Crystal structure of a cAMP-independent form of catabolite gene activator protein with adenosine substituted in one of two cAMP-binding sites.
Catabolite gene activator protein (CAP) in the presence of cAMP stimulates transcription from several operons in Escherichia coli. A cAMP-independent variant, in which Ala-144 is replaced by Thr (CAP91), is activated by analogues of cAMP, such as adenosine, which do not activate the wild-type CAP. In order to test the effect of adenosine on the structure, a crystal of CAP91 grown as a complex with cAMP was soaked in a solution of 10 mM adenosine, and X-ray diffraction data were measured to 3.5-A resolution. The difference Fourier map calculated with phases from the CAP91 structure showed significant negative density at the position of the phosphate of cAMP bound in one subunit of the CAP91 dimer. Adenosine was preferentially substituted for cAMP in the subunit in the "closed" conformation, while the cAMP-binding site of the "open" subunit was apparently still occupied by cAMP. The structure was refined by restrained least-squares methods to an R factor of 20.2%. Adenosine is not bound in exactly the same position as cAMP; instead, the 5'-OH of adenosine is in a new position that allows formation of two hydrogen bonds with Ser-83, replacing two of the three interactions of the phosphate of cAMP with Arg-82 and Ser-83.
Vaney
M C
MC
NCI-Frederick Cancer Research Facility, BRI-Basic Research Program, Maryland 21701.
Gilliland
G L
GL
Harman
J G
JG
Peterkofsky
A
A
Weber
I T
IT
D000241:Adenosine / Q000032:analysis* / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D000242:Cyclic AMP / Q000032:analysis* / Q000494:pharmacology; D002373:Cyclic AMP Receptor Protein / Q000235:genetics; D004563:Electrochemistry; D004926:Escherichia coli / Q000235:genetics*; D015394:Molecular Structure; D010710:Phosphates / Q000032:analysis; D011953:Receptors, Cyclic AMP / Q000032:analysis / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects; D014961:X-Ray Diffraction
D000596:Amino Acids; D002373:Cyclic AMP Receptor Protein; D010710:Phosphates; D011953:Receptors, Cyclic AMP; D000242:Cyclic AMP; D000241:Adenosine