Characterization and crystallization of a minimal catalytic core domain from mammalian type II adenylyl cyclase

Abstract

Adenylyl cyclases play a pivotal role in signal transduction by carrying out the regulated synthesis of cyclic AMP. The nine cloned mammalian adenylyl cyclases all share two conserved regions of sequence, C₁ and C₂, which are homologous to each other and are together responsible for catalytic activity. Recombinant C₁ and C₂ domains catalyze the synthesis of cyclic AMP when they are mixed and activated by forskolin, and C₂ domains alone also manifest reduced levels of forskolin-stimulated enzyme activity. Using limited proteolysis and mass spectrometry, we have mapped the boundaries of a minimal stable and active C₂ catalytic domain to residues 871–1090 of type II adenylyl cyclase. We report the properties and crystallization of this trimmed domain, termed IIC₂-Δ4. Crystals belong to space group P4_n2₁2, where n = 1 or 3; a = b = 81.3, and c = 180.5 Å; and there are two molecules per asymmetric unit related by an approximate body centering operation. Flash-frozen crystals diffract anisotropically to 2.2 Å along the c^* direction and to 2.8 Å along the a^* and b^* directions using synchrotron radiation.