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

Ortega-Roldan, Jose L., Jensen, Malene Ringkjøbing, Brutscher, Bernhard, Azuaga, Ana I., Blackledge, Martin, van Nuland, Nico A. J. (2009) Accurate characterization of weak macromolecular interactions by titration of NMR residual dipolar couplings: application to the CD2AP SH3-C:ubiquitin complex. Nucleic Acids Research, 37 (9). e70. ISSN 0305-1048. E-ISSN 1362-4962. (doi:10.1093/nar/gkp211)

Abstract

The description of the interactome represents one of key challenges remaining for structural biology. Physiologically important weak interactions, with dissociation constants above 100??M, 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 (Kd?=?132?±?13??M) 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.

Item Type: Article
DOI/Identification number: 10.1093/nar/gkp211
Uncontrolled keywords: ubiquitin titration method complex
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Jose Ortega-Roldan
Date Deposited: 31 Jul 2017 15:17 UTC
Last Modified: 29 May 2019 19:16 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/62505 (The current URI for this page, for reference purposes)
Ortega-Roldan, Jose L.: https://orcid.org/0000-0002-6316-4390
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