Structural resolution of ligand-receptor interactions in functional, membrane-embedded receptors and proteins using novel, non-perturbing solid-state NMR methods
Defining structural details for membrane-embedded proteins is limited by the availability of two- or three-dimensional crystals suitable for diffraction studies. This is even more difficult when the structure of a ligand in its binding site is required because difference crystallography would be necessary, and with two-dimensional crystals, extra-membraneous protein detail is often missing and resolution is low in the direction of the membrane normal. Also, many bioactive ligands do not have any structured form in solution (solvents, detergents, etc.), but it is usually accepted that their target site does have structural requirements which define efficacy and potency. To address the question of ligand structure for bound ligands, novel solid-state NMR techniques were used to define molecular constraints at atomic resolution for ligands at their site of action, in fully functional, hydrated proteins under near physiological conditions (4°C or higher), and in a form often used for pharmacological and biochemical characterization. Using the solid-state NMR approach, the structure of retinal has been resolved in the bacterial proton pump, bacteriorhodopsin, and in the 7-TMD G-coupled receptor, mammalian rhodopsin, without any knowledge of the protein structure itself. The first structural details of a widely used drug analogue, an imidazopyridine inhibitor of the gastric H+-K+ATPase, at its site of action, have been determined. Other examples for large integral proteins include the observation and kinetic description of solutes in the antimicrobial target sugar transporter proteins of bacteria, and the resolution of the chemistry of the binding site of acetylcholine when in the binding site of the nicotinic acetylcholine receptor. In addition, small peptide ion channels are amenable to study using solid-state NMR methods, and the state of oligomerization, residues involved in the channel, can now be defined, with the potential for describing functionally significant residues and thus aid inhibitor or modulator design. The information gained and methods developed for small amounts of protein (μmol-nmol of binding sites), open the way to defining structural details of peptide hormones, flexible drugs and other ligands bound at their site of action in a functional system. The results therefore have relevance to the in-vivo situation, and are of direct importance for the understanding of structural requirements for ligand-activated signal transduction and cellular activity. In some cases, the residues involved in binding are also being resolved, thereby giving a complete picture of the vital and highly relevant ligand-binding site structure and environment, in the absence of knowledge of the protein backbone or its structural arrangements.