Structural Resolution of Ligand–Receptor Interactions in Functional, Membrane-embedded Receptors and Proteins using Novel, Non-perturbing Solid-state NMR Methods

Abstract

Derning 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 difrcult when the structure of a ligand in its binding site is required because difference crystallography would be necessary, and with two‐dimensional crystals, extramembraneous 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 derne efrcacy and potency. To address the question of ligand structure for bound ligands, novel solid‐state NMR techniques were used to derne 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 rrst 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 derned, with the potential for describing functionally signircant residues and thus aid inhibitor or modulator design. The information gained and methods developed for small amounts of protein (μmolnmol of binding sites), open the way to derning 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.

Solid-State 17O NMR of Amino Acids

“…A key experimental challenge for biomolecular chemistry is to provide high-quality, detailed, and unambiguous atomic-scale information about the molecular bonding arrangement and changes that occur upon ligand−receptor interaction. Solid-state NMR is one nonperturbing approach which can be used to study such interactions where molecular size is not limiting and crystallinity not a requirement, , and the ubiquity of oxygen throughout living systems should imply that 17 O is an important nucleus for such studies. Oxygen plays a key role in intra- and intermolecular interactions, with hydrogen-bonding important in biological processes so that 17 O could provide detailed information about the dynamics and structure of amino acids both in the solid state and in solution.…”

2H{19F} REDOR for distance measurements in biological solids using a double resonance spectrometer

“…The protein, a 114 kDa relative of the Na þ /K þ -and Ca 2þ P-type ATPases, is involved in a number of diseases such as gastric ulceration or atrophic gastritis, and the development of inhibitors to moderate its function is of great pharmaceutical interest and need. In particular, structural information on the conformation of the inhibitor is desirable to form a rationale basis for drug design [11,[34][35][36][37]. To this aim, an internuclear distance within the inhibitor was investigated here, revealing constraints on its conformation.…”