Accurate characterization of weak macromolecular interactions by titration of NMR residual dipolar couplings: application to the CD2AP SH3-C:ubiquitin complex.

TitleAccurate characterization of weak macromolecular interactions by titration of NMR residual dipolar couplings: application to the CD2AP SH3-C:ubiquitin complex.
Publication TypeJournal Article
Year of Publication2009
AuthorsOrtega-Roldan, J. Luis, Ringkjøbing Jensen M., Brutscher B., Azuaga A. I., Blackledge M., and van Nuland N. A. J.
JournalNucleic Acids Res
Volume37
Issue9
Paginatione70
Date Published2009 May
ISSN1362-4962
KeywordsAdaptor Proteins, Signal Transducing, Cytoskeletal Proteins, Models, Molecular, Nuclear Magnetic Resonance, Biomolecular, src Homology Domains, Titrimetry, Ubiquitin
Abstract

The description of the interactome represents one of key challenges remaining for structural biology. Physiologically important weak interactions, with dissociation constants above 100 muM, are remarkably common, but remain beyond the reach of most of structural biology. NMR spectroscopy, and in particular, residual dipolar couplings (RDCs) provide crucial conformational constraints on intermolecular orientation in molecular complexes, but the combination of free and bound contributions to the measured RDC seriously complicates their exploitation for weakly interacting partners. We develop a robust approach for the determination of weak complexes based on: (i) differential isotopic labeling of the partner proteins facilitating RDC measurement in both partners; (ii) measurement of RDC changes upon titration into different equilibrium mixtures of partially aligned free and complex forms of the proteins; (iii) novel analytical approaches to determine the effective alignment in all equilibrium mixtures; and (iv) extraction of precise RDCs for bound forms of both partner proteins. The approach is demonstrated for the determination of the three-dimensional structure of the weakly interacting CD2AP SH3-C:Ubiquitin complex (K(d) = 132 +/- 13 muM) and is shown, using cross-validation, to be highly precise. We expect this methodology to extend the remarkable and unique ability of NMR to study weak protein-protein complexes.

DOI10.1093/nar/gkp211
Alternate JournalNucleic Acids Res.
PubMed ID19359362
PubMed Central IDPMC2685109
Research group: