Biochemical and Biophysical Research Communications 390 (2009) 441–446

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Biochemical and Biophysical Research Communications

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NMR solution structure of human cannabinoid receptor-1 helix 7/8 peptide:Candidate electrostatic interactions and microdomain formation

Sergiy Tyukhtenko a, Elvis K. Tiburu a, Lalit Deshmukh b, Olga Vinogradova b, David R. Janero a,Alexandros Makriyannis a,*

a Center for Drug Discovery and Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115-5000, USAb Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, CT 06269-3092, USA

a r t i c l e i n f o

Article history:Received 2 September 2009Available online 18 September 2009

Keywords:Cannabinergic ligandG protein-coupled receptorInterhelical microdomainProline kinkSignal transductionStructural biologyTransmembrane protein

0006-291X/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.bbrc.2009.09.053

* Corresponding author. Address: NortheasternDiscovery, 360 Huntington Avenue, Mugar 116, BoFax: +1 617 373 7493.

E-mail address: a.makriyannis@neu.edu (A. Makriy

a b s t r a c t

We report the NMR solution structure of a synthetic 40-mer (T377–E416) that encompasses human can-nabinoid receptor-1 (hCB1) transmembrane helix 7 (TMH7) and helix 8 (H8) [hCB1(TMH7/H8)] in 30%trifluoroethanol/H2O. Structural features include, from the peptide’s amino terminus, a hydrophobic a-helix (TMH7); a loop-like, 11 residue segment featuring a pronounced Pro-kink within the conservedNPxxY motif; a short amphipathic a-helix (H8) orthogonal to TMH7 with cationic and hydrophobicamino-acid clusters; and an unstructured C-terminal end. The hCB1(TMH7/H8) NMR solution structuresuggests multiple electrostatic amino-acid interactions, including an intrahelical H8 salt bridge and ahydrogen-bond network involving the peptide’s loop-like region. Potential cation–p and cation–phenolicOH interactions between Y397 in the TMH7 NPxxY motif and R405 in H8 are identified as candidate struc-tural forces promoting interhelical microdomain formation. This microdomain may function as a flexiblemolecular hinge during ligand-induced hCB1 conformer transitions.

� 2009 Elsevier Inc. All rights reserved.

Introduction

In humans, G protein-coupled receptors (GPCRs) are ubiquitoussignal transduction elements and therapeutically important drugtargets [1]. GPCRs continue to garner intense research and transla-tional interest aimed at defining GPCR activation dynamics, opti-mizing the design of drugs that modulate the function of specificGPCRs, and improving extant GPCR-targeted therapies [2,3]. Tradi-tional GPCRs exhibit three hallmark architectural features: anextracellular amino terminus, an intracellular carboxyl terminus,and seven interconnected transmembrane helices (TMHs) [1,3].The heptahelical, integral-membrane character of GPCRs presentsformidable challenges to their purification in quantities sufficientfor high-resolution structural analysis [3]. Alternatively, homologymodels of some therapeutically interesting GPCRs have been con-structed, mainly by using refined, rhodopsin crystal structures astemplates [3,4]. The limited global sequence homology amongclass-A GPCRs and the ligand-independent nature of rhodopsinactivation compromise the accuracy of rhodopsin-based GPCRcomputational modeling and its utility as a drug-discovery tool

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University, Center for Drugston, MA 02115-5000, USA.

annis).

[2,4]. Consequently, experimental data are avidly sought on thestructural features and functionally-relevant conformationaldynamics of specific GPCRs. In this regard, interactions betweenthe highly-conserved NPxxY motif in transmembrane helix 7(TMH7) and its cytoplasmic extension, helix 8 (H8), appear to par-ticipate in the conformer transitions underlying the activation ofclass-A GPCRs, including rhodopsin and the serotonin 2C (5-HT2C) and insulin-like growth factor-1 receptors [1,5–7]. Theintracellular H8 domain is believed to assist in establishing pro-ductive interactions between GPCRs and soluble cytoplasmic Gproteins requisite for signal transmission [1,3,8].

A component of the endogenous cannabinoid (endocannabi-noid) signaling system, human cannabinoid receptor-1 (hCB1) isa class-A, rhodopsin-like GPCR activatable by intrinsically-pro-duced lipid ligands and exogenous cannabinergic agents [9]. Itsroles in diverse (patho)physiological processes make hCB1 a prom-inent ‘‘druggable” GPCR to which designer ligands are being tar-geted as potential treatments for common health problemsincluding cardiometabolic disorders, substance abuse/drug addic-tion, mood disturbances, and pain management [10]. The currentunavailability of a high-resolution hCB1 crystal structure under-mines hCB1 therapeutic exploitation [9,10]. Mutational, pharmaco-logical, and rhodopsin-based homology-modeling data for hCB1have allowed inferences that TMH7 resides within the plasmamembrane and H8 is juxtaposed to the plasma membrane’s inneraspect as likely participants in ligand-dependent hCB1 activation

Table 1Structural statistics for the 18 hCB1(TMH7/H8) NMR conformers.

Parameter Ensemblea

Distance restrainsAll 1224Short-range (|i � j| 6 1) 574Medium-range (1 < |i � j| < 5) 624Long-range (|i � j| P 5) 26

ViolationsNOE (>0.5 Å) 0r.m.s.d.b (residues 2–14, 16–21, 26–38)Average backbone r.m.s.d. to mean 1.1 ÅAverage heavy atom r.m.s.d. to mean 1.5 ÅVan der Waals energy �92.74 ± 8.67

Ramachandran plotc

Residues in most favored regions 65.2%Residues in additional allowed regions 30.2%Residues in generously allowed regions 4.3%Residues in disallowed regions 0.4%

r.m.s.d. from idealized covalent geometryBonds (Å) 0.011 ± 0.00012Angles (�) 0.76 ± 0.021Impropers (�) 1.18 ± 0.17

a Values given are means ± SEM, wherever applicable.b Residues 1, 15, 22–25, and 39, 40 were excluded from r.m.s.d. calculations due

to the dynamic disorder in these regions.c Calculated by the PSVS Protein Structure software suite (http://psvs-13.nes-

g.org/).

442 S. Tyukhtenko et al. / Biochemical and Biophysical Research Communications 390 (2009) 441–446

[11–13]. In contrast to other GPCRs [3,5–7], however, the potentialfor short-distance, electrostatic interactions within and betweenhCB1 domains and the putative relationship of such interactionsto hCB1 structure and activation have not been characterized.Notably, hCB1 lacks the H8 phenylalanine F313 residue that con-tributes to an activating molecular switch in rhodopsin througharomatic interactions with TMH7 Y306 [12]. For this reason, a struc-turally identical activation mechanism for both hCB1 and rhodop-sin cannot be postulated.

The present work is focused on determining the solutionstructure of the hCB1 TMH7/H8 region and identifying structuralfeatures that might participate in hCB1’s conformational re-sponse to activation. For this purpose, we have studied by nucle-ar magnetic resonance (NMR) spectroscopy a synthetic peptide40-mer (T377–E416) [hCB1(TMH7/H8)], encompasing the con-joined hCB1 TMH7 and H8 regions, in trifluoroethanol (TFE)/H2O [14,15]. Peptides representative of defined polytopic GPCRsegments have proven useful for interrogating GPCR structureand inferring structure–function relationships [16]. Our data de-fine the overall structural signature of hCB1(TMH7/H8) in solu-tion and allow information to be extracted regarding favorableelectrostatic interactions having potential importance to hCB1regional architecture and overall function.

Materials and methods

Peptide synthesis and purification. The numbering system ofBramblett et al. is employed [11]. The 40-mer [377TVFAFCSM-LALLNSTVNPIIYALRSKDLRHAFRSMFPSAE416] peptide, hCB1(TMH7/H8), was synthesized by a standard 9H-fluoren-9-ylmethoxycar-bonyl (Fmoc)-polyamide method at the Molecular Biology CoreFacility, GenScript Corporation (Scotch Plains, NJ, USA). To opti-mize spectral resolution and preclude confounding disulfide for-mation/peptide dimerization, two conservative substitutions(C386A and C415A) were made, as indicated in the above sequence.The peptide was isolated by reverse-phase LC to >95% purityaccording to LC and MALDI-TOF mass spectrometry analyses. Theprocedure yielded 23 mg of purified hCB1(TMH7/H8) peptide witha molecular mass of �4.5 kDa.

Sample preparation and NMR experiments. For 1D and 2DNMR, hCB1(TMH7/H8) (1.0 mM final conc.) was dissolved in30% (v/v) aqueous TFE-d2. 2-Dimethyl-2-silapentane-5-sulfonicacid was added as reference standard. All 1H-NMR spectra wererecorded at 37 �C on a 700-MHz Bruker AVANCE II NMR spec-trometer. Complete sequential assignments were made using2D TOCSY and NOESY experiments. For spin system identifica-tion, TOCSY spectra with mixing times of 70, 80, and 90 msand a decoupling in the presence of scalar interactions (DIPSI)composite pulse sequence for mixing were obtained. NOESYspectra with 150, 200, and 250 ms mixing times were acquiredin phase-sensitive mode. TOCSY and NOESY analyses incorpo-rated the WATERGATE 3-9-19 pulse sequence with gradientsfor water suppression.

hCB1(TMH7/H8) NMR structural analysis. All NMR spectra wereprocessed using Topspin 2.1 (Bruker BioSpin) and visualized usingCARA [17] and CCPN [18] software suites. Nuclear Overhauser ef-fect (NOE) assignments were improved by a KNOWNOE protocol[19]. All resonances were assigned by using interactive interpreta-tion of standard phase-sensitive TOCSY and NOESY NMR spectra[20]. A total of 1224 NOEs were classified as short-, medium-,and long-range (Table 1). Structure calculations were performedby Xplor-NIH [21]. The 18 lowest-energy conformations in explicitwater were obtained by molecular dynamics simulation refine-ment using the Crystallography and NMR System software suite[22,23]. Structures were validated by the PSVS Protein Structuresoftware suite (http://psvs-13.nesg.org/) and visualized with MOL-

MOL [24] and Discovery Studio Visualizer (Accelrys Inc., San Diego,CA, USA). Atomic coordinates have been deposited in the ResearchCollaboratory for Structural Bioinformatics Protein Data Bank (pdbid: 2koe). The NMR data have been deposited in BioMagResBank(accession number 16504).

Results

hCB1(TMH7/H8) NMR solution structure

hCB1(TMH7/H8) dissolved in 30% TFE/H2O yielded 2D TOCSYand NOESY spectra of excellent quality, allowing almost completesequence-specific assignment. There was no evidence of polypep-tide precipitation or change in the NMR spectra during data acqui-sition. The assignment for each individual residue and the NH-a, b,c connectivity for each assigned residue are shown in Fig. 1A. TheNH–NH region of the NOESY spectrum with sequential NOE con-nectivities is shown in Fig. 1B. Identification of only one set of sig-nals is indicative of hCB1(TMH7/H8) structural homogeneity inTFE/H2O. Sequential assignment was completed by analyzingcross-peak patterns in the fingerprint region of the NOESY spec-trum. NOEs calculated from the NOESY spectrum, as well as Hachemical shift indices (CSIs), are shown in Fig. 2. The strongsequential NHi–NHi + 1 connectivities in the V378–T391 region andthe many medium-range aHi–NHi + 3 NOEs, together with a aHi–NHi + 4 and several aHi–bHi + 3 NOEs, are indicative of an a-helicalregion spanning these residues. Evidence for the a-helical nature ofV378-T391 is strengthened by the long continuous bend of negativeCSI values therein. A series of strong sequential NHi–NHi + 1 NOEsand the presence of several aHi–NHi + 4 and aHi–bHi + 3 NOEs indi-cate that D403–R409 is also a-helical. These a-helices represent foot-prints for the predicted hCB1 TMH7 and H8 regions, respectively[11–13]. The initial N-terminal residue (T377), the 11 residues fromV392 to K402, and seven residues at the peptide’s C-terminal end(S410–E416) are nonhelical, as evidenced by the low number of in-ter-residue NOEs in these regions.

The NMR results were used to analyze hCB1(TMH7/H8) struc-tural details. Superimposition of the 18 lowest-energy conformers

Fig. 1. (A) The amide region of the TOCSY spectrum for hCB1(TMH7/H8) in 30% (v/v) aqueous TFE-d2 (mixing time, 70 ms). The NH-a, b, c connectivity and assignment of eachresidue is labeled and color-coded. Residues 2–25 (V378–S401) corresponding to the TMH7 transmembrane region are blue. Residues from 26 to 35 (K402–M411) correspondingto the cytoplasmic region are red. (B) The amide region of the 2D NOESY spectrum for hCB1(TMH7/H8) in aqueous TFE-d2 (mixing time, 250 ms). The lines map the sequentialassignments of the amide N–H resonances starting at V2 (8.63 ppm). The assignments of inter-residue NHi–NHi + 1 cross peaks are labeled and color-coded. Connectivity from2 to 25 (V378–S401) is colored blue, and from 26 to 35 (K402–M411), red.

Fig. 2. Summary of NOE connectivities observed for hCB1(TMH7/H8). Sequential, midrange, and long-range NOE connectivities are linked by line segments. Strength isindicated by box height and line thickness. The chemical shift indices for Ha protons are also shown. Negative values indicate a helical conformation.

S. Tyukhtenko et al. / Biochemical and Biophysical Research Communications 390 (2009) 441–446 443

is shown in Fig. 3A, and the ensemble statistics are summarized inTable 1. In our structure, eight residues predicted [11–13] to behelical in the hCB1 TMH7 a-helix (V392–L399) and the initial threeresidues predicted [11–13] to be helical in the H8 a-helix (R400–K402) are nonhelical, even in the helix-inducing [14] TFE/H2O sol-vent. The NPxxY motif is a component of this 11 amino-acid,loop-like hCB1(TMH7/H8) region, and the proline residue inducesa kink. The two hCB1(TMH7/H8) a-helices are oriented approxi-mately orthogonally to each other at either side of the interposedloop-like region. The ribbon representation of the meanhCB1(TMH7/H8) solution structure (Fig. 3B) depicts the peptide’smajor structural domains: the TMH7 and H8 a-helices; a loop-likesegment between the two a-helices with a pronounced Pro-kink;and an unstructured C-terminal end. The orthogonality betweenTMH7 and H8 suggests the presence of interhelical amino-acidinteractions.

Noncovalent interactions in hCB1(TMH7/H8)

The hCB1(TMH7/H8) NMR solution structure was examined toevaluate potential, short-distance amino-acid interactions residingtherein. The R400–E416 hCB1(TMH7/H8) region containing H8 fea-tures five hydrophilic residues (R400, K402, R405, H406, and R409) ori-ented to form a compact cationic cluster, contralateral to which is ahydrophobic ‘‘face” of clustered nonpolar residues (L404, A407, F408,M411, F412, and P413) (Fig. 4A). These cationic and hydrophobic clus-ters lend an amphipathic character to both H8 and the entirehCB1(TMH7/H8) C-terminus. NOE interactions between the nega-tively-charged D403 (HN, Ha, Hb1, Hb2) and positively-chargedH406 (Hd2) side chains are indicative of a salt bridge between thesetwo proximal (2.6 Å) H8 amino-acid residues (Fig. 4B).

The hCB1(TMH7/H8) NMR solution structure suggests hydro-gen-bonding involving the peptide’s loop-like region. Specifically,

Fig. 3. (A) Eighteen lowest-energy hCB1(TMH7/H8) structures are displayed. Only backbone atoms are shown. (B) Ribbon depiction of the mean hCB1(TMH7/H8) structure.

Fig. 4. (A) Clustering of cationic and hydrophobic residues in the hCB1(TMH7/H8) cytoplasmic region. Cationic residues are blue; hydrophobic residues, yellow; anionicresidues, red; polar residues, purple. (B) The H8 salt bridge between D403 and H406. (C) An interhelical microdomain (circled and expanded) reflects potential cooperativecation–p and cation–phenolic OH interactions (———) between proximal (<4.5 Å) TMH7 Y397 in the NPxxY motif and H8 R405. Potential hydrogen-bonding interations of Y397

with L399, R400, N393 are also indicated (///////).

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hydrogen bonds between the NPxxY Y397 backbone carbonyl andthe backbone amides of both L399 and R400 as well as the side-chainN393 amino group would help position Y397 for electrostatic inter-action between its hydroxyl moiety and the cationic �NH2

+ groupof R405 in H8, thereby establishing a hydrogen-bond network(Fig. 4C). An additional hydrogen-bond may exist between theN389–NH2 and the N393 carbonyl oxygen (not shown).

The hCB1(TMH7/H8) NMR solution structure is also suggestiveof cooperative interhelical cation–p and cation–phenolic OH inter-actions which may define a microdomain (Fig. 4C). The close prox-imity (<4.5 Å) of the p-electron cloud of the Y397 aromatic ringwithin the TMH7 NPxxY motif to the side-chain cationic aminogroup of H8 R405 implies a possible cation–p interaction [25] be-tween these residues. Existence of this cation–p binding force issubstantiated experimentally by the NOESY cross peaks betweenY397 (Hb2 and He) and R405 (Hd2 and Ha), in that order. ThehCB1(TMH7/H8) NMR solution structure further suggests a cat-ion–phenolic OH hydrogen-bond between Y397 and R405. Solventexposure of R405 is considerable, whereas Y397 is both relativelyshielded (�20% maximum solvent-accessible surface) and con-

formationally restricted by the hydrophobic V392, I395, and I396 sidechains. Van der Waals contacts of I395 and I396 with Y397 tend to ori-ent the Y397 phenol moiety toward the R405 guanidinium moietyfor cation–p interaction between Y397 and R405. Guanidiniumgroups in amino-acid residues have a propensity for hydrogen-bond donation to electronegative atoms such as a tyrosine pheno-lic oxygen [26]. Hydrogen-bonding between Y397 and R405 wouldalso help stabilize the orientation of the Y397 aromatic ring andsimultaneously assist in positioning the R405 guanidinium groupperpendicularly to it. In turn, the electron-deficient R405–CH2 di-rectly adjacent to the R405 guanidinium group would reside overthe center of the Y397 aromatic ring, distinguishing the cation–pinteraction between Y397 and R405 in hCB1(TMH7/H8) from thetypical cation–p interaction that centers the arginine guanidiniummoiety over the aromatic ring [25].

Discussion

Important roles in hCB1 activation and hCB1-G protein couplinghave been attributed to the hCB1 TMH7/H8 region in the absence

S. Tyukhtenko et al. / Biochemical and Biophysical Research Communications 390 (2009) 441–446 445

of high-resolution, experimental characterization of this GPCR’sstructure [1,9,11–13]. We have determined the NMR solutionstructure in TFE/H2O of hCB1(TMH7/H8), a synthetic polypeptideencompassing the hCB1 segment that includes TMH7 and a con-joined cytoplasmic extension containing H8. Whereas the generalimportance of electrostatic amino-acid interactions to protein sec-ondary structure, substrate/ligand recognition, and catalysis is wellrecognized [27], their potential occurrence within hCB1 as intrinsicdeterminants of hCB1 conformation/function is not known. Thestructural insights garnered with our model system have also al-lowed information to be extracted regarding potential electrostaticforces within hCB1(TMH7/H8).

Several features of the overall hCB1(TMH7/H8) NMR solutionstructure have been identified: a lengthy hydrophobic a-helicalsegment (TMH7); a short amphipathic a-helix (H8) orientedorthogonally to TMH7 and containing an intrahelical salt bridgeand both cationic and hydrophobic amino-acid clusters; a loop-like segment of 11 residues interposed between the two a-helicesthat features a pronounced Pro-based kink and includes the con-served [1,5–7] NPxxY motif and a hydrogen-bonding network;and an unstructured C-terminal end. The TMH7 NPxxY motif inbovine rhodopsin and the human b2 adrenergic receptor exhibitsa Pro-kink and a non-canonical distortion and has been linked tothe activating structural responses of these two GPCRs [1,3,28–30]. The NMR solution structure of hCB1(TMH7/H8) provides evi-dence for a more extensive, loop-like segment that includes aP394-induced kink and involves up to 11 amino-acid residues con-joining the TMH7 and H8 a-helices. Unlike the comparatively cir-cumscribed molecular motion afforded by NPxxY-associated Pro-kinks considered typical of GPCRs [1,28,29], the additional flexi-bility afforded by this hCB1 loop may be important to the pur-ported involvement of the NPxxY motif and nearby residues inthe conformational dynamics associated with hCB1 activation[11–13]. Hydrogen-bonding potential within the hCB1(TMH7/H8) loop-like region appears extensive enough to act as a localstructural force that may help coordinate ligand-induced hCB1protein motion with the docking of cytoplasmic G protein sub-units. A critical role in rhodopsin’s conformational response toactivation has been attributed to a hydrogen-bonding networkin that GPCR [31].

Prior NMR structural investigations of hCB1 C-terminal poly-peptides have focused specifically on its cytoplasmic tail includingH8 [32–35]. We demonstrate herein that hCB1(TMH7/H8) H8adopts an a-helical solution structure congruent with its confor-mation in other class-A GPCRs [1,3,8] and similar to the NMR con-formation of a short, synthetic C-terminal rat CB1 peptidereconstituted into phospholipid micelles [33]. The salt bridge be-tween D404 and H407 in that rat CB1 peptide [33] is also reminiscentof the H8 salt bridge between D403 and H406 in a correspondinghCB1(TMH7/H8) region. Other features of rat CB1 C-terminal pep-tides reconstituted in phospholipid or detergent environments[34,35] are shared by the H8 region of hCB1(TMH7/H8) in TFE/H2O: i.e., significant helicity, a cationic amino-acid cluster, andamphipathicity. Conservation of key CB1 H8 characteristics in di-verse environments and the almost complete lack of secondarystructure within a hCB1 H8 peptide in water [32] may be indicativeof this region’s general importance as an activation domain fortransmitting receptor-based structural information to intracellularG proteins, perhaps through hydrogen-bonding associations withspecific G protein subunits [12,36]. In this manner, the H8 ionicclustering evident in the hCB1(TMH7/H8) solution structure mayhelp define the specificity of hCB1-G protein interaction [12].

In models of class-A GPCRs including hCB1, H8 has been ori-ented approximately orthogonally to the hydrophobic TMH7 do-main, juxtaposed parallel to the inner (i.e., cytoplasmic) face ofthe plasma membrane [1,3,12,32]. We have previously shown that

the two hCB1(TMH7/H8) a-helical segments are similarly disposedwhen the peptide is reconstituted and mechanically oriented inphospholipid bilayers [15]. As demonstrated herein, orthogonalitybetween TMH7 and H8 is also characteristic of the hCB1(TMH7/H8) NMR solution structure, suggesting that intrinsic hCB1 proper-ties independent of the plasma membrane help define the recep-tor’s structure (and, by inference, functional responses). In turn,as we have shown previously for peptides representing helical re-gions of both principal cannabinoid GPCRs [37,38], conformationaldeterminants encoded within the hCB1 primary structure willlikely be modulated by a local phospholipid matrix.

p–p interactions between TMH7 NPxxY tyrosine and a H8phenylalanine establish an interhelical microdomain that helpscontrol activating structural changes in rhodopsin and the 5-HT2C receptor [5,6]. An identical p–p structural force is precludedin hCB1, for the hCB1 NPxxY(X)5,6L motif possesses a leucine in-stead of a phenylalanine residue [12]. The present work describesan extended interhelical microdomain in hCB1(TMH7/H8) involv-ing electrostatic forces that pair the NPxxY tyrosine (Y397) withan arginine in H8 (R405) through cooperative cation–p and cat-ion–phenolic OH interactions. Identification of such helix–helixinteractions has recently been highlighted [16] as essential to anunderstanding of polytopic GPCR structure/function. By analogyto rhodopsin and the 5-HT2C receptor [5,6], it is tempting tohypothesize that reversible changes in this hCB1 interhelicalmicrodomain may represent an important feature of hCB1 activa-tion, contributing to ligand-induced transitions among distincthCB1 conformers.

In summary, the NMR solution structure of hCB1(TMH7/H8), a40-mer that encompasses hCB1 TMH7 and H8, has been character-ized. In solution, the peptide evidences a hydrophobic a-helix(TMH7) oriented approximately orthogonally to a shorter, amphi-pathic a-helix (H8); an interposed, loop-like segment that featuresa Pro-kink and includes the conserved NPxxY motif; and anunstructured C-terminal end. The hCB1(TMH7/H8) NMR solutionstructure suggests electrostatic amino-acid interactions includingan intrahelical salt bridge in H8 and a hydrogen-bonding networkinvolving the peptide’s loop-like region. Potential cation–p andcation–phenolic OH interactions between Y397 in TMH7 NPxxYand H8 R405 would promote formation of a stable interhelicalmicrodomain which, by analogy to other GPCRs [5,6], could partic-ipate in hCB1 conformer transitions. These data provide the basisfor mutational studies directed at specific hCB1(TMH7/H8) ami-no-acid residues to probe the potential impact of their electrostaticinteractions on hCB1 function [31].

Acknowledgments

This study was supported by NIH/National Institute on DrugAbuse Grants DA009158-10S2 (A.M./E.K.T.) and DA3801 (A.M.).

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