Structural Basis for the Inhibition of Mammalian Membrane Adenylyl Cyclase by 2 ′(3′)-O-(N-Methylanthraniloyl)-guanosine 5 ′-Triphosphate*

  1. Stephen R. Sprang**‡‡
  1. Department of Biochemistry, **Howard Hughes Medical Institute, The University of Texas Southwestern Medical, Dallas, Texas 75390-9050, the §Department of Pharmacology and Toxicology, The University of Kansas, Lawrence, Kansas 66045-7582, and the Center for Biomedical Invention, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9050
  1. ‡‡ To whom correspondence should be addressed: Howard Hughes Medical Institute, Dept. of Biochemistry, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9050. Tel.: 214-648-5008; Fax: 214-648-6336; E-mail: stephen.sprang{at}utsouthwestern.edu.

Abstract

Membrane-bound mammalian adenylyl cyclase (mAC) catalyzes the synthesis of intracellular cyclic AMP from ATP and is activated by stimulatory G protein α subunits (Gαs) 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 Gαs·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 Ki 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 Mg2+, 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.

  • Received August 9, 2004.
  • Revision received December 6, 2004.
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  1. The Journal of Biological Chemistry 280, 7253-7261.
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