Protein conformational change and nucleotide binding involved in regulation of sigmaF in Bacillus subtilis.
We have studied the ability of three mutant forms of SpoIIAA, containing amino acid substitutions at the site of phosphorylation (serine 58), to interact with SpoIIAB. Native gel analysis revealed that SpoIIAAS58A could form a complex with SpoIIAB in the presence of ADP and more strongly in the presence of ATP. SpoIIAAS58N did not form a complex with SpoIIAB in the presence of ADP but displayed some interaction with SpoIIAB in the presence of ATP. SpoIIAAS58D was unable to form a complex with SpoIIAB in the presence of either ADP or ATP. Corresponding differences were found in the behavior of the three mutant proteins when studied by gel permeation with high-performance liquid chromatography and limited proteolysis. SpoIIAAS58A behaved like the wild-type SpoIIAA, SpoIIAAS58D like SpoIIAA-P, and SpoIIAAS58N in a way that was intermediate between the behaviors of SpoIIAA and SpoIIAA-P. Limited proteolysis was also used to show that on binding of ADP or ATP SpoIIAB undergoes a shift in conformation. The affinity of SpoIIAB for ADP and ATP was determined by limited proteolysis in the presence of a wide range of nucleotide concentrations. The results indicated that SpoIIAB has approximately equal affinity for ADP and for ATP.
Lord
M
M
Microbiology Unit, Department of Biochemistry, University of Oxford, United Kingdom.
Magnin
T
T
Yudkin
M D
MD
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D012808:Sigma Factor / Q000378:metabolism*; D014157:Transcription Factors
D001426:Bacterial Proteins; C064971:FliA protein, Bacteria; D012808:Sigma Factor; D014157:Transcription Factors; C092468:spoIIR protein, Bacillus subtilis; C030069:spore-specific proteins, Bacillus; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate