The crystal structure of a soluble, catalytically active form of adenylyl cyclase in a complex with its stimulatory heterotrimeric G protein α subunit (G _{s α} ) and forskolin was determined to a resolution of 2.3 angstroms. When P-site inhibitors were soaked into native crystals of the complex, the active site of adenylyl cyclase was located and structural elements important for substrate recognition and catalysis were identified. On the basis of these and other structures, a molecular mechanism is proposed for the activation of adenylyl cyclase by G _{s α} .

The phosphorylation of heptahelical receptors by heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptor kinases (GRKs) is a universal regulatory mechanism that leads to desensitization of G protein signaling and to the activation of alternative signaling pathways.We determined the crystallographic structure of bovine GRK2 in complex with G protein β ₁ γ ₂ subunits.Our results show how the three domains of GRK2–the RGS (regulator of G protein signaling) homology, protein kinase, and pleckstrin homology domains–integrate their respective activities and recruit the enzyme to the cell membrane in an orientation that not only facilitates receptor phosphorylation, but also allows for the simultaneous inhibition of signaling by Gα and Gβγ subunits.

The crystal structure of G _{s α} , the heterotrimeric G protein α subunit that stimulates adenylyl cyclase, was determined at 2.5 Å in a complex with guanosine 5′- O -(3-thiotriphosphate) (GTPγS). G _{s α} is the prototypic member of a family of GTP-binding proteins that regulate the activities of effectors in a hormone-dependent manner. Comparison of the structure of G _{s α} ·GTPγS with that of G _{i α} ·GTPγS suggests that their effector specificity is primarily dictated by the shape of the binding surface formed by the switch II helix and the α3-β5 loop, despite the high sequence homology of these elements. In contrast, sequence divergence explains the inability of regulators of G protein signaling to stimulate the GTPase activity of G _{s α} . The βγ binding surface of G _{s α} is largely conserved in sequence and structure to that of G _{i α} , whereas differences in the surface formed by the carboxyl-terminal helix and the α4-β6 loop may mediate receptor specificity.

Adenylyl cyclase (AC) converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate, a ubiquitous second messenger that regulates many cellular functions. Recent structural studies have revealed much about the structure and function of mammalian AC but have not fully defined its active site or catalytic mechanism. Four crystal structures were determined of the catalytic domains of AC in complex with two different ATP analogs and various divalent metal ions. These structures provide a model for the enzyme-substrate complex and conclusively demonstrate that two metal ions bind in the active site. The similarity of the active site of AC to those of DNA polymerases suggests that the enzymes catalyze phosphoryl transfer by the same two-metal-ion mechanism and likely have evolved from a common ancestor.