Nucleic Acids Research Advance Access published online on April 9, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp211
Methods Online |
Accurate characterization of weak macromolecular interactions by titration of NMR residual dipolar couplings: application to the CD2AP SH3-C:ubiquitin complex
1Departamento de Química Física e Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, Fuentenueva s/n, 18071 Granada, Spain, 2Protein Dynamics and Flexibility by NMR, Institut de Biologie Structurale Jean-Pierre Ebel, CEA; CNRS; UJF UMR 5075, 41 Rue Jules Horowitz, Grenoble 38027, France, 3Laboratoire de RMN, Institut de Biologie Structurale Jean-Pierre Ebel, CEA; CNRS; UJF UMR 5075, 41 Rue Jules Horowitz, Grenoble 38027, France and 4Structural Biology Brussels, VIB Department of Molecular and Cellular Interactions, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel, Belgium
*To whom correspondence should be addressed. Tel: +33 438789554; Fax: +33 438785494; Email: martin.blackledge{at}ibs.fr
Correspondence may also be addressed to Malene Ringkjøbing Jensen. Email: malene.ringkjobing-jensen{at}ibs.fr
Received February 10, 2009. Revised March 13, 2009. Accepted March 16, 2009.
The description of the interactome represents one of key challenges remaining for structural biology. Physiologically important weak interactions, with dissociation constants above 100 µM, 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 µM) 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.