Identification of the protein kinase A regulatory RIα-catalytic subunit interface by amide H/2H exchange and protein docking
- Ganesh S. Anand*,†,‡,
- Dennis Law‡,§,
- Jeffrey G. Mandell†,¶,
- Aaron N. Snead†,
- Igor Tsigelny§,
- Susan S. Taylor*,†,
- Lynn F. Ten Eyck†,§, and
- Elizabeth A. Komives†,∥
- *Howard Hughes Medical Institute and †Department of Chemistry and Biochemistry, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0378; and §San Diego Supercomputer Center, P.O. Box 85608, San Diego, CA 92186-5608
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Edited by John Kuriyan, University of California, Berkeley, CA (received for review April 16, 2003)
Abstract
An important goal after structural genomics is to build up the structures of higher-order protein–protein complexes from structures of the individual subunits. Often structures of higher order complexes are difficult to obtain by crystallography. We have used an alternative approach in which the structures of the individual catalytic (C) subunit and RIα regulatory (R) subunit of PKA were first subjected to computational docking, and the top 100,000 solutions were subsequently filtered based on amide hydrogen/deuterium (H/2H) exchange interface protection data. The resulting set of filtered solutions forms an ensemble of structures in which, besides the inhibitor peptide binding site, a flat interface between the C-terminal lobe of the C-subunit and the A- and B-helices of RIα is uniquely identified. This holoenzyme structure satisfies all previous experimental data on the complex and allows prediction of new contacts between the two subunits.
Footnotes
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↵ ∥ To whom correspondence should be addressed. E-mail: ekomives{at}ucsd.edu.
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↵ ‡ G.S.A. and D.L. contributed equally to this work.
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↵ ¶ Present address: Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215.
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This paper was submitted directly (Track II) to the PNAS office.
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Abbreviations: MALDI-TOF, matrix-assisted laser desorption ionization/time-of-flight; R, regulatory; C, catalytic; TFA, trifluoroacetic acid.
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Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.rcsb.org (PDB ID code 1PVK).
- Copyright © 2003, The National Academy of Sciences
