NMR of drugs and ligands bound to membrane receptors Anthony Watts NMR methods are now able to give detailed structural, dynamic of these still not identified or classed into families [4] and electronic information about drugs and ligands while NMR of such macromolecules themselves, whether pur constrained at their site of action in membrane-embedde fied or in situ in isolated membrane fragments, is not receptors, information which is essential for mechanist routine because of their slow, anisotropic motion, giving descriptions of their action and design of new ligands, Using solid rise to broad resonances. Conventional solution state NM state NMR methods, a peptic ulcer drug analogue has been and the use of nuclear Overhauser effects(NOEs), which described at atomic resolution (to 0.3 A between two atoms)at can give intra- and inter-molecular distance information its site of action in the gastric H*/K+-ATPase, and the aromaticity can be performed on fragments of relevant loops of recep- of the agonist binding site of the nicotinic acetylcholine receptor tors, that fold in solution in ways thought to be has been demonstrated, with both targets in functionally physiologically relevant 15, 6]. Solid-state NMR methods competent membranes under conditions similar to those used in on the other hand, can give a more direct approach to lig- screening assays Gprotein-coupled receptor ligands and and-receptor interactions [7-9], usually by enhancement prosthetic groups are also being resolved using NMR methods. of sensitivity, resolution and assignments with specifically incorporated NMR isotopes(for example, 2H, 13C, ISN and 19F). The spectral anisotropy of certain nuclei(2H,15N Biomembrane Structure Unit, Bio rtment,University of 9F)in static solid state NMR gives orientational con Oxford, Oxford, OX1 3QU. UK inch. ox ac uk Current Opinion in Biotechnology 1999, 10: 48-53 [8, 10,11. Magic angle sample spinning (MASS)solid state NMR methods have been applied to determine spin coupled distances through dipolar coupling determinat C Elsevier Science Ltd ISSN 0958-1669 [12" to high resolution(+0.3 A), and chemical shifts ods have been developed in model systems in which th component of the complement system multiple spins can be resolved for structural determin GPCR G-protein-coupled recept tions [14 and in which spectral editing permits selective NoE nuclear Overh observation of specific ligands within a complex system [15], such that these approaches are now available for Introduction defining drug-receptor interactions for large targets, when Biotechnological exploitation of NMR to define the they become available lved in binding of drugs, and their structure and dynamics while at their site of action in membrane A major hurdle yet to be overcome, as in all biomolecular receptors, is now being realised. Although difference elec- structure studies but perhaps more so with membrane tron density maps for receptors with and without ligand receptors, is the production of the protein of interest in suf- from X-ray crystallographic studies would provide an ideal ficient amounts and functionally competent with way to get some of this information, many drug targets, associated lipids. The gulf between conducting biochemi- especially those that are membrane bound and not readi- cal assays of receptor activity to resolve the mechanism of ly available in a purified form, are not amenable to routine action and in direct structural investigations is being over- crystallographic analysis at the present time. For small come through improvement in expression technology, but (M, >30,000)complexes and are often NMR to give several (25 )atomic constraints for the open multi-subunit. Even though in cach cell they may be pre- and closed liganded form. 'This was ahead of the crystal sent at only a few copies, membrane components are structure for the open form [16, 17.1, and allowed Imerous and make up 30040% of the products of open ligand-target structural detail to bc extracted at high atom- reading frames of known genomes [3], with a vast majority ic resolution(better than + 0.3 A in some cases)
NMR of drugs and ligands bound to membrane receptors Watts 4 Here, the recent progress in the use of NMR, usually in con- mutagenisis and other studies used to define the residues junction with isotope enrichment, to define bound drugs and involved in the C5a-receptor interaction [33] nds in membrane receptors will be the molecular details for several ligands, including prosthet- The interactions of C5a with the amino-terminal domar ic groups and now one pharmaceutically relevant drug, have of the CSa receptor were examined using solution state been resolved using these methods, and so the future holds NMR of recombinant human C5a molecules and three promise for defining pharmacophores and binding Sites in peptide fragments (hCSaRF-1-34, hCSaRF-13-34,and embrane receptors hC5aRF-19-31)derived from human C5a receptor 134" All three receptor peptides studied responded to the bind G-protein-coupled receptors ng of C5a through the 21-30 amino acid region containing Most of the transmembrane signal transduction in re either hydrophobic, polar, or positively charged residues. to hormones and neurotransmitters occurs through such as Thr24, Pro25, Val26, Lys28, Thr29, and Ser30 tein-coupled receptors(GPCRs)[18, 19]. There is a Binding induced resonance perturbations in the NMR lack of detailed knowledge about receptor-ligand complex spectra (chemical shift and line-width changes) interactions [20], despite some extensive computer model- receptor fragments and the C5a molecules, indicated that ling[21 andheuristic'approaches, such as those applied to the isolated amino-terminal domain or residues 1-34 of the the neurotensin receptor[22 Some 2000 GPCRs are now C5a receptor retain specific binding to C5a and to a C5a known,which account for about 1% of the human genome. antagonist analogue(Figure 1 Primary sequence hydropathy profiles show a common seven transmembrane helix(7 IMD)and rationalisation of Interestingly, all three receptor fragments were found to the large amount of functional information has given rise to have a partially folded conformation in solution. The exis- over 100 individual agonist-defined subfamilies 23 tence of non-sequential NOEs, along with many see NH-NH NOE contacts, indicated that the free hCsaRF. Recently, a low resolution(6 A)electron diffraction struc- 1-34 peptide may have locally folded conformations (a ure of rhodopsin, a model GPCR, was elucidated showing 3o helices) within residues 20-30 or the carboxy-terminal the position and orientation of the seven transmembrane region of the peptide in solution. It seems that in some a helices 124125]. Expression methods are now avalable cases perhaps, for the C5a receptor [34] and rhodopsin for GPCRs [26"l, with purification methods permitting [5, 6l solution state NMR studies of drug-receptor interac Efficient protein for biophysical studies to be produced tions may be possible if extramembranous loops fold in a way that is similar to that for the full membrane-bound model for this important class of membranc receptor. until full 3D high resolution structures are available. uS/y Rhodopsin is still, therefore, the best 7TMD GPCR receptor, although this cannot be proved unambig Although the activating 'hormone' is light, the sequence of protein-protein interactions that follow receptor acti- Some peptide hormones, for example, are thought to pa ation are similar for all GPCRs [27]. In the absence of a tion into the cell membrane and then traverse to the high resolution receptor structure, deuterium solid-state membrane-bound TTMD GPCR, with structures that are nMR has been used to define the structure of the reti- relevant for receptor binding To study the hormone-mem nal chromophore bound within the protein, in both dark brane interaction, isotropic solvents or micelles are used as ground state, and in the light-activated (meta I)form mimetics in which the hormone has been shown to have [281. The detail available from such studies will sup- stable conformations, as shown for a pl-opoid receptor ago plement functional descriptions that may be more nist, leucine enkephalin, which forms a stable type IV elusive in higher, or even lower resolution structure B-turn structure in dodecylphosphocholine micelles [3.51 maps,unless they become available for all intermediate Another peptide hormone, dynorphin A(1-17), which is an forms of the activation process. Double quantum, solid- agonist of the K-opoid receptor, has been shown in similar state NMR recoupling methods. for example, the C7 micelles to form a family of structures containing an a-heli pulse sequence, and related ones have given torsional cal region over 6-7 residues(3-10) followed, after some angles for specific parts of the ISC labelled retinal poly- unstructured residues, by a B-curn(14-17)[361 ene chain while in its receptor binding site [151 Isotropic homogenous mimetics are most probably not An alternative approach to defining ligand-receptor inter- good substitutes for complex receptor binding sites, and actions is to study direct NOEs between a ligand and a major challenge is now to exploit the unique power of putative membrane receptor loop region in solution. The solid-state NMR methods to define ligand structures at C5a anaphylatoxin is a 74-residue glycoprotein derived their site of action, define the residues at the binding site. from the fifth component of the complement system(C5) as well as the known conformational changes that occur upon proteolytic activation [29 and plays an important role upon activation, such as helix orientational changes and in host defence against invading microorganisms or tumour helix loop exposure in 7I'MD GPCRs, when they become cells. The receptor for C5a is a TIMD and a GPCR [30] available for study. ' These GPCRs need to be embedded in and has been cloned [31,32], with site-directed a membrane environment for full functional competence
50 Analytical biotechnology Figure 1 A model for the inter delled 7TMD GPCr from solution state N-terminal between Cys109 in the first extracellular loop is shown by -ss- The C5a protein core only contacts residues 21-30 of the receptor Residues 1-18 (in particular (-)(-)(-) erminus may interact with the positively charged and hydrophobic stretch of residues extracellular loop shown here)of the C5a Extraceilular bind to and activate the receptor. Reproduced from [34] with permission membrane rather than a crystal. Extending this challenge still further, of inhibitors of the gastric ATPase. Using these approach and taking the ligand-GPCR-G-protein interaction es, substituted imidazo(1, 2-a)pyridines have gained process one step further, the 'empty pocket' formed pharmaceutical importance. Recent low resolution struc- between activated GPCR and g protein seems an attrac- tural data shows a protein with two 4-helix bundles and tive localised area of a large membrane-associated protein two further helices and very little lumenal density in which complex for study by selective NMR approaches, as do the to accommodate a drug binding site [37] terfaces between the protein components [27], to help drug design directed at this important family of proteins, Using rotational resonance solid-state NMR spectroscopy the precise(to+ 0.3 A)distance between two 13C-labelled Gastric peptic ulcer targets sites in a substituted imidazo(1, 2-a)pyridine, TMPIP Inhibition of gastric acid production by the H+/K+-ATPase while bound at its high-affinity binding site(0. 3 umoles of in peptic ulcer treatment is of major commercial s,o d in native membrane fragments, has been determined tance. This inhibition can be achieved either by use K+-competitive inhibitors or covalently modifying com- [38]. The structural analysis of the enzyme-inhibitor pounds alone, or together with antibacterial agents against complex revealed that the flexible moiety of TMPIP Helicobacter pylori to prevent acid hydrolysis of suitable adopts a syn conformation at its site of action, which may antibiotics. Being a readily available target, much bio- involve Glu820 from studies with the closely related chemical data exists on ATPases in general, and SCH28080 [39, 40] and Cys892 for binding of omeprazole ombinatorial chemistry methods have given a multitude [411, both of which are in the putative helix H5-H6 loop
NMR of drugs and ligands bound to membrane receptors Watts 51 and in which Phc818 may undergo T-T bond interactions modulation of nervous impulses across the synapses [42] with the benzylic moiety of the TMPIP(Figure 2) These receptors are recognised targets for a variety of phar maceutical agents for a range of neurological diseases, such as Hence, the conformation of an inhibitor has been resolved schizophrenia, AlzZheimer's disease and some epilepsies [43] directly under near-physiological conditions, providing a sound experimental basis for rational design of many active The introduction of 13C labels into the receptor agonist, compounds of pharmaceutical interest. Chemically restrain- acetylcholine, at the N-methy ing the flexible moiety of compounds such as TMPIP in the [3CH3I3-acetylcholine) permits the direct observ syn binding conformation was found to increase activity by the bound agonist(40 nmoles of N+-[ 13CH3l3-acetyl- over two orders of magnitude. Such information is normally choline to 40 mg of nicotinic acetylcholine recepto only available after extensive synthesis of related com- membranes)in its binding site on the membrane bound ounds and multiple screening approaches functionally competent nicotinic acetylcholine receptor [131. By exploiting the favourable cross polarization Nicotinic acetylcholine receptor characteristics of motionally constrained membranc sys- The nicotinic acetylcholine receptor, a ligand-gated ion tems, due to close contact between ligand and the binding channel, is a member of the four transmembrane superfami. site, in contrast to those in free solution, only co, of ago- ly of receptors, which includes GABa, glycine and 5-HT3 agonist by the protein is observed. The specificity receptors and play a key role in the transmission and nist-receptor interaction has been demonstrated specific inhibitor of agonist binding, a-bungarat Figure 2 The observed resonance of the bound agonist at 52.3 shifted 1.6 ppm upfield(p< 0.05)compared to that in free solution and crystalline solid(53.9 ppm), indicating that the ligand experiences a different non- aqueous electrostatic environment when in the receptor binding site. This chemical shift change is in contrast to sugars observed selectively in their translocation site within membrane bound transporters, which have identi cal chemical shifts to aqucous solute and can be displaced from their binding site specifically by antimicrobial agents, as shown by solid-state NMR methods[44 Carbon-13 chemical shifts are sensitive to molecular confor- mation, local charged environment and ring currents induced by local aromatic groups. It is suggested that the agonist From information from chemical cross labelling approaches, the binding site for the drug analogue K+-competitive TMPIP is thought to be in the putative helix H5-H6 loop, involving t-n bond sharing with Phe818, and charged residues Glu820 and Glu822 to the rotational resonance, solid-state NMR methods have defined the in 10 A of the substrate stance between two 13C sites in the TMPIP to 4.2+0.3 A while at the target binding site in fully functionally competent, gastric ure, consistent with the concept that substrate binding is H+-K+-ATPase membranes at 4@C [38.1 dominantly through cation-Tt interactions [45]
2 Analytical biotechnology acetylcholine binds to its site on the receptor not through a 3. Wallin E, von Heijne G: Genome-wide analysis of integ simple charge-charge interaction (which would produce a archaean, and eukaryotic organisms. Protein Sci 1998, 7: 1029-1038 mall chemical shift of 1 ppm), but at a site rich in aromat- 4 ic residues, thereby causing the large observed chemical shift protein families with a given number of helices? Proteins Struct which would be ring current induced [13l, probably Dr of the significant numbers of mono- and through a cation-T interaction. Similar interactions are sug- proteins which are membrane associated and have for the acetylcholin as though we know about 2000 members of this family already and the a useful model system for the receptor, as the known crystal focus of much study, be a dominant family structure [451 shows a high concentration of aromatic Yeagle PL, Alderfer JL, Albert AD: Three dimensione residues in the binding pocket( Figure 3). Although the chemical shifts are not vet known for the other nuclei in the Biochemistry 1997,36:96499654. n receptor rhodopsin ure of bound agonist, the significant electronic contribution to the 6. Yeagle PL, Alderfer JL, Salloum AC, Ali L, Albert AD: The first and binding of the N-trimethyl group probably reflects the low dependently form beta turns. Biochemistry 1997, 36: 3864-3869 degree of chemical tolerance of this group to modification in 7. Watts A, Ulrich AS, Middleton DA: Membrane protein structu other ligands required to cause receptor activation bution and potential of novel solid state NMR approaches o! Membr bio1995,12233-246 Conclusion and future prospects pooner PJR. Williamson PTF: Structural descriptions of ligands in Most of the tools for resolving rather precise details of lig physiological conditions. Eur Biophys /1998, 28:84.90 ands in receptors are in place. Higher magnetic fields are proving to give improved resolution and increased sensitivi membrane-embedded receptors and proteins using ty in solid-state NMR, in common with solution-state perturbing solid state NMR methods, Pharm Pharmaco/ NMR Expression technology is being improved, and the quirements of biophysical approaches, including NMr 4(su 10. Opella S: NMR and membrane proteins. Nat Struct Bio/ 199 screening of drugs bound to receptors, need to be met. A recent summary of how solid-state NMR is being used as a powerful Isotopic enrichment of ligands and proteins is still necessary, teins in the highly complex and heterogeneous environment of a membrane it seems. Some dipolar recoupling methods are giving reso- 11. Kim Y, Valentine K, Opella s, Schendel SL, Cramer WA: Solid-state tion close or even better to that in X-ray crystallography NMR studies of the membrane-bound closed state of the co able in ideal cases. Very soon, the complete structure of A descnpthannel c with the added benefit of dynamic information being avail- important drugs in the heterogeneous environment of fully other similar functionally competent, membrane-bound receptors will be peptides whik in MAS spectra of biological solids. ds available for dete ining dipolar couplings, and hence distance measurements between spe- will identify points of structural and electronic importance cific spins, which can also be applied to membrane systems for a bound ligand, helping in the design of potentially use ful new ligands. A new area of exploration is that of 13. Williamson PTE c KW. Watts A n the nicotinic acetylcholin embrane ion channels, and with solid-state NMR meth ochemistry1998,30:1085410859 ods giving new and detailed structural information for The first identification of a vital peptides [10, 11] and for peptide-peptide associations site of action in its membrane-embedded receptor under [461, the channel itself will be a focus for modulation and resolved, thereby helping in descriptions of its actic control by drugs ic-angle spinnin Acknowledgements llowship. Thanks to Feng Ni and Zhigang Chem Phys Letts 1995, 242: 304-309. he text, as is David Jack for his foresight and stimulating encouragemen for us to develop solid stare NMR to study drugs bound in their receptors 16. Hing Aw, Ti Schaefer J, Ho C: An investigation l-echo double resonance NMR References and recommended reading 17. Klug CA, Tasaki K, Tjandra N, Ho C. Schae · of special interest Following on from the resolution of inter- and intra atomic coordinates for the of outstanding interest ar dipolar recoupling NMR methods, in advance of the crystal structure, this paper describes the structure of the and diffusion-edited NMR method for screening compounds that closed form of the protein. 18. Ji TH, Grossman M, Ji l: G protein-coupled receptors. L. atter M, Fesi sw: Use of JBo/Chem1998,278:1 overcoming a sizeable problem. Stru 6,4:12451249 nd well presented review emphasising diversity of GPCRs from the functional aspect
NMR of drugs and ligands bound to membrane receptors Watts 53 19. Gether U, Kobilka BK: G n-coupled receptors Il Mechanism routine NOE approaches. If the folding extramembranous loops are of agonist activation J Bio/ Chem 1998 erived from site. 20. Strader CD, Fong TM, Tota MR, Underwood D, Dixon RA: Structure and ev of G protein- coupled receptors. An etail obtained 1994,63:101-132 DA: Structure of Leu-5-enkephalin 1. ygh chen 1g96. 273 n upsd receptors minireview series. In NMR 2. Pang YP, Cusack B, Roshan K, Richelson E: Proposed ligand 36, Tessmer MR, Kallick DA: NMR and structural model of dynorphin A (1-17) bound to dodecylphosphocholine micelles. Biochemistry 23. Vaughan M: G protein-coupled receptors. Bio/ Chem 1998, 273:17297 rough G, Kuehlbrandt w: Three-dimensional map of he plasma membrane H+-ATPase in the open conformation. 24. Baldwin JM, Schertler GF, Unger VM: An alpha-carbon template for Naue1998,392840843. o/1997,272 38. Middleton DA, Robins R, Feng X, Levitt MH, Spiers ID, Schwalbe CH The most comprehensive model for mammalian rhodopsin, the standard unavailable for any other GPcR des a range of experimental data that is gastric proton pump. FEBS Lett 1997, 410: 269-274 5. Unger VM, Hargrave PA, Baldwin JM, Schertler GF: Arrangement of rhodopsin transmembrane alpha helices Nature 1997, 647: 203-206 portant, and this new information about the drug is now available corporation into drug design, and shows how the methods can be used for 26. Grisshammer R, Tucker J: Expression in Escherichia coli and large H, De Pont J]: Involvement of glutamic acid system, E coli, which shor expresson of GPCRs in the versatile expression 820 in K+ and SCH 28080 binding to gastric H+, K(+)-ATPase Ann duction and labelling with NMR isotopes. H+ K+ATPase are 27. Hamm HE: The many faces of G protein signaling. J Bio/ Chem 5-6he elix loop at residues Glu795 and GluB20 using si 1998,273:6696 the binding site using solid 28. Grobner G, Choi G, Burnett L, Glaubitz C. Verdegem P, Lugtenburg state NmR (data in 38 n 40, Asano S, Matsuda S, Tega Y, Shimizu K se nstraints Ca etermined for ligands and prosthetic groups in membrane-bound G 272:17668-17674 Further evidence, from site directed mutagen using solid state NMR the helix 5-6 the site of action for the SCH28080 drug analogue in the H+, K ughi ATPase target. functions of C3a, C4a, and C5a Crt Rey Immuno/ 1981.1: 321-366 41. Besancon M, Simon A, Sachs G, Shin JM: Sites of reaction of the gastric H, K-ATPase with extracytoplasmic thiol for the C5a re hem19972722243822446 ein Eng 199 ard C5a 42. Galzi JL, Revah F, Bessis A, Change oxin. Nature1991,349:614617 ic acetylcholine receptor: from the electric organ and 32. boulay f Mery L L vignais P: Expression crenulate 43. Cooper JR, Bloom FE, Roth RH: Biochemical Basis of 60 cells. Biochemistry 1991, 30: 2993-2999 teut Spooner PJR, O'Reilly WJ, Homans SW, Rutherford NG Henderson PJF Watts A: Weak substrate bine proteins studied by NMR. Biophys /1998, 75 C5a receptor. Arg 206 determines high-affinity binding sites of C5a receptor. Eur J Biochem 1996. 235: 82-90 45. Sussman JL, Harel M, Frolow F, Oefner C, Goldman A, Toker L. Silman I: Atomic structure of acetylcholinesterase from Torpedo 34. Chen Z Zh ionnella NC, Pellas TC, Boyar WC, Ni F: lon of the prototypic acetylc 991,253:872879 46. Smith SO, Bormann BJ: Determination of helix-helix Solution-state NMR in In in membranes by rotational resonance NMR. Proc Na ix interactions loops of the receptor have been synthesised and studied in solution using USA1995,92:48849