Analogs of cyclic AMP that elicit the biochemically defined conformational change in catabolite gene activator protein (CAP) but do not stimulate binding to DNA

Abstract

We have measured the effects on catabolite gene activator protein (CAP) of 22 synthetic analogs of cAMP. Each analog was assayed to test three parameters: (1) binding to CAP; (2) induction of the conformational change in CAP; and (3) activation of transcription. Thus we have identified seven cAMP analogs that bind to CAP as well or better than does cAMP, cause the assayed conformational change in CAP, yet exhibit no ability to activate transcription. We designate these analogs class D. The conformational change elicited in CAP by the class D analogs was further investigated by: (1) sensitivity to the proteolytic enzymes chymotrypsin, Staphylococcus aureus V8 protease, subtilisin and trypsin; (2) formation of inter-subunit covalent crosslinks by 5,5'-dithiobis(2-nitrobenzoic acid); and (3) degree of labeling of cysteine by [3H]N-ethylmaleimide. These experiments failed to detect a conformational difference between the CAP-class D and CAP-cAMP complexes. Filter binding and nuclease protection experiments indicate that the class D analogs do not efficiently support the binding of CAP to DNA. From these results, we suggest that there exists a hitherto undetected event dependent on cAMP, and required for CAP to bind to DNA. We suggest that this event involves a change that takes place in proximity to the N6 atom of cAMP. Three possible interpretations are discussed.