Molecular Basis for P-Site Inhibition of Adenylyl Cyclase^,

Abstract

P-site inhibitors are adenosine and adenine nucleotide analogues that inhibit adenylyl cyclase, the effector enzyme that catalyzes the synthesis of cyclic AMP from ATP. Some of these inhibitors may represent physiological regulators of adenylyl cyclase, and the most potent may ultimately serve as useful therapeutic agents. Described here are crystal structures of the catalytic 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 yet exhibit non- or uncompetitive patterns of inhibition. While most P-site inhibitors require pyrophosphate (PP(i)) as a coinhibitor, 2',5'-dd-3'-ATP is a potent inhibitor by itself. The crystal structure reveals that this inhibitor exhibits two binding modes: one with the nucleoside moiety bound to the 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.PP(i) complex. A single metal binding site is observed in the complex with 2'-d-3'-AMP, whereas two are observed in the complex with 2', 5'-dd-3'-ATP. Even though P-site inhibitors are typically 10 times more potent in the presence of Mn(2+), the electron density maps reveal no inherent preference of either metal site for Mn(2+) over Mg(2+). 2',5'-dd-3'-ATP binds to the catalytic core of adenylyl cyclase with a K(d) of 2.4 microM in the presence of Mg(2+) and 0.2 microM in the presence of Mn(2+). Pyrophosphate does not compete with 2',5'-dd-3'-ATP and enhances inhibition.

Differential Inhibition of Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by Purine and Pyrimidine Nucleotides

“…2). However, the MANT group at the 2Ј-O-position did not confer high potency to compounds 21 and 22 because a polyphosphate chain is important for high inhibitor potency as well (25,27). MANT-ATP and MANT-ADP (compare compounds 6 and 8) were similarly potent C1⅐C2 inhibitors, whereas MANT-GTP was ϳ70-fold more potent than MANT-GDP (compare compounds 1 and 10).…”

“…However, ACT was the only cyclase studied that exhibited a higher V max in the presence of Mg 2ϩ than in the presence of Mn 2ϩ . In addition, the available literature evidence suggested that inhibitor sensitivity of cyclases in general is higher in the presence of Mn 2ϩ than in the presence of Mg 2ϩ (12,23,27,30,37,41). Therefore, we conducted the systematic analysis of the structure/activity relationships of inhibitors in the presence of Mn 2ϩ .…”

“…These compounds are also referred to as P-site inhibitors. P-site inhibitors are noncompetitive or uncompetitive AC inhibitors that bind to the AC-PP i conformation (26,27). The most potent P-site inhibitors presently available are 2Ј,5Ј-dd-3Ј-ATP (IC 50 for rat brain AC, 40 nM) and 2Ј,5Ј-dd-3Ј-A 4 P (IC 50 for rat brain AC, 7.4 nM) (24,25).…”

Identification of Guanylate Cyclases and Related Signaling Proteins in Sperm Tail from Sea Stars by Mass Spectrometry

“…Modeling was performed with SWISS-MODEL (Kopp and Schwede 2006) and Phyre (Kelley et al 2000). The extracellular, kinase-like, and guanylate cyclase domains were modeled using a hormone-bound atrial natriuretic peptide (ANP) receptor (NPR)-A extracellular domain (Protein Data Bank ID code 1t34; Ogawa et al 2004), Raf proto-oncogene serine/threonine-protein kinase (1uwj; Wan et al 2004), and type II adenylyl cyclase (1cul; Tesmer et al 2000) as their templates, respectively. The root mean square of distances between the template (real structure) and target (model structure) were 1.4, 0.5, and 1.3 Å (10 −10 m) for extracellular, kinase-like, and guanylate cyclase domains, respectively.…”

Ligand-gated Ion Channels

“…Adenosine and its derivatives (e.g. 2′,5′-dideoxyadenosine), acting through the P-site, appear to be physiological inhibitors of adenylyl cyclase activity (Tesmer et al, 2000). (Tang et al, 1991), PKC-evoked phosphorylation (Jacobowitz et al, 1993) Gbg (Taussig et al, 1993), PKC-evoked phosphorylation (Chen and Iyengar, 1993;Lustig et al, 1993) Ca 2+ /CaM (Choi et al, 1992), PKC-evoked phosphorylation (Jacobowitz et al, 1993) Gbg (Gao and Gilman, 1991) PKC-evoked phosphorylation (Kawabe et al, 1994) Endogenous inhibitors…”