Solution NMR Studies of Interactions of Ligands With Plasma Proteins

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Plasma proteins, although well known as carriers of serum solutes (e.g. fatty acids), have been recently shown to function also as extra-cellular chaperones, which inhibit the aggregation of unfolded proteins and amyloidogenic peptides (e.g. Aß). The long-term goal of our research program is to define fundamental molecular mechanisms governing ligand binding and aggregation inhibition by plasma proteins. My laboratory has made significant contributions to the current understanding of how the prototypical human serum albumin (HSA) acts as the most potent inhibitor of Aß fibrillization in plasma. Using a multidisciplinary combination of nuclear magnetic resonance (NMR) spectroscopy and other biophysical and biochemical approaches (e.g. fluorescence, dynamic light scattering, electron microscopy and site-directed mutagenesis), we have shown that HSA prevents the Aß peptide from forming insoluble amyloid fibrils by selectively binding to soluble Aß oligomers and competing with the further addition of Aß monomers. We have determined the stoichiometry and the affinities of the HSA - Aß oligomer complexes and through comparative mutational analyses we have shown that HSA recognizes the Aß oligomers through sites that are unique and distinct from those interacting with low molecular weight (MW) ligands. Our preliminary data suggest the hypothesis that the critical contacts with the Aß oligomers are mediated by flexible loops of HSA with partial sequence homology to Aß. However, the current knowledge about the location of the Aß oligomer binding sites within HSA is at best scant. Our first short-term objective will therefore be to: (i) Map at single-residue resolution the binding sites within HSA for the Aß oligomers, by combining the multidisciplinary approach of our previous publications with new NMR experiments designed to probe reversible interactions with the Aß oligomers. This will be the first time that binding sites of a protein for oligomers of an amyloidogenic peptide are mapped at single-residue resolution. In the long term, this objective will be extended to a plethora of other amyloidogenic peptides and other extracellular chaperones. We will also elucidate the mechanisms underlying the other primary function of plasma proteins, i.e. ligand transport. The structures of ligand-bound HSA reveal the HSA architecture and the location of the binding sites for low MW ligands. However, structures alone cannot address several outstanding questions about ligand binding. How do ligands access buried binding sites? How does conformational entropy drive ligand binding? How are the apo conformational pre-equilibria allosterically coupled to ligand binding? To address these fundamental questions our second objective will be to: (ii) Comparatively analyze the dynamics of HSA in the absence and presence of fatty acids and other ligands, benefiting from recent NMR advances. These include an approach we pioneered to map otherwise elusive allosteric networks through the covariance analysis of NMR chemical shifts. We will start with the analysis of isolated HSA domains and will expand to progressively longer HSA constructs to reveal how dynamics modulates ligand binding, gating and allostery. The preliminary data available so far indicate that both objectives are feasible in terms of available materials and of experimental methods, capitalizing on the NMR approaches employed in our past publications. The resulting program will have a broad impact that goes well beyond HSA, as it will reveal general molecular mechanisms for the extra-cellular chaperones and for the allosteric coupling of ligand binding to protein dynamics. The impact will be both scientific and educational, as the proposed program provides a unique HQP training opportunity.