structural studies were carried out using an engineered hkor construct ( see methods and supplementary fig . 1 ) and crystallized in cholesterol - doped monoolein lipidic cubic mesophase ( see methods ) .
the construct used displayed pharmacological behavior similar to that of a native receptor expressed in hek 293-t cells ( supplementary tables 2 and 3 ) . data collection and refinement statistics are shown in supplementary table 1 .
the structure of hkor - jdtic was determined at 2.9  in the p 212121 space group .
the asymmetric unit ( asu ) consists of a two receptors forming a parallel dimer ( fig .
the dimer interface with ~1100  buried surface area is formed through contacts between helices i , ii and viii ( fig .
parallel receptor dimers have been identified in crystal structures of activated rhodopsin ( involving helices i , ii and viii ) , 2 adrenergic receptor ( 2ar ; cholesterol mediated ) and cxcr4 ( involving helices iv , v and vi ) .
consistent with these crystallographic data , recent biochemical studies have suggested the existence of two dimerization interfaces : along helices iv and v - sensitive to receptor activation , and along helix i  insensitive to the state of activation .
while the orientations of the two t4-lysozyme ( t4l ) copies in the receptor monomers in one asu differ by ~ 60 rotation , both copies of the receptor are highly similar ( fig .
the remaining residues of the n- and c - termini in the hkor / jdtic structure are disordered as in other class a gpcrs .
the main fold of the hkor consists of a canonical 7tm bundle of  helices followed by an intracellular helix viii that runs parallel to the membrane .
structural comparison with other gpcrs suggests that hkor has striking similarities in the ecl region with cxcr4 , another peptide binding receptor in the  subfamily . in the 7tm region , however , the hkor structure is closer to aminergic receptors belonging to the  subfamily ( rmsdc ~2.3  for 2ar , ~1.9  for dopamine d3 receptor ( d3r ) , and ~2.7 for cxcr4 ) .
the structure reveals distinctive features of hkor , including : ( i ) conformation of the extracellular end of helix i that deviates from the position observed in cxcr4 , where the tip of helix i is pulled towards the tm bundle by a disulfide bond between the n - terminus and ecl3 .
( ii ) ecl2 , the largest extracellular loop of hkor , forms a -hairpin similar to that observed in cxcr4 , despite the low sequence similarity in this domain between the two receptors .
conservation of this feature between these peptide receptors suggests that the -hairpin could be a common motif in the ecl2 of other  subfamily receptors , where interactions between ecl2 and their endogenous peptide ligands are deemed important for ligand recognition and selectivity .
( iii ) unlike other solved non - rhodopsin class a gpcrs which have more than one disulfide bond , hkor has only one formed between cys131 ( superscripts indicate residue numbering using the ballesteros - weinstein nomenclature ) and cys210 , bridging ecl2 to the end of helix iii . these two cysteines are conserved in all ors and this disulfide bond is the canonical one shared by all other solved class a gpcrs .
( iv ) intracellular loop 2 ( icl2 ) adopts slightly different structures in the two hkor molecules in the asu , involving a two - turn  helix in molecule b , and only a one - turn  helix in molecule a ( supplementary fig .
( v ) ecl3 of hkor is disordered . of the approximately eleven residues in this loop (
residues 300310 ) , six residues in molecule a and three in molecule b do not have interpretable electron density . a common feature of the class a gpcrs is the presence of a conserved sequence motif asp / glu - arg - tyr ( d / ery ) located at the cytoplasmic end of helix iii .
a salt bridge interaction between arg and asp / glu from the cytoplasmic end of helix vi constitutes an  ionic lock  , which is thought to stabilize the inactive conformation of rhodopsin and other rhodopsin - like class a gpcrs , while its absence can enhance constitutive activity .
although hkor lacks either of the acidic residues asp / glu at position 6.30 , arg156 forms a hydrogen bond to another helix vi residue , thr273 ( supplementary fig .
3a ) in this inactive hkor structure , thereby conceivably stabilizing the inactive receptor conformation .
the npxxy motif located at the cytoplasmic side of helix vii , which is composed of asn326 , pro327 , ile328 , leu329 and tyr330 in hkor , is another highly conserved functional motif that is proposed to act as one of the molecular switches responsible for class a gpcr activation .
comparison of hkor with inactive 2ar and a2a adenosine receptor ( a2aar ) structures ( supplementary fig .
3b ) reveals a similar conformation of this motif in these receptors , thereby supporting the hypothesis that the observed hkor - jdtic complex structure corresponds to the inactive state . to further establish that jdtic stabilizes an inactive conformation
, we evaluated its ability to modulate gi - mediated and -arrestin - mediated signaling in transfected hek293-t cells .
we found that jdtic was devoid of agonist activity at both canonical and non - canonical pathways and completely blocked the effects of the prototypic agonist u69593 ( supplementary fig .
the hkor ligand binding pocket displays a unique combination of key characteristics both shared with and distinct from those in the chemokine and aminergic receptor families . while the hkor binding pocket
is comparatively large and partially capped by the ecl2 -hairpin , as in cxcr4 , it is also much narrower and deeper than in cxcr4 ( fig .
in addition to a different set of side chains lining the pocket , the shape differences result from an approximately 4.5  inward shift of the extracellular tip of helix vi in hkor as compared to cxcr4 .
6 ) , which reaches deep into the pocket to form ionic interactions with the asp138 side chain ( fig .
the asp residue is conserved in all aminergic gpcrs , thereby playing a critical role in the selectivity of aminergic receptors toward protonated amine - containing ligands .
asp is conserved in all ors and for which modeling and mutagenesis studies suggest an essential role in anchoring positively charged hkor ligands .
jdtic , developed as a derivative of the trans-(3r,4r)-dimethyl-4-(3-hydroxyphenyl ) piperidine scaffold , has exceptionally high affinity ( ki = 0.32 nm ) , potency ( ki = 0.02 nm in gtps assays ) , long duration of action and a more than 1000-fold selectivity for hkor as compared to other or subtypes .
extensive structure  activity relationship ( sar ) analyses performed on jdtic analogues have yielded important insights into key determinants of jdtic activity , although reliable identification of the interaction mode(s ) and contact residues of these ligands has not been feasible without a receptor crystal structure .
jdtic shows a tight fit of the ligand in the bottom of the binding cleft ( fig .
2a ) , forming ionic , polar , and extensive hydrophobic interactions with the receptor ( fig .
the protonated amines in both piperidine and isoquinoline moieties of the ligand form salt bridges to the asp138 side chain ( 3.0 and 3.1  n  o distances , respectively ) .
the piperidine amine is a part of the original trans-(3r,4r)-dimethyl-4-(3-hydroxyphenyl)piperidine scaffold , and is essential for binding .
sar studies of jdtic analogues show that the isoquinoline nitrogen can be replaced by carbon , oxygen or sulfur atoms with only ~10- to 50-fold reduction in affinity .
similar to the observed jdtic conformation in the hkor - jdtic complex , a v - shaped conformation was found in the crystal structure of jdtic by itself and which showed its amino groups coordinating a water molecule ( supplementary fig .
7a ) . while several rotatable bonds within the jdtic molecule allow for the sampling of different conformations ( see supplementary fig .
7b ) and facilitate the ligand passage through the narrow binding pocket entrance , the anchoring - type interaction of two amino groups with asp138 likely fixes the ligand in this characteristic v - shape .
sar studies have also underscored the importance of the distal hydroxyl groups on both the piperidine and isoquinoline moieties of jdtic , the removal of which did result in about a 100-fold reduction of affinity .
a much smaller effect was observed upon methylation of these hydroxyls or their replacement by other polar groups .
these sar results suggest the importance of water - mediated interactions between these two hydroxyl groups and the receptor . indeed ,
while the crystal structure does not show direct hydrogen bonding with the receptor for both hydroxyl groups , there is clear electron density for several structured water molecules that mediate their polar interactions ( supplementary fig .
the structure provides important clues for understanding the structural basis for the exceptional subtype selectivity of jdtic . among many extensive contacts , jdtic interacts with four residues of the binding pocket that differ in other closely related ors , which are thought to contribute to the subtype selectivity of jdtic and other kor - selective ligands ( human mor ( hmor ) and human dor ( hdor ) amino acids shown in parentheses , respectively ) : val108 ( ala and ala ) , val118 ( asn and lys ) , ile294 ( val and val ) , and tyr312 ( trp and leu ) ( fig .
analysis of jdtic binding into hkor - based hmor and hdor homology models , as well as jdtic sar results ( supplementary fig .
9 ) , suggest that all described residues can contribute to the jdtic selectivity profile .
thus , changes in the val118 side chain , where larger hydrophilic residues , asn and lys are found in hmor and hdor , respectively , are likely to introduce unfavorable contacts with jdtic . additionally , changing tyr312 to the trp and leu residues found in hmor and hdor , respectively , are likely to result in the loss of an important polar interaction with the jdtic amide .
the remaining two hydrophobic side chains replacements , val to ala at position 2.53 and ile to val at position 6.55 , may cause a reduction of the hydrophobic contact between jdtic and the receptor . the isopropyl group from jdtic reaches deep in the orthosteric pocket to form a hydrophobic interaction with a conserved trp287 side chain ( aka the  rotamer toggle switch  ) , possibly playing a critical role in the pharmacological properties of this ligand .
trp is thought to be a key part of the activation mechanism in many class a gpcrs including rhodopsin and a2aar , and similar hydrophobic contacts have been implicated in blocking activation - related conformational changes in the dark state visual rhodopsin by 11-cis retinal , and by inverse agonists in the a2aar and d3r .
prior mutagenesis and modeling studies suggested that many small molecule opioid ligands can interact with kor , as well as with mor and dor , by forming a salt bridge with the highly conserved asp ( ref ) .
this is consistent with our mutagenesis studies ( supplementary table 3 ) and flexible docking of a series of morphine analogues , including selective kor antagonists nor - bni and gnti ( fig . 3 and supplementary fig .
10 ) . to assess the compatibility of these bulky and rigid ligands with the observed hkor protein backbone conformation , we performed global energy optimizations of nor - bni and gnti in the binding cavity of hkor , keeping side chains of the binding pocket fully flexible .
multiple independent runs consistently resulted in low energy conformations with essentially identical poses and receptor contacts for the common naltrexone moieties of both nor - bni and gnti ( rmsd = 0.85  ) .
in addition to a highly complementary van der waals interface , both compounds formed an amino group salt bridge to the asp138 side chain and a hydrogen bond to the tyr139 side chain , both of which are important anchoring points for binding of morphine - based ligand , as supported by previous mutagenesis studies .
moreover , unlike jdtic , both nor - bni and gnti compounds have a second basic moiety located more than 10  away from the first amino group ( the other morphine in nor - bni and the guanidine moiety in gnti ) . in the predicted models of hkor- nor - bni / gnti complexes , these additional amino groups of both ligands form a salt bridge with glu297 located at the entrance to the ligand binding pocket , which was previously characterized as a residue critical for subtype selectivity of hkor - selective morphinan derivatives .
this interaction is also supported by our mutagenesis results ( supplementary table 3 ) , where a glu297ala mutation induced a significant drop in both nor - bni and gnti binding , but did not affect jdtic binding .
hydrophobic interactions at the kor - specific residue ile294 were also found for both nor - bni and gnti ; consistent with our mutagenesis results ( supplementary table 3 ) and suggesting that ile294 may also be important for developing kor subtype selective morphinan derivatives .
additional polar interactions with hkor - specific residues , glu209 and ser211 in ecl2 , are found for nor - bni , which may further enhance hkor - selectivity of this bulky ligand . another side chain of the pocket , his291 , which is involved in the highly conserved aromatic cluster around trp and thought to play a critical role in the receptor activation process , forms hydrophobic contacts with jdtic , nor - bni and gnti .
his291 can be mutated to another aromatic residue , phenylalanine , without disrupting binding of these antagonists ( supplementary table 3 ) .
the non - conservative his291lys mutation , however , totally abolished binding of all tested ligands , likely because of the disruption of the aromatic cluster induced by the lysine side chain .
interestingly , the cyclopropyl moiety of both nor - bni and gnti in these binding poses has the same position as the isopropyl moiety of jdtic , making hydrophobic contact with the conserved residue trp287 .
this cyclopropyl moiety is generally implicated in conversion of opioid agonists into antagonists ( e.g. agonist oxymorphone into antagonist naltrexone ) , and this effect may be partially explained by a direct interaction with the trp287 side chain .
overall , these structure - based docking results support the  message - address model in applications to morphine - based ligands nor - bni and gnti , which points to glu297 as a key side chain that controls hkor selectivity by anchoring the 
, however demonstrates that even in lieu of  address  interaction with glu297 more than 1000-fold subtype - selectivity to hkor can be achieved for jdtic and some of its derivatives .
importantly , then , the  message - address hypothesis does not uniformly apply to all hkor - selective antagonists .
salvinorin a ( sala ) , a naturally occurring diterpene from the widely abused hallucinogenic plant salvia divinorum , represents an exceedingly potent ( ec50 = 1 nm ) and selective kor agonist ( > 1000-fold ) .
sala is unique compared to other kor ligands in that it lacks a charged or polar nitrogen atom to anchor it in the binding pocket .
extensive site - directed mutagenesis , scam ( substituted cysteine - accessibility mutagenesis ) and sar studies on sala and its analogues have been performed , indicating ( among others ) that the 2-acetoxy moiety interacts with cys315 ( ref ) .
possible modes of interaction between the cysteine - reactive and ultra - potent agonist and sala analog 22-thiocyanatosalvinorin a ( rb-64 ; ki = 0.59 nm ; ec50 = 0.077 nm ) , and the hkor structure were thus evaluated .
exposure of hkor to rb-64 produces irreversibly - bound , wash - resistant adducts that are tethered to cys315 ( ref ) . as the thiocyanate group contains two electrophilic centers ,
two distinct adducts may be formed , increasing the mass by either 463 or 431 amu .
docking studies using gold predict that the salvinorin 2-position can access cys315 while maintaining many of the interactions implicated by site - directed mutagenesis for sala , providing a possible mechanism for the formation of the kor 
additionally , the docking results serve as a model of the initial recognition process of sala - related agonists of the hkor in an inactive state , although additional studies will be needed to fully elucidate the nature of the sala - induced activation mechanism .
the jdtic - hkor crystal structure has uncovered a combination of key features shared with chemokine and aminergic gpcrs along with unique structural details characteristic of the opioid subfamily .
the hkor was crystallized as a parallel - dimer with contacts involving helices i , ii and viii . while the existence of gpcr dimers in vivo and their physiological relevance remain highly debatable , several distinct potential dimer interfaces are starting to emerge from crystallographic and biochemical studies . such multiple dimerization interfaces may serve to support different functional pathways , as well as to promote oligomeric assembly of gpcrs .
analysis of the ligand - receptor interactions has revealed important molecular details responsible for the exceptionally high affinity and subtype selectivity of jdtic  a small molecule antagonist with a broad therapeutic potential .
the elucidation of a large binding cavity with a multitude of potential anchoring points begins to explain both the extreme structural diversity of hkor drugs and differences in their receptor interaction modes , as supported by differential effects of various site - directed mutations on the binding properties of chemically diverse prototypic ligands .
the structure clearly provides a long anticipated molecular framework for understanding opioid drug action , and thereby affords valuable new opportunities for a structure - based discovery of new drugs with ideal pharmacological properties .
sf9 cells were solubilized using 1% ( w / v ) n - dodecyl--d - maltopyranoside ( ddm ) and 0.2% ( w / v ) cholesteryl hemisuccinate ( chs ) , and purified by immobilized metal ion affinity chromatography ( imac ) , followed by reverse imac after cleaving n - terminal flag-10xhis tags by his - tagged tobacco etch virus ( tev ) protease .
the purified protein was mixed with monoolein and cholesterol in a ratio of 40%:54%:6% ( w / w ) to form lipidic cubic phase ( lcp ) from which the receptor was crystallized .
crystals were grown at 20 c in 45 nl protein - laden lcp boluses overlaid by 800 nl of precipitant solutions as described in methods .
x - ray diffraction data were collected on the 23id - b / d beamline ( gm / ca cat ) at the advanced photon source , argonne , il using a 10 m minibeam at wavelength of 1.0330  .
modeling of jdtic analogues and hkor - selective morphine derivatives nor - bni and gnti was performed using icm - pro ; sybyl - x 1.3 and gold suite 5.1 were used to model rb-64 complexes , as described in methods .
full methods and any associated references are available in the online version of the paper at www.nature.com/nature .
 opioid receptors ( ors ) mediate the actions of endogenous and exogenous opioids for many essential physiological processes including regulation of pain , respiratory drive , mood , and , in the case of -opioid receptors ( kor ) , dysphoria and psychotomimesis . here 
 we report the crystal structure of the human kor ( hkor ) in complex with the selective antagonist jdtic , arranged in parallel - dimers , at 2.9 angstrom resolution . 
 the structure reveals important features of the ligand binding pocket that contribute to jdtic s high affinity and subtype - selectivity for hkor . 
 modeling of other important kor - selective ligands , including the morphinan - derived antagonists nor - bni and gnti , and the diterpene agonist salvinorin a analog rb-64 , reveals both common and distinct features for binding these diverse chemotypes . 
 analysis of site - directed mutagenesis and ligand structure - activity relationships confirms the interactions observed in the crystal structure , thereby providing a molecular explanation for hkor subtype - selectivity along with insight essential for the design of hkor compounds with new pharmacological properties . 
Overall architecture of hKOR
hKOR ligand binding pocket
Structural basis of JDTic selectivity
Binding of KOR-selective morphinans
Binding of Salvinorins
Conclusions
Methods Summary
Supplementary Material