Molecular basis for P-site inhibition of adenylyl cyclase

P-site inhibitors are adenosine and adenine nucleotide analogues that inhib it adenylyl cyclase, the effector enzyme that catalyzes the synthesis of cy clic AMP from ATP. Some of these inhibitors may represent physiological reg ulators of adenylyl cyclase, and the most potent may ultimately serve as us eful therapeutic agents. Described here are crystal structures of the catal ytic core of adenylyl cyclase complexed with two such P-site inhibitors, 2' -deoxyadenosine 3'-monophosphate (2'-d-3'-AMP) and 2',5'-dideoxyadenosine 3 '-triphosphate (2',5'-dd-3'-ATP). Both inhibitors bind in the active site y et exhibit non- or uncompetitive patterns of inhibition. While most P-site inhibitors require pyrophosphate (PPi) as a coinhibitor, 2',5'-dd-3'-ATP is a potent inhibitor by itself. The crystal structure reveals that this inhi bitor exhibits two binding modes: one with the nucleoside moiety bound to t he nucleoside binding pocket of the enzyme and the other with the beta and gamma phosphates bound to the pyrophosphate site of the 2'-d-3'-AMP PPi com plex. A single metal binding site is observed in the complex with 2'-d-3'-A MP, whereas two are observed in the complex with 2',5'-dd-3'-ATP. Even thou gh P-site inhibitors are typically 10 times more potent in the presence of Mn2+, the electron density maps reveal no inherent preference of either met al site for Mn2+ over Mg2+. 2',5'-dd-3'-ATP binds to the catalytic core of adenylyl cyclase with a K-d Of 2.4 muM in the presence of Mg2+ and 0.2 muM in the presence of Mn2+. Pyrophosphate does not compete with 2',5'-dd-3'-AT P and enhances inhibition.