Tuesday June 16 2009
CEA
.jpg)
The Role of Conformational Flexibility in Weak Protein-Protein Interactions: An NMR Study
CEA
Following the successful development of structural genomic initiatives dedicated to the determination of the three dimensional conformation of a large number of proteins, attention is now turning to the characterization of the multitude of interactions between these proteins that control cellular processes and biological function. This paradigm, the description of the molecular basis of the interactome, is expected to provide a comprehensive portrayal of the overall interaction structure of an organism's proteome and thereby represents one of the major challenges for structural biology in the coming decade. The Protein Dynamics and Flexibility group at the IBS/DSV, is developing novel techniques to study and understand the importance of molecular flexibility in protein-protein interactions at atomic resolution, even in the most challenging experimental systems.
Physiologically important weak interactions, with dissociation constants above 0.1 mM, are remarkably common, they are important for many cellular processes, such as transcription and replication, signal transduction, transient formation of encounter complexes and assembly of protein complexes. Despite their importance, they remain beyond the reach of most of structural biology, due to the experimental difficulty to isolate the complex. In a recent article in Nucleic Acids Research (1), a novel approach was developed, using NMR spectroscopy, and in particular residual dipolar couplings (RDCs), combined with specific isotopic labeling schemes, to provide crucial conformational constraints on intermolecular orientation in weak molecular complexes. This approach was applied to the determination of the conformational basis of the interaction between Ubiquitin and SH3, that plays an important role in molecular signaling.
In parallel the same group is developing methods to determine the nature and extent of protein motions on timescales ranging from the pico- to the milli-second. Until recently the exact role of protein dynamics in molecular interaction has remained obscure, due to the experimental difficulty in probing motions on functionally important timescales at atomic resolution. The Protein Dynamics and Flexibility group have published, in the review Angewandte Chemie International Edition (2), a novel approach, again based on NMR RDCs, that provides absolute and quantitative determination of protein dynamics in solution at each amino acid in the protein Ubiquitin. The results, combined with observations made in the study of the Ubiquitin/SH3 complex (1) imply an important role for conformational selection as a driving force for protein-protein interactions: While most of the protein is found to be rigid on slower timescales (ns-ms), surface loops exhibit intrinsic concerted motions in regions implicated in the interaction with SH3, suggesting that appropriate structural states are selected from a broader intrinsic conformational distribution, facilitating molecular interaction with the partner protein.
These combined analyses provide unprecedented insight into the conformational basis of protein flexibility and will have important consequences for our understanding of the mechanisms governing molecular recognition and function.
Determination of the conformational basis of weakly interacting protein complexes (1) .jpg)
Quantitative analysis of RDCs identifies local slow correlated motions involved in interaction (2)
__________
Reference
1 - Accurate Characterization of Weak Macromolecular Interactions by Titration of NMR Residual Dipolar Couplings: Application to the CD2AP SH3-C:Ubiquitin complex. J.L. Ortega-Roldan, M. Ringkjøbing Jensen*, B. Brutscher, A. I. Azuaga, M. Blackledge* and N. A.J. van Nuland.
2 - Protein Conformational Flexibility from Structure-Free Analysis of NMR Dipolar Couplings: Quantitative and Absolute Determination of Backbone Motion in Ubiquitin. L. Salmon, G. Bouvignies, P. Markwick, N. Lakomek, S. Showalter, D.W. Li, K. Walter, C. Griesinger, R. Brüschweiler and M. Blackledge* Angewandte Chemie International Edition. 48, 4154-4157 (2009).
__________
Notes
- [1] Interactions faibles : Elles sont caractérisées par des constantes de dissociation au-dessus de 0,1 mM.
- [2] SH3 = domaine protéique retrouvé dans de nombreuses protéines, qui joue un rôle important dans la signalisation moléculaire.
- [3] Marquages isotopiques différentiels : dans une protéine les atomes de C14 sont remplacés par du C13, dans l’autre c’est le N12 qui est remplacé par le N15, ce qui permet de différencier les deux protéines)
- [4] Conformation : structure d’une protéine en 3 dimensions
__________
To find out more about this research : http://www.ibs.fr/spip.php?article792
Read-around resources : Protein dynamics from disorder - Nature - Vol 459|25 June 2009