Identification of new residues involved in intramolecular signal transmission in a prokaryotic transcriptional repressor.
TtgV is a member of the IclR family of transcriptional regulators. This regulator controls its own expression and that of the ttgGHI operon, which encodes an RND efflux pump. TtgV has two domains: a GAF-like domain harboring the effector-binding pocket and a helix-turn-helix (HTH) DNA-binding domain, which are linked by a long extended helix. When TtgV is bound to DNA, a kink at residue 86 in the extended helix gives rise to 2 helices. TtgV contacts DNA mainly through a canonical recognition helix, but its three-dimensional structure bound to DNA revealed that two residues, R19 and S35, outside the HTH motif, directly contact DNA. Effector binding to TtgV releases it from DNA; when this occurs, the kink at Q86 is lost and residues R19 and S35 are displaced due to the reorganization of the turn involving residues G44 and P46. Mutants of TtgV were generated at positions 19, 35, 44, 46, and 86 by site-directed mutagenesis to further analyze their role. Mutant proteins were purified to homogeneity, and differential scanning calorimetry (DSC) studies revealed that all mutants, except the Q86N mutant, unfold in a single event, suggesting conservation of the three-dimensional organization. All mutant variants bound effectors with an affinity similar to that of the parental protein. R19A, S35A, G44A, Q86N, and Q86E mutants did not bind DNA. The Q86A mutant was able to bind to DNA but was only partially released from its target operator in response to effectors. These results are discussed in the context of intramolecular signal transmission from the effector binding pocket to the DNA binding domain.
Department of Environmental Protection, Consejo Superior de Investigaciones Científicas, Granada, Spain.
Carlos
C
Molina-Santiago
Abdelali
A
Daddaoua
Sandy
S
Fillet
Tino
T
Krell
Bertrand
B
Morel
Estrella
E
Duque
Juan L
JL
Ramos
D051046:Amino-Acid N-Acetyltransferase; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D055550:Protein Stability; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D051046:Amino-Acid N-Acetyltransferase