48

NMR methods are now able to give detailed structural, dynamicand electronic information about drugs and ligands whileconstrained at their site of action in membrane-embeddedreceptors, information which is essential for mechanisticdescriptions of their action and design of new ligands. Using solidstate NMR methods, a peptic ulcer drug analogue has beendescribed at atomic resolution (to ± 0.3 Å between two atoms) atits site of action in the gastric H+/K+-ATPase, and the aromaticityof the agonist binding site of the nicotinic acetylcholine receptorhas been demonstrated, with both targets in functionallycompetent membranes under conditions similar to those used inscreening assays. G-protein-coupled receptor ligands andprosthetic groups are also being resolved using NMR methods.

AddressesBiomembrane Structure Unit, Biochemistry Department, University ofOxford, Oxford, OX1 3QU, UK; e-mail: awatts@bioch.ox.ac.uk

Current Opinion in Biotechnology 1999, 10:48–53

http://biomednet.com/elecref/0958166901000048

© Elsevier Science Ltd ISSN 0958-1669

Abbreviations7TMD seven transmembrane helixC5 fifth component of the complement systemGPCR G-protein-coupled receptorNOE nuclear Overhauser effect

IntroductionBiotechnological exploitation of NMR to define theresidues involved in binding of drugs, and their structureand dynamics while at their site of action in membranereceptors, is now being realised. Although difference elec-tron density maps for receptors with and without ligandfrom X-ray crystallographic studies would provide an idealway to get some of this information, many drug targets,especially those that are membrane bound and not readi-ly available in a purified form, are not amenable to routinecrystallographic analysis at the present time. For small(Mr < 30,000) soluble, highly purified drug targets all ofthe sophisticated multi-dimensional solution-state NMRapproaches are being applied, in the most successfulcases, to resolve drug structure and ligand binding sites,even to facilitate drug screening (see this issue, Roberts,pp 42–47). Adaptation of this screening approach to muchlarger macromolecular membrane targets and receptorsmay become a reality [1,2], but currently the lack of suffi-cient amounts of protein is usually limiting.

Many drug receptors, however, are membrane-boundmacromolecular (Mr >> 30,000) complexes and are oftenmulti-subunit. Even though in each cell they may be pre-sent at only a few copies, membrane components arenumerous and make up 30–40% of the products of openreading frames of known genomes [3], with a vast majority

of these still not identified or classed into families [4••].NMR of such macromolecules themselves, whether puri-fied or in situ in isolated membrane fragments, is notroutine because of their slow, anisotropic motion, givingrise to broad resonances. Conventional solution state NMRand the use of nuclear Overhauser effects (NOEs), whichcan give intra- and inter-molecular distance information,can be performed on fragments of relevant loops of recep-tors, that fold in solution in ways thought to bephysiologically relevant [5,6]. Solid-state NMR methods,on the other hand, can give a more direct approach to lig-and–receptor interactions [7–9], usually by enhancementof sensitivity, resolution and assignments with specificallyincorporated NMR isotopes (for example, 2H, 13C, 15N and19F). The spectral anisotropy of certain nuclei (2H, 15N,19F) in static solid state NMR gives orientational con-straints of labelled groups in ligands and peptides[8,10••,11•]. Magic angle sample spinning (MASS) solidstate NMR methods have been applied to determine spin-coupled distances through dipolar coupling determinations[12••] to high resolution (± 0.3 Å), and chemical shifts todefine the ligand binding environment [13••]. New meth-ods have been developed in model systems in whichmultiple spins can be resolved for structural determina-tions [14], and in which spectral editing permits selectiveobservation of specific ligands within a complex system[15], such that these approaches are now available fordefining drug–receptor interactions for large targets, whenthey become available.

A major hurdle yet to be overcome, as in all biomolecularstructure studies but perhaps more so with membranereceptors, is the production of the protein of interest in suf-ficient amounts and functionally competent withassociated lipids. The gulf between conducting biochemi-cal assays of receptor activity to resolve the mechanism ofaction and in direct structural investigations is being over-come through improvement in expression technology, butthere are currently severe limitations in obtaining suffi-cient amounts (usually tens of milligrams) of a target fordirect study.

In the best cases, expression systems can be amplified and,for NMR studies, even be modified to label selectivelyspecific residues, or at least all residues (usually a limitednumber) of one type by construction of an auxotroph. Forexample, all the tryptophans in the glutamine-binding pro-tein expressed in Escherichia coli have been 19F-labelledand the protein uniformly 15N labelled and studied byNMR to give several (>25) atomic constraints for the openand closed liganded form. This was ahead of the crystalstructure for the open form [16,17•], and allowedligand–target structural detail to be extracted at high atom-ic resolution (better than ± 0.3 Å in some cases).

NMR of drugs and ligands bound to membrane receptorsAnthony Watts

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NMR of drugs and ligands bound to membrane receptors Watts 49

Here, the recent progress in the use of NMR, usually in con-junction with isotope enrichment, to define bound drugs andligands in membrane receptors will be presented. Alreadythe molecular details for several ligands, including prosthet-ic groups and now one pharmaceutically relevant drug, havebeen resolved using these methods, and so the future holdspromise for defining pharmacophores and binding sites inmembrane receptors.

G-protein-coupled receptorsMost of the transmembrane signal transduction in responseto hormones and neurotransmitters occurs through G-pro-tein-coupled receptors (GPCRs) [18••,19]. There is a distinctlack of detailed knowledge about receptor–ligand complexinteractions [20], despite some extensive computer model-ling [21] and ‘heuristic’ approaches, such as those applied tothe neurotensin receptor [22]. Some 2000 GPCRs are nowknown, which account for about 1% of the human genome.Primary sequence hydropathy profiles show a commonseven transmembrane helix (7TMD) and rationalisation ofthe large amount of functional information has given rise toover 100 individual agonist-defined subfamilies [23].

Recently, a low resolution (6 Å) electron diffraction struc-ture of rhodopsin, a model GPCR, was elucidated showingthe position and orientation of the seven transmembraneα helices [24••,25]. Expression methods are now availablefor GPCRs [26••], with purification methods permittingsufficient protein for biophysical studies to be produced.

Rhodopsin is still, therefore, the best 7TMD GPCRmodel for this important class of membrane receptor.Although the activating ‘hormone’ is light, the sequenceof protein–protein interactions that follow receptor acti-vation are similar for all GPCRs [27]. In the absence of ahigh resolution receptor structure, deuterium solid-stateNMR has been used to define the structure of the reti-nal chromophore bound within the protein, in both darkground state, and in the light-activated (meta I) form[28••]. The detail available from such studies will sup-plement functional descriptions that may be moreelusive in higher, or even lower resolution structuremaps, unless they become available for all intermediateforms of the activation process. Double quantum, solid-state NMR recoupling methods, for example, the C7pulse sequence, and related ones have given torsionalangles for specific parts of the 13C labelled retinal poly-ene chain while in its receptor binding site [15].

An alternative approach to defining ligand–receptor inter-actions is to study direct NOEs between a ligand and aputative membrane receptor loop region in solution. TheC5a anaphylatoxin is a 74-residue glycoprotein derivedfrom the fifth component of the complement system (C5)upon proteolytic activation [29] and plays an important rolein host defence against invading microorganisms or tumourcells. The receptor for C5a is a 7TMD and a GPCR [30]and has been cloned [31,32], with site-directed

mutagenisis and other studies used to define the residuesinvolved in the C5a–receptor interaction [33].

The interactions of C5a with the amino-terminal domainof the C5a receptor were examined using solution stateNMR of recombinant human C5a molecules and threepeptide fragments (hC5aRF-1–34, hC5aRF-13–34, andhC5aRF-19–31) derived from human C5a receptor [34••].All three receptor peptides studied responded to the bind-ing of C5a through the 21–30 amino acid region containingeither hydrophobic, polar, or positively charged residues,such as Thr24, Pro25, Val26, Lys28, Thr29, and Ser30.Binding induced resonance perturbations in the NMRspectra (chemical shift and line-width changes) of thereceptor fragments and the C5a molecules, indicated thatthe isolated amino-terminal domain or residues 1–34 of theC5a receptor retain specific binding to C5a and to a C5aantagonist analogue (Figure 1).

Interestingly, all three receptor fragments were found tohave a partially folded conformation in solution. The exis-tence of non-sequential NOEs, along with many sequentialNH–NH NOE contacts, indicated that the free hC5aRF-1–34 peptide may have locally folded conformations (α or310 helices) within residues 20–30 or the carboxy-terminalregion of the peptide in solution. It seems that in somecases perhaps, for the C5a receptor [34••] and rhodopsin[5,6] solution state NMR studies of drug–receptor interac-tions may be possible if extramembraneous loops fold in away that is similar to that for the full membrane-boundreceptor, although this cannot be proved unambiguouslyuntil full 3D high resolution structures are available.

Some peptide hormones, for example, are thought to parti-tion into the cell membrane and then traverse to themembrane-bound 7TMD GPCR, with structures that arerelevant for receptor binding. To study the hormone–mem-brane interaction, isotropic solvents or micelles are used asmimetics in which the hormone has been shown to havestable conformations, as shown for a µ-opoid receptor ago-nist, leucine enkephalin, which forms a stable type IVβ-turn structure in dodecylphosphocholine micelles [35].Another peptide hormone, dynorphin A (1–17), which is anagonist of the κ-opoid receptor, has been shown in similarmicelles to form a family of structures containing an α-heli-cal region over 6–7 residues (3–10) followed, after someunstructured residues, by a β-turn (14–17) [36].

Isotropic homogenous mimetics are most probably notgood substitutes for complex receptor binding sites, and amajor challenge is now to exploit the unique power ofsolid-state NMR methods to define ligand structures attheir site of action, define the residues at the binding site,as well as the known conformational changes that occurupon activation, such as helix orientational changes andhelix loop exposure in 7TMD GPCRs, when they becomeavailable for study. These GPCRs need to be embedded ina membrane environment for full functional competence,

bta110.qxd 11/29/1999 3:53 PM Page 49

rather than a crystal. Extending this challenge still further,and taking the ligand–GPCR–G-protein interactionprocess one step further, the ‘empty pocket’ formedbetween activated GPCR and G protein seems an attrac-tive localised area of a large membrane-associated proteincomplex for study by selective NMR approaches, as do theinterfaces between the protein components [27], to help indrug design directed at this important family of proteins.

Gastric peptic ulcer targetsInhibition of gastric acid production by the H+/K+-ATPasein peptic ulcer treatment is of major commercial impor-tance. This inhibition can be achieved either by use ofK+-competitive inhibitors or covalently modifying com-pounds alone, or together with antibacterial agents againstHelicobacter pylori to prevent acid hydrolysis of suitableantibiotics. Being a readily available target, much bio-chemical data exists on ATPases in general, andcombinatorial chemistry methods have given a multitude

of inhibitors of the gastric ATPase. Using these approach-es, substituted imidazo(1,2-a)pyridines have gainedpharmaceutical importance. Recent low resolution struc-tural data shows a protein with two 4-helix bundles andtwo further helices and very little lumenal density in whichto accommodate a drug binding site [37].

Using rotational resonance solid-state NMR spectroscopy,the precise (to ± 0.3 Å) distance between two 13C-labelledsites in a substituted imidazo(1,2-a)pyridine, TMPIP,while bound at its high-affinity binding site (0.3 µmoles ofdrug were detected) in the intact H+/K+-ATPase embed-ded in native membrane fragments, has been determined[38••]. The structural analysis of the enzyme–inhibitorcomplex revealed that the flexible moiety of TMPIPadopts a syn conformation at its site of action, which mayinvolve Glu820 from studies with the closely relatedSCH28080 [39•,40•] and Cys892 for binding of omeprazole[41], both of which are in the putative helix H5–H6 loop

50 Analytical biotechnology

Figure 1

A model for the interaction of C5a with itsmodelled 7TMD GPCR from solution stateNMR studies of the NOEs from C5a tosoluble loops of the receptor. A disulfide bondbetween Cys109 in the first extracellular loopand Cys189 in the second extracellular loopis shown by –s-s–. The C5a protein core onlycontacts residues 21–30 of the receptoramino terminus. Residues 1–18 (in particularresidues 10–18) of the receptor aminoterminus may interact with the positivelycharged and hydrophobic stretch of residueswithin the extracellular loops (the secondextracellular loop shown here) of the C5areceptor. The C5a protein molecule is held ina proper position by the amino terminus of thereceptor, allowing its carboxy-terminal tail tobind to and activate the receptor. Reproducedfrom [34••] with permission.

ST

K

D

V

P TN

L D LT

D K D D Y H G Y D

30

25

2015 10

(–)(–)(–)(–)+

+ +

+ + +

N-terminal

Extracellular

Plasmamembrane

Intracellular

C5a

ss

12

3 4

5

67

C-terminal

bta110.qxd 11/29/1999 3:53 PM Page 50

and in which Phe818 may undergo π−π bond interactionswith the benzylic moiety of the TMPIP (Figure 2).

Hence, the conformation of an inhibitor has been resolveddirectly under near-physiological conditions, providing asound experimental basis for rational design of many activecompounds of pharmaceutical interest. Chemically restrain-ing the flexible moiety of compounds such as TMPIP in thesyn binding conformation was found to increase activity byover two orders of magnitude. Such information is normallyonly available after extensive synthesis of related com-pounds and multiple screening approaches.

Nicotinic acetylcholine receptorThe nicotinic acetylcholine receptor, a ligand-gated ionchannel, is a member of the four transmembrane superfami-ly of receptors, which includes GABA, glycine and 5-HT3receptors and play a key role in the transmission and

modulation of nervous impulses across the synapses [42].These receptors are recognised targets for a variety of phar-maceutical agents for a range of neurological diseases, such asschizophrenia, Alzheimer’s disease and some epilepsies [43].

The introduction of 13C labels into the receptor agonist,acetylcholine, at the N-methyl position (N+-[13CH3]3-acetylcholine) permits the direct observation ofthe bound agonist (40 nmoles of N+-[13CH3]3-acetyl-choline to 40 mg of nicotinic acetylcholine receptormembranes) in its binding site on the membrane bound,functionally competent nicotinic acetylcholine receptor[13••]. By exploiting the favourable cross polarizationcharacteristics of motionally constrained membrane sys-tems, due to close contact between ligand and the bindingsite, in contrast to those in free solution, only constrainedagonist by the protein is observed. The specificity of ago-nist–receptor interaction has been demonstrated using thespecific inhibitor of agonist binding, α-bungaratoxin.

The observed resonance of the bound agonist at52.3 ppm is shifted 1.6 ppm upfield (p < 0.05) comparedto that in free solution and crystalline solid (53.9 ppm),indicating that the ligand experiences a different non-aqueous electrostatic environment when in the receptorbinding site. This chemical shift change is in contrast tosugars observed selectively in their translocation sitewithin membrane bound transporters, which have identi-cal chemical shifts to aqueous solute and can be displacedfrom their binding site specifically by antiomicrobialagents, as shown by solid-state NMR methods [44].

Carbon-13 chemical shifts are sensitive to molecular confor-mation, local charged environment and ring currents inducedby local aromatic groups. It is suggested that the agonist

NMR of drugs and ligands bound to membrane receptors Watts 51

Figure 2

From information from chemical cross labelling approaches, thebinding site for the drug analogue K+-competitive TMPIP is thoughtto be in the putative helix H5–H6 loop, involving π−π bond sharingwith Phe818, and charged residues Glu820 and Glu822 to theimidazole moeity. Structural constraints at high resolution usingrotational resonance, solid-state NMR methods have defined thedistance between two 13C sites in the TMPIP to 4.2 ± 0.3 Å while atthe target binding site in fully functionally competent, gastricH+-K+-ATPase membranes at 4°C [38••].

Figure 3

Diagram showing the acetylcholine binding site within theacetylcholinesterase. Aromatic residues within 10 Å of the substrate-binding site have been highlighted to demonstrate its predominantlyaromatic nature, consistent with the concept that substrate binding ismediated predominantly through cation–π interactions [45].

bta110.qxd 11/29/1999 3:54 PM Page 51

52 Analytical biotechnology

acetylcholine binds to its site on the receptor not through asimple charge–charge interaction (which would produce asmall chemical shift of < 1 ppm), but at a site rich in aromat-ic residues, thereby causing the large observed chemical shiftwhich would be ring current induced [13••], probablythrough a cation–π interaction. Similar interactions are sug-gested to occur for the acetylcholinesterase, which serves asa useful model system for the receptor, as the known crystalstructure [45] shows a high concentration of aromaticresidues in the binding pocket (Figure 3). Although thechemical shifts are not yet known for the other nuclei in thebound agonist, the significant electronic contribution to thebinding of the N-trimethyl group probably reflects the lowdegree of chemical tolerance of this group to modification inother ligands required to cause receptor activation.

Conclusion and future prospectsMost of the tools for resolving rather precise details of lig-ands in receptors are in place. Higher magnetic fields areproving to give improved resolution and increased sensitivi-ty in solid-state NMR, in common with solution-stateNMR. Expression technology is being improved, and therequirements of biophysical approaches, including NMRscreening of drugs bound to receptors, need to be met.Isotopic enrichment of ligands and proteins is still necessary,it seems. Some dipolar recoupling methods are giving reso-lution close or even better to that in X-ray crystallography,with the added benefit of dynamic information being avail-able in ideal cases. Very soon, the complete structure ofimportant drugs in the heterogeneous environment of fullyfunctionally competent, membrane-bound receptors will beresolved to near atomic resolution using appropriate NMRmethods, as a matter of course. In addition, chemical shiftswill identify points of structural and electronic importancefor a bound ligand, helping in the design of potentially use-ful new ligands. A new area of exploration is that ofmembrane ion channels, and with solid-state NMR meth-ods giving new and detailed structural information forpeptides [10••,11•] and for peptide–peptide associations[46], the channel itself will be a focus for modulation andcontrol by drugs.

AcknowledgementsThe author wishes to thank BBSRC for their support of a Senior ResearchFellowship. Thanks to Feng Ni and Zhigang Chen for Figure 1 and PhilWilliamson and Jude Watts for producing Figures 2 and 3. Members of thegroup are acknowledged for their helpful comments and critical reading ofthe text, as is David Jack for his foresight and stimulating encouragementfor us to develop solid state NMR to study drugs bound in their receptors.

References and recommended readingPapers of particular interest, published within the annual period of review,have been highlighted as:

• of special interest•• of outstanding interest

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