Binding Mechanisms and Conformational Equilibria in Biomacromolecular Interactions

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Understanding how biological macromolecules recognize and bind to their cognate ligands is essential for the rational design of drugs, biocatalysts, and for explaining biology at the atomic level. However, developing rigorous, quantitative descriptions of these processes is extremely challenging for a number of reasons. Firstly, biological macromolecules such as proteins and DNA are inherently flexible and populate ensembles of different conformations at equilibrium. In the presence of a cognate ligand, the relative populations of the conformers can be dramatically redistributed. This coupling between ligand binding and conformational changes can potentially make large contributions to the affinities of biomacromolecular interactions and can mediate allosteric communication between distant binding sites. However, it remains difficult to characterize in detail their conformational heterogeneity and harder still to quantify the contributions of structural and dynamical changes to the thermodynamics of binding. Secondly, the kinetics of molecular recognition events depend upon weakly-populated intermediate configurations of binding partners. In many cases the nature of the kinetic bottleneck is not well understood. For example, the access of ligands to deeply buried binding sites in proteins often requires some degree of conformational rearrangement of the protein in order to permit passage. However the extent of the structural distortions and the associated energetic costs are not well understood. The aim of this proposal is to gain a deeper understanding of biomacromolecular recognition at the atomic level by developing new experimental tools for characterizing binding events and applying them to model systems that exemplify the complexity of the phenomenon.Our experimental approach is based on combining high-field solution nuclear magnetic resonance (NMR) spectroscopy with calorimetry and mutagenesis. NMR spectroscopy is exquisitely sensitive to biomacromolecular structure and dynamics, making it a powerful tool for characterizing how conformational sampling is perturbed by ligand binding. Many NMR measurements can be interpreted quantitatively in terms of exchange rates or thermodynamic differences between conformational states, such as folded and unfolded or ligand-free and ligand-bound forms. In this regard, biological NMR data are highly complementary to those of isothermal titration calorimetry and differential scanning calorimetry. These methods directly quantify the thermodynamics of binding and folding reactions, respectively. Furthermore, mutagenesis permits the selective removal or introduction intra- and inter-molecular contacts. Using NMR, calorimetry, and mutagenesis, we will dissect binding pathways with atomic resolution in terms of specific chemical moieties on the macromolecule and ligand. In addition, we propose to develop new NMR approaches to characterize the structural and orientational heterogeneity of bound ligands. Ligand dynamics in the bound state can potentially have a significant impact on the free energy of binding, particularly on the entropic component, however it has typically been challenging to address experimentally.We will apply this combined approach to study ligand binding by guanine quadruplex DNA, a promising target in cancer therapeutics. We will determine the impact of coupled local folding and binding on DNA recognition by a homeodomain. We will elucidate how ligands gain access to a buried site in an odorant binding protein and characterize their dynamics once bound. This research program is unique world-wide in its approach and scope and will provide a detailed and rigorous new perspective on how biological macromolecules recognize and bind to their targets.