Structural basis for the inhibition of mammalian membrane adenylyl cyclase by 2′(3′)-O-(N-methylanthraniloyl)-guanosine 5′-triphosphate

Membrane-bound mammalian adenylyl cyclase (mAC) catalyzes the synthesis of intracellular cyclic AMP from ATP and is activated by stimulatory G protein α subunits (Gα_s) and by forskolin (FSK). mACs are inhibited with high potency by 2′(3′)-O-(N-methylanthraniloyl) (MANT)-substituted nucleotides. In this study, the crystal structures of the complex between Gα_s·GTPγS and the catalytic C1 and C2 domains from type V and type II mAC (VC1·IIC2), bound to FSK and either MANT-GTP·Mg²⁺ or MANT-GTP·Mn²⁺ have been determined. MANT-GTP coordinates two metal ions and occupies the same position in the catalytic site as P-site inhibitors and substrate analogs. However, the orientation of the guanine ring is reversed relative to that of the adenine ring. The MANT fluorophore resides in a hydrophobic pocket at the interface between the VC1 and IIC2 domains and prevents mAC from undergoing the "open" to "closed" domain rearrangement. The K_i of MANT-GTP for inhibition of VC1-IIC2 is lower in the presence of mAC activators and lower in the presence of Mn²⁺ compared with Mg ²⁺, indicating that the inhibitor binds more tightly to the catalytically most active form of the enzyme. Fluorescence resonance energy transfer-stimulated emission from the MANT fluorophore upon excitation of Trp-1020 in the MANT-binding pocket of IIC2 is also stronger in the presence of FSK. Mutational analysis of two non-conserved amino acids in the MANT-binding pocket suggests that residues outside of the binding site influence isoform selectivity toward MANT-GTP.