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The chemokine receptor CXCR4 controls cell migration duing immune surveillance and development of the cardicular hematopoietic and central nervous systems Like many other chemokine receptors CKR ID: 941980

chemokine cxcr4 fig vmipii cxcr4 chemokine vmipii fig receptor cxcl12 cxc medlinedoi residues structure doi medline terminus binding interactions

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earch Articles The chemokine receptor CXCR4 controls cell migration duing immune surveillance and development of the cardicular, hematopoietic, and central nervous systems ). Like many other chemokine receptors (CKRs), CXCR4 cotributes to inflammatory diseases and cancer (). It also functions as one of two coreceptors that facilitate entry of HIV into host immune cells (). Despite the importance of CXCR4 and CKRs Crystal structure of the chemokine receptor CXCR4 in complex with a viral chemokine Ling Qin,* Irina Kufareva,Lauren G. Holden,* Chong Wang,Yi Zheng,Chunxia Zhao,Gustavo Fenalti,Huixian Wu,Gye Won Han,Vadim Cherezov,‡ Ruben Abagyan,Raymond C. Stevens,2†‡Tracy M. Handel1†University of California, San Diego, Skaggs School of Pharmacy and Pharmaceutical Sciences, La Jolla, CA 92093, USA. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. sciencemag.org/content/early/recentJanuary/ Page 10.1126/science.1261064 CXCR4(D187C):vMIPII(W5C) for crystallization in lipidic cubic phase (LCP) (), and determined the structure at 3.1 Å resolution. Data collection and refinement statistics are shown in table S1.Overall complex geometryIn complex with vMIPII, CXCR4 possesses the typical seven TM helical topology. Whereas previous dimeric structures of CXCR4 suggested that chemokines might bind receptors in a 2:1 CKR:chemokine stoichiometry (), the present structure demonstrates that the stoichiometry is 1:1, in agreement with a recent study (). The chemokine interacts via its globular core with the receptor N terminus [chemkine recognition site 1, CRS1 ()] and via its N terminus with the receptor TM pocket (CRS2) (Fig. 1C). Clear electron density is observed for the entire chemokine N terminus, including the CXCR4(D187C):vMIPII(W5C) disulfide bond,which adopts a favorable geometry (Fig. 1D). Residues 1of the receptor are not visible in the density, consistent with the moderate stability of the CRS1 interaction between CXCR4 and vMIPII as suggested by disulfidetrapping eperiments (fig. S2) and prior mutagenesis studies (Molecular interactions between CXCR4 and vMIPThe CXCR4:vMIPII interaction is mediated by an extensive (1330 Å) contiguous interface, with every residue in the chemokine N terminus and Nloop (1LGASCHRPDKCCLGYQ16) contacting the receptor (Fig. 2 and table S2). Although parts of the interface can be classfied as CRS1 or CRS2, the absence of a distinct boundary prompted introduction of an intermediate region, CRS1.5 (Fig. 2, A and B). The CRS1 interaction involves CXCR4 Nterminal residues 23SMKEP27 packing against the chemkine Nloop (residues 13LGYQ16) and its third strand (residues 49QVC51) (Fig. 2, C and D, and table S2). This interaction continues toward CRS1.5 where receptor resdues 27PCFRE31 bind to chemokine residues 8PDKCC12 (Fig. 2, C and D) and form an antiparallel sheet. In CRS2, the chemokine N terminus makes hydrogen bonds to recetor residues D972.63, D2626.58and E2887.39and numerous van der Waals packing interactions (Fig. 2, C and D, and tab

le S2). Most of the interacting CXCR4 residues are known dterminants of either vMIPII binding (table S3) or CXCL12 binding and activation (). The dominant role of the vMIPII N terminus is supported by the fact that an isolated vMIPII(121) peptide binds CXCR4 with appreciable affinity [190 nM () versus 615 nM for wildtype vMIPII ()], which is dramatically reduced by mutations L1A, R7A, and K10A () (table S3). Notably, a W5A mutation has only a moderate effect (). Disulfidetrapping studies also support the role of the chemokine Nloop (fig. S2).Comparison of CXCR4:vMIPII with previous struturesThe conformation of the observed part of the receptor N terminus differs significantly from previous smallmolecule and peptidebound structures (), in that it adopts an orentation almost perpendicular to the membrane to form a sheet interaction in CRS1.5 with chemokine residues C11C12 (Fig. 3, A and B). To accommodate this change as well as binding of the chemokine N terminus in the TM pocket, the extracellular half of helix I is laterally shifted outwards by ~2.4 Å, forming an extra helical turn and bending at the top (Fig. 3A). ECL2 forms a hairpin as in other CXCR4 structures but is more closed onto the binding pocket (Fig. 3A), bringing D181 and D182 of CXCR4 in close proximity with K10 of vMIPII (Fig. 2, C and D).The binding pocket of CXCR4 is open and negatively charged (Fig. 3C), and can be separated into a major and minor subpocket (). Similar to the small molecule antaonist, IT1t, the chemokine N terminus makes the majority of contacts in the minor subpocket and makes polar interations with D972.63and E2887.39(Fig. 3, C and D). By contrast, the spatial overlap between the vMIPII N terminus and CVX15 is moderate, with common recognition determinants including D187ECL2and D2626.58(Fig. 3, C and E). The limited overlap between CVX15 and the chemokine N terminus may enable the design of modulators that simultaneously occupy the minor andmajor subpockets; in fact, a series of CXCR4 ligands obtained by grafting the N terminus of CXCL12 onto a peptide analog of CVX15 () may bind CXCR4 in this manner.As in five earlier structures (), CXCR4 forms a dimer in the vMIPIIbound form (Fig. 4A). The preservation of simlar dimerization patterns in all CXCR4 structures (Fig. 4B) suggests possible physiological relevance and is consistent with numerous reports of CXCR4 homoand heterodimerzation in cells [() and references therein]. The structure also suggests that a receptor dimer can accommodate two monomeric chemokine ligands.Structure comparisons, bioinformatics, and homology modeling insights into the specificity of CC and CXC chemokine recognition by CKRsWith the exception of atypical CKRs, human CC and CXC chemokines generally pair exclusively with CKRs from the same subfamily. To gain insight into this specificity, as well as the noncanonical pairing of a human CXC receptor (CXCR4) with a viral CC chemokine (vMIPII), structural andsequence analyses (fig. S4) were complemented by mlecular modeling (). A complex between CXCR4 and its endogenous CXC chemokine, CXCL12, as well as a complex between vMIPII a

nd another human CKR, CCR5, were chsen for analysis due to available structural and mutagenesis information.An initial systematic analysis of chemokine structures revealed conformational differences between CC and CXC motifs of the respective chemokines: while in CC chemkines, this region is straight and forms sheet interactions within chemokine dimers, it is bent in CXC chemokines and sciencemag.org/content/early/recentJanuary/ Page 10.1126/science.1261064 forms no substantial proteinprotein interface contacts (fig. S4A). This difference is reflected in the CRS1.5 interactions of the structure and the modeled complexes (Fig. 5, A to C). In the CXCR4:CXCL12 model (Fig. 5A), the bend directs the chemokine N terminus toward receptor helices V/VI and enables hydrogen bonding between chemokine R8 (highly conserved as a base in CXC but not CC chemokines) (fig. S4B) and receptor D2626.58(highly conservedas an acid in CXC but not CC CKRs) (fig. S4C). By contrast, in the CCR5:vMIPII model (Fig. 5C), the straightened confomation of the chemokine CC motif directs the chemokine N terminus along the receptor N terminus toward helix I, aied by interactions with receptor K22 in position C+2 (where C is the conserved Nterminal cysteine) and with D2767.32Notably a base in position C+2 and an acid in position 7.32 are both highly conserved in CC but not CXC CKRs (fig. S4C). Furthermore, mutation of K22 or D2767.32in CCR5 arogates binding to vMIPII, CCL3, and CCL5 (). Interest-ingly, both vMIPII and CXCR4 possess features that are atypical for their respective classes; vMIPII has three basic residues (H6, R7, K10) in its proximal N terminus (fig. S4B) and CXCR4 has a base (R30) at C+2 (fig. S4C), which may partially explain the unusual coupling between CXCR4 and vMIPII.Relevant differences between CC and CXC families are also observed in the predicted CRS1 interactions. The preence of sulfotyrosines sY14and sY15 () in proximity of the conserved Nterminal cysteine in CCR5 (fig. S4C) facilitates interactions with basic residues in the vMIPII Nloop (K17 and R18) and loop (R46 and R48) (Fig. 5C). When evaluated familywide, high acidity and sulfotyrosine cotent of the proximal N terminus are characteristic of CC but not CXC receptors (fig. S4C), whereas the basic nature of Nand loops distinguishes CC from CXC chemokines (fig. S4B). It appears therefore, that even when sulfotyrosines in theN terminus of CXC receptors contribute to chemokine affinity, they do not engage the Nor loops of CXC chemokines. Consistent with this notion, CXCR4 sY21 is predicted to interact with the CXCL12 Nloopstrand junction (Fig. 5A) instead of the neutral Nand loops, similar to positions of sulfate groups in multiple CXCL12 structures (). The cleft defined by the Nand loops of CXCL12 is occupied by the backbone of CXCR4 reidues S23M24, which closely mimic the interaction of a small molecule CXCR4:CXCL12 inhibitor (). CXCR4 is a rare CXC receptor that possesses a sulfotyrosine in the proimal N terminus (position C7) (fig. S4C), which may eplain its unique ability to engage a CC chemokine (vMIPII) via its basic Nloops. This engag

ement is further assisted by a 4residue epitope in the chemokine strand that is strictly conserved between vMIPII (48RQVC51) and CXCL12 (47RQVC50) and that interacts with receptor D22 and E26 (Fig. 5B), both of which are importantfor vMIPII and CXCL12 recognition (The CXCR4:vMIPII structure can also explain why CXC ) but not CC () chemokines bind and activate their rceptors as dimers. CC chemokines dimerize by sheet iteractions between the straight CC motifs anterminal residues (Fig. 6A). This largely coincides with the CRS1.5 interaction in the CXCR4:vMIPII structure, making it stercally impossible for a CC chemokine to simultaneously bind its dimer partner and a receptor (Fig. 6B). By contrast, CXC chemokines dimerize by their strands (Fig. 6C), which are not involved in receptor interactions and therefore compaible with the geometry of the CKR:chemokine complexes (Fig. 6D). This model also suggests that CXC chemokine dimers likely bind to single receptor subunits (Fig. 6E) and not to both subunits in a dimer as previously hypothesized Modelingbased insights into agonist versus antagonist chemokine binding to CXCR4CXCL12 can be converted into a potent antagonist of CXCR4 by as little as a single Nterminal aminoacid substitution (P2G) (). To investigate the basis for this dramatic change in pharmacology, modeling of CRS2 interactions for both CXCL12 and CXCL12(P2G) with CXCR4 was performed. With both chemokine variants, the four distal Nterminal residues were predicted to bind in the minor subpocket of CXCR4 in a manner similar to vMIPII (Fig. 5, D and E). The terminal and sidechain amines of chemokine K1 were predicted to form hydrogen bonds to receptor residues D972.63and E2887.39respectively, while chemokine residues S4 (in CXCL12) and Y7 [in both CXCL12 and CXCL12(P2G)] hydrogenbond to D187ECL2. Notably, K1 in CXCL12 and D972.63, D187, E2887.39in CXCR4 are all critical for receptor interaction and activation (). In CXCL12, the sidechain of P2 was found in proximity of receptor residue Y1163.32(Fig. 5D) whose direct interaction with agonists is frequently involved in activation of GPCRs (). By contrast, due to its greater flexibility and smaller steric volume, the S4 region of CXCL12(P2G) packed differently (Fig. 5E), avoiding interaction with Y1163.32and potentially explaining the inability of CXCL12(P2G) to activate CXCR4. However, because docking was performed with an inactive receptor conformation, further structural studies will be necessary to fully understand activation mechanisms.Chemokine receptor plasticity, promiscuity, and implcations for drug designCXCR4 is remarkable in its ability to recognize multiple urelated small molecules, peptides, and proteins. While egaging a conserved set of binding determinants, the ligands occupy different regions of the binding pocket due to recetor conformational plasticity involving receptor sidechain and backbone adjustments. Such versatility may allow the receptor to accommodate ligands of different classes, icluing both CC and CXC type chemokines as well as allsteric inhibitors. The growing number of chemokine receptor structures wit

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L.G.H. is supported by a 2012 Postdoctoral Fellowship in Pharmacology/Toxicology from the Pharmaceutical Research and Manufacturers of America (PhRMA) Foundation. The GM/CACAT beamline (23ID) is supported by the National Cancer Institute (Y11020) and the National stitute of General Medical Sciences (Y11104).UPPLEMENTARY MATERIALSwww.sciencemag.org/cgi/content/full/science.1261064/DC1Materials and MethodsFigs. S1 to S4Tables S1 to S3References (4110 September 2014; accepted 6 January 2015Published online 22 January 201510.1126/science.1261064 sciencemag.org/content/early/recentJanuary/ Page 10.1126/science.1261064 Fig. 1. Design and crystallization of a disulfide - trapped CXCR4:vMIP - II complex. ( A ) Nonreducing SDS - PAGE and Western blot of CXCR4(D97C) (left) and CXCR4(D187C) (right) coexpressed with cysteine mutants of vMIPII (residues 17). Uncomplexed CXCR4 and disulfidetrapped complexes have molecular weights of approximately 45 kDa and 55 kDa, respectively. Band identities were confirmed by Western blot using antibodies against the FLAG and HA tags at the Nand Ctermini of CXCR4 and vMIPII, respectively (2nd and 3rd row). The 55 kDa band was labeled by antiFLAG and antiHA antibodies (2nd4th row); the band at 45 kDa was only labeled by the antiFLAG antibody (2nd and 4th row). ( B ) Thermal stability of the complexes measured by a CPM assay () are shown as mean ± SEM measurements performed in triplicate. ( C ) Overallstructure of the CXCR4:vMIPII complex (gray:magenta ribbon and transparent mesh). ( D ) Zoomed view of the vMIPII N terminus in the CXCR4 pocket showing the CXCR4(D187C):vMIPII(W5C) disulfide bond. The 2mFoDFc electron density map around the N terminus is contoured at 1.0 and colored blue. sciencemag.org/content/early/recentJanuary/ Page 10.1126/science.1261064 Fig. 2. Interactions between CXCR4 and vMIP - II. ( A and B ) The interaction is mediated by a contiguous interface containing CRS1 (green), CRS2 (red) and CRS1.5 (blue). (A) The receptor is shown as a cutopen surface, the chemokine is shown as a ribbon, chemokine residues making substantial contacts with the receptor are shown as sticks. (B) The receptor is shown as a ribbon, receptor residues making substantial contacts with chemokine are shown as sticks, and vMIPII is shown as a surface mesh. ( C ) Key residues (gray sticks) from CXCR4 (ribbon) that bind vMIPII (surface representation). ( D ) Key residues (magenta sticks) from vMIP(white ribbon) that bind CXCR4 (cutopen surface). Noncarbon atoms are red (O), blue (N), and yellow (S); carbon stick color intensity is indicative of residue contact strength (table S2). sciencemag.org/content/early/recentJanuary/ Page 10.1126/science.1261064 Fig. 3 . Comparison between CXCR4:vMIP - II and earlier CXCR4 structures. ( A ) Overlay of CXCR4 in the vMIPII complex (gray), the IT1t complex (PDB ID 3ODU; cyan), and the CVX15 complex (PDB ID 3OE0; pale green). vMIPII is shown as a gray transparent mesh. ( B ) CRS1 interaction between CXCR4 (gray) and vMIPII (magenta), in comparison with IT1tbound (cyan) and CVX15bound (green) structu

res. Keyresidues mediating the CXCR4:vMIPII interactions are shown as sticks. ( C ) Binding modes of vMIPII, IT1t and CVX15 to CXCR4. CXCR4 is shown as a cutopen surface, colored by electrostatic potential; the bound ligands are shown as spheres. The white dotted line represents the boundary between the major and minor subpockets. ( D and E ) Comparison of CRS2 interactions of vMIP - II (magenta) with IT1t (cyan) and CVX15 (green). sciencemag.org/content/early/recentJanuary/ Page 10.1126/science.1261064 Fig. 4. Crystallographic dimer of CXCR4:vMIP - II. ( A ) The overall dimer geometry i s similar between CXCR4:vMIPII (gray:magenta), CXCR4:IT1t (PDB ID 3ODU, light cyan:dark cyan), and CXCR4:CVX15 (PDB ID 3OE0, light green:green). () In all CXCR4 complexes solved thus far, the interaction between two CXCR4 molecules (ribbon) is mediated by the top halves of helix V, with additional contacts provied by either intracellular parts of helices III and V, or the top halves of helix VI (spheres). Views of the dimer interface on one monomer are shown for CXCR4:vMIPII, CXCR4:IT1t, and CXCR4:CVX15plexes. Residues that contribute to dimer formation are shown as spheres; color intensities represent contact strength. sciencemag.org/content/early/recentJanuary/ Page 10.1126/science.1261064 Fig. 5 . Molecular models of homologous receptor:chemokine complexes: CXCR4:CXCL12 ( A and D ), CXCR4:vMIPII ( B ), CCR5:vMIPII ( C ), and CXCR4:CXCL12(P2G) ( E ). Panels (A) to (C) focus on CRS1 and CRS1.5 interactions while panels (D) and (E) show predicted CRS2 interactions. The dotted line indicates the approximate extracellular membranesolvent boundary. (B) A model of WT CXCR4 sY2F304 (gray) is built in complex with WT vMIPII (magenta). Despite the absence of the D187CW5C disulfide bond, the predicted interactions coincide precisely with those observed in the xray structure. vMIPII W5 provides additional packing interaction with ECL2 of CXCR4. (A and C) Models of CXCR4:CXCL12 (gray:orange) and CCR5:vMIPII (navy:magenta). (D and E) Models of CXCR4:CXCL12 (gray:orange) and CXCR4:CXCL12(P2G) (gray:green). sciencemag.org/content/early/recentJanuary/ Page 10.1126/science.1261064 Fig. 6 . Implications of the CXCR4:vMIP - II structure for understandin g the stoichiometry of receptor:chemokine recognition. ( A ) vMIPII (shown, PDB ID 2FHT) and other CC chemokine dimers are stabilized by sheet formation between the CC region and neighboring residues. ( B ) Superposition of the vMIPII dimer onto the CXCR4:vMIPII structure shows that binding of a CC dimer to the receptor is sterically impossible. ( C ) CXCL12 (shown, PDB ID 3GV3) and other CXC chemokine dimers are stabilized by sheet formation between their strands. ( D ) Superposition of the CXCL12 dimer onto the CXCR4:vMIPII structure shows that binding of a dimer is feasible. ( E ) If the receptor dimer geometry is relevant, and the vMIPII orientation is predictive of CXC chemokine binding, CXC dimers do not simultaneously bind both receptors in a dimer. sciencemag.org/content/early/recentJanuary/ Page 10.1126/science.12610