Molecular recognition dynamics

Molecular recognition dynamics

While solution NMR is now established as the method of choice for studying local dynamics on picosecond to nanosecond timescales, dynamics occurring on slower time-scales are also of particular interest because many biologically important processes, such as enzymatic catalysis, signal transduction, ligand binding and allosteric regulation are expected to occur in this range. In our group we use residual dipolar couplings, combined with advanced analytical and simulation methods, to develop a precise model of slower motions occurring in proteins.

We have thus developed approaches to study functionally important slower intrinsic motions in proteins directly from experimental data. However until now no molecular simulation methods have been available to aid in the interpretation of these data due to the very long time scale motions involved in the averaging mechanism. This situation has potentially dramatically changed with the development of accelerated molecular dynamics (AMD) approaches that provide enhanced access to rare conformational events. This opens up remarkable perspectives that we will continue to explore, to enhance our understanding of slower motional modes in proteins. We have applied enhanced sampling techniques for the first time to the identification of slow molecular motions detected using RDCs (Figure 1).

Figure 1. Residual dipolar couplings, combined with analytical determination of peptide plane orientation and motional amplitudes (3D-GAF) or independently with accelerated molecular dynamics (AMD) simulation, provide unique insight into the nature of nanosecond to microsecond dynamics occurring in the SH3C domain of the protein CD2AP.

More recently, we have developed an approach termed SUPERNOVA (sub-state populations on potential-energy surfaces using restraints from NMR spectroscopy and conformational oversampling) to map the free-energy landscape occupied by folded proteins in solution, determining populations of accessible conformational sub-states contributing to the dynamic equilibrium. The method initially exploits multi-level AMD simulation, flooding the conformational space available to the protein as completely as possible to sample different sub-states, which are combined to provide an extensive pool of conformers, comprising both high-and low-energy conformations (Figure 2). Boltzmann-weighted ensembles are then assembled by comparison with experimental NMR data. Ensemble selection is achieved using model-free interpretation of residual dipolar couplings combined with a specifically designed genetic algorithm (ASTEROIDS).

Figure 2. Illustration of the SUPERNOVA approach that employs a combination of over-sampling of conformational space using AMD and ensemble selections using a genetic algorithm (ASTEROIDS).

Almost all cellular mechanisms are controlled by protein-protein interactions, and the dynamic and kinetic properties of these interactions determine their function. Weak or transient protein-protein interactions, with equilibrium dissociation constants approaching millimolar ranges, are known to control numerous biological processes. NMR is an essential tool for the study of molecular complexes, due to its extraordinary sensitivity to interactions whose affinities vary over many orders of magnitude. Although NMR can in theory be used to study ultra-weak complexes, they remain the least well characterized in terms of molecular structure and dynamics becasue the weakness of the interaction precludes the measurement of parameters that originate uniquely from the bound form of either protein when working at experimentally accessible concentrations.
We have recently presented a titration approach for determining residual dipolar couplings from experimentally inaccessible complexes and have recently extended this approach to the measurement of 15N spin relaxation rates, demonstrating that this can provide long-range structural, dynamic and kinetic information about these elusive complexes (Figure 3).

Figure 3. Structure, dynamics and kinetics of weak protein-protein interactions by measurements of 15N spin relaxation rates in different titration mixtures.

Related publications:

P. Guerry, L. Salmon, L. Mollica, J.L. Ortega Roldan, P. Markwick, N. A. van Nuland, J. A. McCammon and M. Blackledge
Mapping the population of protein conformational energy sub-states from NMR dipolar couplings.
Angew. Chem. 52, 3181-3185 (2013)

L. Salmon, L. Pierce, A. Grimm, J. L. Ortega Roldan, L. Mollica, M.R. Jensen, N. van Nuland, P. R. Markwick, J. A. McCammon and M. Blackledge
Multi-timescale conformational dynamics of the SH3 domain of CD2-associated protein using NMR spectroscopy and accelerated molecular dynamics.
Angew. Chem. 51, 6103-6106 (2012)

L. Salmon, G. Bouvignies, P. Markwick and M. Blackledge
Nuclear magnetic resonance provides a quantitative description of protein conformational flexibility on physiologically important time scales.
Biochemistry 50, 2735-2747 (2011)

L. Salmon, J. L. Ortega Roldan, E. Lescop, A. Licinio, N. van Nuland, M.R. Jensen and M. Blackledge
Structure, dynamics, and kinetics of weak protein-protein complexes from NMR spin relaxation measurements of titrated solutions.
Angew. Chem. 50, 3755-3759 (2011).

P. R. Markwick, G. Bouvignies, L. Salmon, J. A. McCammon, M. Nilges AND M. Blackledge
Toward a unified representation of protein structural dynamics in solution.
J. Am. Chem. Soc. 131, 16968-16975 (2009)

L. Salmon, G. Bouvignies, P. Markwick, N. Lakomek, S. Showalter, D. W. Li , K. Walter, C. Griesinger, R. Brüschweiler and M. Blackledge
Protein conformational flexibility from structure-free analysis of NMR dipolar couplings: quantitative and absolute determination of backbone motion in ubiquitin.
Angew. Chem. 48, 4154-4157 (2009)

P. Markwick, G. Bouvignies and M. Blackledge
Exploring multiple timescale motions in protein GB3 using accelerated molecular dynamics and NMR.
J. Am. Chem. Soc. 129, 4724-4730 (2007)

G. Bouvignies, R.Bruschweiler and M. Blackledge.
Simultaneous Determination of Protein Structure and Dynamics using Residual Dipolar Couplings.
J. Am. Chem. Soc. 128, 15100-15101 (2006).

G. Bouvignies, P. Bernado, S. Meier, K. Cho, S. Grzesiek, R. Bruschweiler and M. Blackledge
Identification of slow correlated motions in proteins using residual dipolar and hydrogen-bond scalar couplings.
Proc. Natl. Acad. Sci. 102, 13885-13890 (2005)