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College Park, Maryland      June 6 - 10 , 2004

WP5: SANS Reveals that Protein Kinase A RIa Dimer Undergoes a Large Conformational Change upon Binding C Subunits

W. T. Heller (Condensed Matter Sciences Division, Oak Ridge National Laboratory), D. Vigil, S. Brown (Department of Chemistry and Biochemistry and Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92037), D. K. Blumenthal (Departments of Pharmacology and Toxicology and Biochemistry, University of Utah, Salt Lake City, UT 84112), S. S. Taylor (Department of Chemistry and Biochemistry and Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92037), J. Trewhella (Bioscience Division Los Alamos National Laboratory)

Small-angle neutron scattering with contrast variation was used to study the type Ia isoform of the cAMP-dependent protein kinase A (PKA) holoenzyme in solution. PKA consists of two catalytic (C) subunits bound to a regulatory (R) subunit dimer. Information on the shapes and dispositions of the C subunits and R subunit homodimer within a holoenzyme reconstituted with deuterated R subunits was derived from the data. The study demonstrates for the first time that a large-scale conformational change occurs within the R subunit homodimer upon binding the C subunits. The results infer that inhibition of C subunit activity is a multi-step process involving local conformational changes both in the cAMP-binding domains and the linker region of the R subunit that impact the global structure of the R subunit dimer. The scattering data also show that, despite extensive sequence homology between the isoforms, the type Ia holoenzyme adopts a significantly more compact conformation than the type IIα isoform. A model of the type Ia holoenzyme was constructed to fit the SANS data using available structures of the component subunits and domains. In this model, the type Ia holoenzyme forms a flattened V shape with the R subunit dimerization domain at the point of the V and the R subunit cAMP-binding domains and the C subunits at the ends.

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