W0059

Quaternary Structure of the cAMP-Dependent Protein Kinase by Neutron Scattering. Jinkui Zhao, Elaine Hoyer*, Sharon Boylen*, Donal A. Walsh*, Jill Trewhella, Los Alamos National Laboratory, Los Alamos NM 87545 and *University of California, Davis CA 95616

We will present the solution structure of the cAMP-dependent protein kinase (PKA). PKA has served as the central model for signal transduction and cellular regulation by phosphorylation since its discovery 30 years ago. Understanding its structure thus not only provides insights into PKA function, but also into cellular regulation by phosphorylation in general. PKA has two catalytic (C, 40 kDa) and two regulatory (R, 45 kDa) subunits. The R subunits each have an N-terminal dimerization domain and two cAMP binding sites. Binding of cAMP to R results in dissociation of the C subunits and their subsequent activation. Our neutron scattering data from reconstituted type II-[alpha] PKA holoenzyme with R deuterated show the shapes and dispositions of the R and C subunits. We have fit the crystal structures of C (Knighton et al. Science 253, 407, 1991) and a truncation mutant of R (Su et al. Science 269, 807, 1995) into the neutron determined molecular boundaries. The resultant model shows that the individual C subunits have the same overall shape as in the crystal structure. Their centers of mass are separated by ~110 Å and there is no surface contact between them. The R subunits associate to form a highly extended dumbbell shaped dimer. It appears that the dimerization domains of each R form the "bar," while the cAMP binding domains form the two lobes. These lobes are separated similarly to the C subunits (~89 Å separation of their centers of mass). Each lobe of the R dimer interacts with only one C subunit. The separation of the centers of mass of each R from its neighboring C is ~46 Å. Binding of cAMP to R must change the binding interface between the R and C subunits thus facilitating the release of C.