Gene Expression in Xanthomonas campestris
(2010-02-26)
Chin, Ko-Hsin
;
Lee, Yen-Chung
;
Tu, Zhi-Le
;
Chen, Chih-Hua
;
Tseng, Yi-Hsiung
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. (C) 2009 Elsevier Ltd. All rights reserved.