Genetic and biochemical analysis of the interaction of Bacillus subtilis CodY with branched-chain amino acids.
Bacillus subtilis CodY protein is a DNA-binding global transcriptional regulator that responds to branched-chain amino acids (isoleucine, leucine, and valine) and GTP. Crystal structure studies have shown that the N-terminal region of the protein includes a GAF domain that contains a hydrophobic pocket within which isoleucine and valine bind. This region is well conserved in CodY homologs. Site-directed mutagenesis was employed to understand the roles of some of the residues in the GAF domain and hydrophobic pocket in interaction with isoleucine and GTP. The F40A, F71E, and F98A forms of CodY were inactive in vivo. They were activatable by GTP but to a much lesser extent by branched-chain amino acids in vitro. The CodY mutant R61A retained partial repression of target promoters in vivo and was able to respond to GTP in vitro but also responded poorly to branched-chain amino acids in vitro unless GTP was simultaneously present. Thus, the GAF domain includes residues essential for full activation of CodY by branched-chain amino acids, but these residues are not critical for activation by GTP. Binding studies with branched-chain amino acids and their analogs revealed that an amino group at position 2 and a methyl group at position 3 of valine are critical components of the recognition of the amino acids by CodY.
Villapakkam
Anuradha C
AC
Graduate Program in Molecular Microbiology, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA 02111, USA.
Handke
Luke D
LD
Belitsky
Boris R
BR
Levdikov
Vladimir M
VM
Wilkinson
Anthony J
AJ
Sonenshein
Abraham L
AL
D000597:Amino Acids, Branched-Chain / Q000737:chemistry / Q000378:metabolism*; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D018983:DNA Footprinting; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D007532:Isoleucine / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D017433:Protein Structure, Secondary
D000597:Amino Acids, Branched-Chain; D001426:Bacterial Proteins; D007532:Isoleucine; D006160:Guanosine Triphosphate