article: Chemokine receptors, a subclass of G protein coupled receptors (GPCRs), play essential roles in the human immune system, they are involved in cancer metastasis as well as in HIV-infection. A plethora of studies show that homo- and heterodimers or even higher order oligomers of the chemokine receptors CXCR4, CCR5, and CCR2 modulate receptor function. In addition, membrane cholesterol affects chemokine receptor activity. However, structural information about homo- and heterodimers formed by chemokine receptors and their interplay with cholesterol is limited. Here, we report homo- and heterodimer configurations of the chemokine receptors CXCR4, CCR5, and CCR2 at atomistic detail, as obtained from thousands of molecular dynamics simulations. The observed homodimerization patterns were similar for the closely related CC chemokine receptors, yet they differed significantly between the CC receptors and CXCR4. Despite their high sequence identity, cholesterol modulated the CC homodimer interfaces in a subtype-specific manner. Chemokine receptor heterodimers display distinct dimerization patterns for CXCR4/CCR5 and CXCR4/CCR2. Furthermore, associations between CXCR4 and CCR5 reveal an increased cholesterol-sensitivity as compared to CXCR4/CCR2 heterodimerization patterns. This work provides a first comprehensive structural overview over the complex interaction network between chemokine receptors and indicates how heterodimerization and the interaction with the membrane environment diversifies the function of closely related GPCRs.
  G protein coupled receptors (GPCRs) constitute one of the largest groups of cell surface transmembrane receptors [1]. All members of this protein family share the typical architecture of seven transmembrane helices (TM1-7) connected via three intra- and three extracellular loops (ICL and ECL, respectively). In general, the receptors sense external signals with their extracellular segment, while G proteins couple to the intracellular domain. Usually, an additional amphipathic helix 8 (H8) is formed by residues between the last transmembrane helix (TM7) and the intracellular C-terminus [2]. A small subgroup of class A GPCRs is formed by so-called chemokine receptors [3]. These receptors cater for the targeted migration of cells belonging to the immune system and play an essential role in the inflammatory response [4]. Consequently, chemokine receptors became intriguing candidates for therapeutic targets in inflammatory diseases [5]. In addition, certain chemokine receptors have been reported to play important roles in tumor progression as well as metastasis [6] and were shown to function as co-receptors for the human immunodeficiency virus (HIV) during cell infection [7]. Depending on the relative positions of the N-terminal cysteine residues of the agonist chemokine, typical chemokine receptors can be divided into four subfamilies: XCR1, CCR (1-10), CXCR (1-6), and CX3CR1 [8]. As reported for a continuously growing number of GPCRs [9], several chemokine receptors have been indicated to function as homo- or heterodimers or even oligomers of higher order [10–12]. The assembly of GPCRs was indicated to have important functional consequences, it may e. g. affect the cooperativity between ligand binding sites, alter the intracellular transport of GPCRs, or activate different signaling pathways [13–16]. For class A GPCRs, the interactions between receptors are mainly formed by their transmembrane helices. Consequently, certain membrane components, e. g. cholesterol or polyunsaturated lipids, have been reported to modulate the function and association of GPCRs [17–22]. Notably, cholesterol is frequently required in the crystallization process of GPCRs and several X-ray crystal structures show cholesterol molecules bound between adjacent protomers under crystal conditions [21,23–26]. Especially three chemokine receptors, namely CCR2, CCR5, and CXCR4 have been shown repeatedly to homo- and heterodimerize and the association was indicated to modulate receptor function [27–48]. Homo- and heterodimerization were reported as either ligand-promoted [49–51] or constitutive [35,52,53]. This discrepancy could be coupled to interpretational difficulties since differences in the measured dimerization data—e. g. from Förster or bioluminescence resonance energy transfer studies [54] (FRET or BRET, respectively) —could result from conformational changes in preexisting dimers upon ligand treatment instead of alterations of the binding affinity between receptors [14,21,35]. Furthermore, the modulation of chemokine receptor function by membrane cholesterol has been reported in several experimental studies [53,55–63]. We recently studied the spontaneous assembly of CXCR4 receptors in different membrane compositions [47] using ensembles of molecular dynamics (MD) simulations, in order to unravel the effects of cholesterol on the receptor dimerization at atomistic resolution. Our results revealed that cholesterol affects the homodimerization via binding between TM1 and TM7, thus preventing these helices from engaging in dimer interactions as observed in cholesterol-free membranes. In turn, the intercalation of cholesterol between monomers induced a symmetric interface including TM3 and TM4 of both receptors (TM3,4/TM3,4 dimer) [47]. Interestingly, peptides derived from TM4 of CXCR4 were shown before to affect the dimerization and further to strongly weaken the activity of the receptor [35,53]. The homodimerization of CCR5 was shown in various studies and suggested to impair HIV infection [28]. CCL5 (RANTES), an agonist chemokine of CCR5, was suggested to stabilize receptor dimers [28,32], however, constitutive agonist-independent assembly of CCR5 was reported as well [31,32,37]. In addition, monoclonal antibodies (mAb) were constructed for CCR5 that can stabilize homodimers [28,30]. Ile52 on TM1 and Val150 on TM4 have been proposed as crucial for both, CCR5 dimerization and signaling, since double mutants of the receptor showed neither dimerization nor function [32]. However, these findings were challenged by other experiments [64]. The chemokine receptor CCR2 is closely related to CCR5, the receptors share ≈ 75% overall sequence identity and their transmembrane segments show up to 92% identity (see S1 Fig) [38]. As reported for CXCR4 and CCR5, CCR2 was shown to homodimerize [29,46] and ligand stimulated receptors altered the dimer configuration [35]. Furthermore, similar to CCR5, residues Val64 on TM1 and Ile164 (according to Uniprot—ID: P41597—Ile163) on TM4 were reported to be involved in CCR2 dimerization since double mutants were unable to trigger signaling [32]. In addition, a heptapeptide containing the residues Met61—Leu67 of the TM1 of CCR2 was shown to inhibit CCR2-mediated cell migration by impeding the receptor homodimerization [65]. Besides homodimerization, heterodimerization of CXCR4, CCR5, and CCR2 was observed in numerous experiments and could be connected to modulations of ligand binding and receptor function [12,44]. The constitutive heterodimerization or -oligomerization of these chemokine receptors was reported in several studies [36,38,40,42,66] and ligand-binding to the receptor complexes was described to induce conformational changes in heterodimers [35,40,42,46]. Negative ligand-binding cooperativity of allosteric nature across chemokine receptor heteromers was reported, suggesting that usually only one ligand binds to a complex of receptors which induces conformational changes at the ligand-binding site of the associated receptor [12,38,39,41]. Additionally, heteromerization was reported to enable chemokine receptors to couple to different kinds of G proteins as compared to receptor homomers and monomers [12,29,38,41,46,67]. Especially the assembly of CXCR4 and CCR5 gained a great deal of attention since both receptors can act as co-receptors for HIV. It was shown that each receptor can associate with the transmembrane glycoprotein cluster determinant 4 (CD4) and that these associates are used by HIV to enter the cell. On the other hand, CD4/CXCR4/CCR5 heterotrimers revealed negligible binding to the virus envelope [45]. In addition, a mutant of CCR2 (Val64Ile) was revealed to delay the development of AIDS in HIV-infected individuals potentially due to heterodimerizing with CXCR4 or CCR5 [68]. However, more recent BRET studies could not indentify different heterodimerization affinites between CXCR4 and wildtype CCR2 or the CCR2 Val64Ile variant [35]. Furthermore, inducing the oligomerization of CCR2 with CCR5 and CXCR4 with a specific monoclonal antibody was reported to block HIV-entry [34]. In addition, the depletion of cholesterol from cellular membranes was reported to reduce the ability of HIV to fuse with host cells, likely by affecting the clustering of coreceptors at cell membranes [57,60]. Based on the impact of GPCR dimerization on receptor function, novel therapies are developed to target protein association [13]. Especially the construction of bivalent ligands that are able to bind both receptors in homo- or heterodimeric GPCR complexes have been proposed as potent targets to regulate GPCR function [69–71]. However, the rational design of new drugs targeting GPCR dimers is subject to the availability of receptor complex structures [72]. So far, structural information regarding GPCR homodimer interfaces is most frequently derived from available crystal structures [2]. During the last years, more and more human class A GPCRs have been solved with X-ray crystallography and different dimeric contacts could be observed [21,47]. Thereby, different receptors revealed distinct homodimer interfaces and the aggregation of receptors is likely dependent on the crystallization conditions [73–75]. In addition, certain receptors (e. g. the β2-adrenergic receptor) could be crystallized in different dimeric configurations, indicating a promiscuous association of GPCRs [23,76]. The structures of the chemokine receptors CXCR4, CCR5, and CCR2 have also been determined with X-ray crystallography during the last years [77–80]. CXCR4 bound to either a small antagonist (IT1t), a cyclic antagonist (CVX15), or a viral antagonist chemokine (vMIP-II) revealed a symmetric TM5,6/TM5,6 dimer interface as the dominant interaction mode under crystallization conditions [77,78]. However, the crystal packing also revealed TM1/TM5-7 and TM1/TM1 contacts [47]. CCR5 in complex with the HIV drug maraviroc showed dimer contacts between TM1,7 and H8 of one monomer and TM4 and TM5 of an adjacent monomer [79]. Interestingly, the previously described residues Ile52 (TM1) and Val150 (TM4) that were suggested to be crucial for CCR5 dimerization and signaling [32] are located on the contacting helices, however, neither residue is directly involved in the crystallographic dimer interface. Recently, the crystal structure of CCR2 in complex with an orthosteric (BMS-681) and allosteric (CCR2-RA-[R]) antagonist was determined [80]. Here, no physiological relevant dimer interface could be observed since the protomers are exclusively oriented antiparallel in the crystal packing. In addition, the chemokine receptor CCR9 revealed a symmetric TM4/TM4 interface under crystallization conditions [81]. Thus far, no crystallographic data on GPCR heterodimers is available due to the enormous complexity of the crystallization process. However, in silico methods such as molecular dynamics (MD) simulations have proven useful to provide molecular information about the dimerization of transmembrane proteins [82–85]. Especially coarse-grained (CG) simulations are a commonly used method to study GPCR assembly, since they allow to explore biological systems on the micro- to millisecond timescale [47,82,86–93]. Dimer and oligomer conformations of several GPCRs could be determined and the influence of membrane properties such as bilayer thickness [86,87] or the presence of specific membrane components were investigated [90]. The interactions between certain lipid types [90,94–97] or cholesterol and GPCRs were also studied in CG and atomistic simulations and specific cholesterol binding spots on GPCR monomers and dimers have been found for several receptors [47,96,98–102]. Besides the spontaneous association of GPCRs, the binding strength of different dimer interfaces can be approximated with CGMD simulations [103–108]. From these studies, energetically favored dimer interfaces and possible energy barriers for dimer formations resulting from trapping of lipids at the interfaces could be elucidated [91,105]. In this work, the spontaneous homo- and heterodimerization of the chemokine receptors CXCR4, CCR5, and CCR2 is investigated using ensembles of molecular dynamics simulations. Our results reveal that the closely related CC chemokine receptors CCR5 and CCR2 share common features with regard to dimerization and interactions with cholesterol that differ from the previously reported characteristics of CXCR4 [47]. In addition, we provide first structural information about chemokine receptor heterodimers and elucidate the role of cholesterol in differentiating between the heterodimerization of CXCR4 and the CC chemokine receptors.
  In the coarse-grained simulation ensembles, receptors were considered as dimers if the interaction energy (calculated as the sum of the Lennard-Jones and Coumbic interaction energies) between the transmembrane segments reached -50 kJ/mol, indicating direct protein-protein contacts (see Materials and methods Fig 8). Chemokine receptor dimerization was observed at rates of 0. 2–0. 3 μs−1 in pure POPC and 0. 05–0. 08 μs−1 in POPC/30% cholesterol membranes. A comparable number of dimers for these different lipid environments was obtained for simulation lengths of 3 μs and 8 μs, respectively (compare Fig 1 and Table 1). Interestingly, the homo- and heterodimerization between CC chemokine receptors in POPC revealed lower rates as compared to the homo- and heterodimerization involving CXCR4. The strongest effect of cholesterol on the dimerization rate was observed for the homodimerization of CXCR4, that was reduced by 80% in presence of cholesterol. Lower bound estimates for the binding free energy between chemokine receptors in coarse-grained simulations could be derived from the ratio between the total simulation time spent in monomeric states following the dissociation of a spontaneously assembled dimer complex, and the total time the system spent in dimeric states (see Materials and methods, S2c Fig and [47]). The binding free energies were computed in a range from -25 to -17 kJ/mol for the different chemokine receptor dimers (see Table 1). As depicted in Fig 1b and S2d Fig, the dissociation was significantly enhanced for the homo- and heterodimerization involving CC chemokine receptors in pure POPC membranes as compared to the homodimerization of CXCR4 [47] (by a factor of 3–5). Presence of cholesterol increased the dissociation propensity for chemokine receptor homo- and heterodimers. Interestingly, this clear trend was mitigated for compact dimer interfaces (i. e. for higher interaction energies between the receptor transmembrane segments), indicating that cholesterol complicates the formation of compact dimers from inital protein-protein contacts (see S3 Fig). Especially, the dissociation propensity for the homodimerization of CXCR4 was increased by a factor of ≈ 3, resulting in a pronounced decrease in binding affinity by 4 kJ/mol in cholesterol-rich membranes, while the homo- and heterodimerization of all other receptor combinations were less affected. 30% cholesterol content decreased the receptor diffusion by ≈ 50% as compared to the diffusion in pure POPC (see S1 Table). In contrast, the dimerization rates were decelerated by approx. 70-80% in cholesterol-rich lipid bilayers. Thus, assuming a simple hit-and-dimerize model, cholesterol affected the dimerization of chemokine receptors not only by slowing down the protein self-diffusion, but as well by modulating the protein association process [47]. Therefore, specific binding of cholesterol to monomeric chemokine receptors is analyzed in detail in the following Section. Based on the observation that cholesterol modulates the association of chemokine receptors, interactions between the studied GPCRs and the sterol were analyzed based on simulations of the different receptors in monomeric form in membranes at 10% cholesterol content. Specific binding sites are highlighted by the spatial distribution densities of cholesterol around the receptor combined with the contact time of helix residues with cholesterol (Fig 2). As described previously [47], cholesterol strongly bound to CXCR4 between the TM1 (red) and TM7 (green) helices close to the intracellular water-membrane interface, covered the extracellular half of TM1, and padded the rugged surface on TM5 (see Fig 2a, TM5 in purple). Interestingly, the cholesterol binding patterns on the closely related CC chemokine receptors largely coincided but differed substantially from CXCR4. The common binding spots on the CC chemokine receptors included regions between the extracellular-facing halves of TM6 and TM7, intracellular-facing residues on TM5, both intra- and extracellular halves of TM1 and the intracellular part of TM4 (see Fig 2b and 2c). For both CC chemokine receptors it was observed that cholesterol molecules reached with their polar headgroups into a lipid entry portal between the extracellular halves of TM1 and TM7. Furthermore, each CC chemokine receptor revealed an additional cholesterol binding spot albeit with lower density: In detail, on CCR5 an additional cholesterol molecule occupied the cytosolic half of TM5 and CCR2 bound an extra cholesterol between TM3 and TM4. In case of CCR5, the intracellular-facing binding spot on TM1 includes the previously discussed Ile52, indicated to play an important role in receptor function [32]. Similarly, in the corresponding binding site on CCR2, the experimentally addressed Val64 [32,68] showed a (moderate) cholesterol occupancy. Interestingly, the residues Met61—Leu67, used for the design of CCR2 homodimerization blocking heptapeptides [65], construct the cholesterol binding spot at the intracellular half of CCR2. Additionally, the residues Val150 (CCR5) or Ile163 (CCR2), recognized as important for both function and dimerization [32], contributed to the cholesterol binding spot on TM4. How binding of cholesterol to chemokine receptors modulates CC chemokine receptor homodimer interfaces is discussed in the following Section. Chemokine receptor dimerization patterns were analyzed from the relative orientations of the receptor monomers in spontaneously formed dimer configurations (described in detail in [47], see also Materials and methods, and Supplementary Information). Relative binding angles between protomers at dimer interfaces can be assigned to individual transmembrane helices (see Fig 3), thus allowing to compute the relative involvement of helices at dimer interfaces. The closely related CC chemokine receptors shared very similar homodimerization patterns (see Fig 3a), likely due to the high sequence identity within their transmembrane domains (see S1 Fig). As shown in Fig 3, while TM1, TM5,6 and 7 of CXCR4 displayed equally high binding densities in homodimers in pure POPC, the presence of cholesterol reduced the involvement of TM1 but increased the binding density on TM4 [47]. In case of CC chemokine receptors, especially TM1 was revealed as a homodimerization hotspot. This observation is in good agreement with previous experimental studies, where mutations on TM1 or heptapeptides derived from the sequence of TM1 hampered CC chemokine receptor homodimerization [32,65]. Furthermore, the presence of cholesterol did not affect the binding position densities of CC chemokine receptors as strongly as previously reported for CXCR4 [47]. The homodimer populations of CCR2 and CCR5 are distinct from dimer configurations reported previously for CXCR4 [47] (see Fig 4a). Overall, three main homodimer interfaces could be observed for the CC chemokine receptors: TM1, H8/TM1, H8, TM1, H8/TM4,5 and TM1, H8/TM5-7 (see Fig 4b). In case of CXCR4, the interface formed by interacting TM1 and TM5-7 helices was most abundant in cholesterol-free membranes, whereas the TM1/TM1 and TM1/TM4,5 motifs that were strongly populated for CC chemokine receptors did not contribute significantly to the homodimerization pattern of CXCR4. Oppositely, symmetric TM4/TM4 or TM5/TM5 dimer interfaces as observed for CXCR4 were barely obtained for CC chemokine receptors (see S4a Fig for representative structures of less populated CC chemokine receptor homodimers). Notably, H8 contributed to the main CC chemokine receptor homodimer interfaces. However, helix 8 was not resolved in any currently available crystal structure of CXCR4 [77,78]. Especially in pure POPC membranes, CCR5 and CCR2 homodimers showed very similar configurations (see relative binding angles of monomers in homodimers in S5b and S5c Fig). Due to the observation, that the Martini coarse-grained model may overestimate the aggregation between proteins [109], we cannot exclude artificial dimer configurations among the less populated dimer interfaces. However, as also shown in previous studies [47,83], highly populated dimer configurations observed in simulation ensembles compare well to experimental findings. The CCR5 TM1, H8/TM4,5 dimer configuration deviates by 6. 7 Å (backbone RMSD of transmembrane region) from the dimer configuration observed in the CCR5 crystal structure [79]. Under crystallization conditions, the proteins in the complex are aligned parallel in periodic chains, while the spontaneously formed TM1/TM4,5 dimer interface derived from simulations adopted an overall more compact configuration (see S6 Fig). The binding position between the monomers of the corresponding self-assembled dimer was shifted by 35° as compared to the crystal complex, allowing for significantly more contacts between the TM1 and TM4 helices at the interface and an accordingly increased buried surface area at the interface of 22. 5nm2 compared to only 14. 7nm2 for the crystal complex. In addition, the above described CC homodimer configurations are indirectly supported by a mutation study: Val150 of CCR5 and Ile163 of CCR2 (both on TM4) were suggested as important for homodimerization and function of the respective receptors [32]. Both residues are centrally located at the dimerization interface of the self-assembled TM1, H8/TM4,5 dimer (see Fig 4b). In turn, Val150 barely contributes to the crystal dimer interface of CCR5 (see S6b Fig). Apart from homodimerization of chemokine receptors, also heterodimerization between CXCR4, CCR5, and CCR2 was observed in experiments and their importance for function discussed [48]. Here, we report the first structural data for chemokine receptor heterodimers obtained from CG simulations.
  During the last decade, an increasing number of studies revealed the role of homo- and heterodimerization of chemokine receptors in modulating protein function by affecting their chemokine sensitivity or altering their G protein coupling mechanisms [43,48]. Besides protein function, the ability of the HI-Virus to infect T cells abusing either CXCR4 or CCR5 as a coreceptor was shown to depend on homo- and heterodimerization of the chemokine receptors [28,34,45,68]. Consequently, targeting the association of chemokine receptors moved into the focus of modern drug discovery. The development of either bivalent ligands [71] or TM peptides [27,32,35,53,65] to regulate chemokine receptor dimerization and function is largely based on structural information of receptor dimer structures. Since structural detail of GPCR dimer complexes is rather limited due to experimental restrictions, we employed extensive molecular dynamics simulations to investigate the dimerization process and gain first molecular insight into homo- and heterodimer structures of chemokine receptors in lipid bilayers. POPC lipid bilayers serve as a robust membrane mimetic to study the interactions between membrane components in a controlled environment [20]. Based on the observation that cholesterol plays an important role in regulating chemokine receptor function and dimerization [53,55–63], the effect of cholesterol on the protein association was additionally studied. Using ensembles of simulations, similar homodimerization patterns were observed for the closely related CC chemokine receptors CCR5 and CCR2, distinct from the homodimerization interfaces of CXCR4 [47]. Especially TM1 of CC chemokine receptors served as a homodimerization hotspot as suggested before by experiments [32,65]. TM1, H8/TM1, H8 interactions formed the only highly populated symmetric CC chemokine receptor homodimer interface, while other dimers were formed mostly via asymmetric TM1, H8/TM4,5 or TM1, H8/TM5-7 contacts. Cholesterol molecules bound to homologous regions on the CC chemokine receptors that differed from the main cholesterol binding sites on CXCR4. Residues indicated by experiments to play crucial roles in regulating CC chemokine receptor dimerization and function, namely Ile52 and Val150 on CCR5 as well as Val64 and Ile163 on CCR2 [32], were both located at the spontaneously formed dimer interfaces and within cholesterol binding sites on the receptor surfaces. These findings substantiate earlier experimental studies [32,53,58,65], that residues which are not directly involved at the ligand- or G protein binding sites but exposed at the receptor surface can modulate GPCR function either via dimerization or interactions with the membrane environment. Interestingly, the presence of cholesterol showed distinct effects on the homodimerization patterns of the three chemokine receptors: CXCR4 homodimer configurations involving TM1 were impaired in cholesterol-rich membranes, while TM4 was more frequently involved at dimer interfaces [47]. Even though cholesterol showed a smaller influence on the homodimerization patterns of both CC chemokine receptors as compared to CXCR4, the dimerization response differed between the closely related receptors. Thus, cholesterol can increase the dynamic range and versatility of associations of closely related transmembrane receptors. Besides homodimerization of chemokine receptors, heterodimerization was studied in cholesterol-free and -rich membranes. The CXCR4/CC chemokine receptor heterodimerization revealed mostly asymmetric dimer configurations involving mainly TM1, TM4 and TM5-7 helices of each protomer at the interface but also symmetric TM1/TM1 dimer configurations. Interestingly, despite the high sequence identity between CCR5 and CCR2, differences were observed for their heterodimerization with CXCR4. Involvement of TM2 of CXCR4 in CXCR4/CCR2 heterodimer interfaces was enhanced as compared to CXCR4/CCR5 complexes. For the latter, CCR5 predominantly bound to areas involving TM1 and TM7 of CXCR4. Experiments conducted by Percherancier et al. [35] using TM peptides derived from CXCR4 showed that TM2 and TM4 peptides strongly reduced SDF-1-promoted conformational changes in preexisting CXCR4/CCR2 heterodimers, indicating that these helices are indeed involved at heterodimeric interfaces. The subtle differences between CXCR4/CC chemokine receptor heterodimer configurations regarding the involvement of TM2 of CXCR4 at dimer interfaces with CCR2 turned out to have a significant impact on the cholesterol-sensitivity of the receptor association: binding of cholesterol to the main cholesterol binding site on CXCR4 at the intracellular halves of TM1 and TM7 hampered the formation of CXCR4/CCR5 heterodimer interfaces formed around TM1 of CXCR4, while it did not impede corresponding CXCR4/CCR2 conformations. Interestingly, the heterodimerization response of CXCR4/CCR5 complexes to cholesterol showed the same mechanism as described for CXCR4 homodimers [47], i. e. interfaces involving TM1 were reduced while the ratio of configurations including TM4 of CXCR4 increased. As discussed in the Introduction, CXCR4 and CCR5 have both been observed to require cholesterol for proper function [55–59]. Furthermore, the HIV-entry via virus-binding to CD4 and either of the coreceptors CXCR4 or CCR5 was reduced upon depletion of cholesterol [57,60,61] and upon heterooligomerization between CXCR4, CCR5 and CD4 in T cells [45]. Our data suggest a cholesterol-sensitive heterodimerization of CXCR4 and CCR5. It appears intriguing to hypothesize that the CXCR4/CCR5 heterodimer interfaces predominantly formed in cholesterol-containing membranes, i. e. TM4,5/TM1, H8 and TM5-7/TM1, H8 dimers, interact with CD4 and display resistance to HIV-entry. In conclusion, associations between the chemokine receptors CXCR4, CCR5 and CCR2 and their interaction with cholesterol, were reported to play essential roles in diversifying receptor function. Here, we provide a structural basis for the versatile interplay between the receptor homo- and heterodimerization and its modulation by cholesterol, aimed to contribute to the deciphering of the complex regulation network of chemokine related signaling pathways and diseases.
  The protein structure of CXCR4, based on the crystal structure 3OE0 [77] was taken from our previous study [47]. The preparation of the CC chemokine receptors CCR5 and CCR2 followed a similar protocol. The crystal dimer structure of the chimera protein containing two copies of CCR5, rubredoxin, and the antagonist drug maraviroc [79] was downloaded from the Protein Data Bank (PDB entry 4MBS). One monomer, maraviroc, rubredoxin, and other non-protein atoms were removed. The thermostabilizing mutations in the CCR5 crystal structure—Tyr58Cys, Asn163Gly, Asp233Ala and Glu303Lys—were mutated back to the wildtype sequence and missing residues of the third intracellular loop, namely Cys224, Arg225, and Asn226, were added to the protein structure (using MODELLER [110]). The final structure contained residues ranging from Pro19 to Phe312, the terminal parts of the protein were not resolved in the crystal structure. The isoform B of CCR2 was crystallized in a chimeric complex with T4-lysozyme, an orthosteric and an allosteric antagonist (BMS-681 and CCR2-RA-[R], respectively) [80]. Thereby, the wildtype residues ranging from Leu226 to Arg240 were removed and replaced with a sequence derived from the M2 muscarinic acetylcholine receptor in order to fuse with the T4-lysozyme at the position of the third intracellular loop [80]. Here, during the structure preparation, both antagonists, the T4-lysozyme and other non-protein atoms were removed from the crystal structure obtained from the Protein Data Bank (PDB entry 5T1A). Next, the third intracellular loop was modelled according to the native sequence, i. e. Leu226—KTLLRCRNEKKRHR—Arg240. Since both termini were not resolved in the crystal structure, the resulting CCR2 structure contained residues from Val37 to Phe320. Finally, both CC chemokine receptor structures were minimized using the CHARMM36 force field [111]. Subsequently, the protein structures were converted to the Martini2. 2 coarse-grained force field [112] using martinize [112]. In order to enforce the secondary and tertiary structure, an elastic RubberBands force network was applied. RubberBands as established in Wassernaar et al. [82] introduce additional weak bonds (with force F) between all backbone bead pairs (i, j) within a distance (dij) of 0. 9 nm following the kernel like function [113]: F i j = f · e - a d i j 2 p. Here, f was set to 500 kJ/mol/nm2, a was set to 3, and p to 6. Thereby, beads that are by default connected via bonds and angles within the Martini force field, i. e. i − i + 1 and i − i + 2 backbone bead pairs were excluded from the RubberBand network. RubberBands is, as well as ElNeDyn [114], implemented in martinize [112] and was chosen here, since it allows to run simulations using the standard Martini protein force fields [112,115]. The application of RubberBands on the coarse-grained structures did not enhance the protein rigidity artificially as it can be seen from a comparison of the RMSD values between CG and atomistic simulations in Fig 7.
summary: G protein coupled chemokine receptors are proteins embedded in the cell membrane. They play essential roles in the human immune system. Moreover, chemokine receptors are also involved in various diseases including cancer metastasis and HIV infection leading to AIDS. In case of the chemokine receptors CXCR4, CCR5, and CCR2, recent studies revealed that the proteins associate to so-called dimers, consisting of two receptors. This dimerization was shown to regulate protein function. Consequently, the association of chemokine receptors increasingly gained attention for modern drug design. However, structural information about chemokine receptor dimers is scarce due to experimental limitations. Here, we present first atomistic insight into chemokine receptor dimer structures consisting of either two equal receptors (homodimers) or of two different receptors (heterodimers). To this end, we employed thousands of molecular dynamics simulations of the receptor association process. The simulations revealed similar homodimerization patterns for the closely related receptors CCR5 and CCR2, and pinpointed the dimerization hotspots on both proteins. Cholesterol was found to differentiate the association patterns between CXCR4 and the related CC chemokine receptors, indicating that both heterodimerization and the interplay with cholesterol play important roles in fine-tuning chemokine-related signaling pathways.
keywords: dimers (chemical physics)
  cell motility
  ccr5 coreceptor
  crystal structure
  condensed matter physics
  membrane receptor signaling
  crystallography
  coreceptors
  physical chemistry
  chemical properties
  lipids
  solid state physics
  dimerization
  chemistry
  chemotaxis
  cholesterol
  physics
  biochemistry
  biochemical simulations
  signal transduction
  cell biology
  chemokines
  biology and life sciences
  physical sciences
  computational biology
  chemical physics
  cell signaling
title: Closely related, yet unique: Distinct homo- and heterodimerization patterns of G protein coupled chemokine receptors and their fine-tuning by cholesterol