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Design and Receptor Interactions of Obligate Dimeric Mutant of Chemokine Monocyte Chemoattractant Protein-1 (MCP-1)*

Open AccessPublished:March 06, 2012DOI:https://doi.org/10.1074/jbc.M111.334201
      Chemokine-receptor interactions regulate leukocyte trafficking during inflammation. CC chemokines exist in equilibrium between monomeric and dimeric forms. Although the monomers can activate chemokine receptors, dimerization is required for leukocyte recruitment in vivo, and it remains controversial whether dimeric CC chemokines can bind and activate their receptors. We have developed an obligate dimeric mutant of the chemokine monocyte chemoattractant protein-1 (MCP-1) by substituting Thr10 at the dimer interface with Cys. Biophysical analysis showed that MCP-1(T10C) forms a covalent dimer with similar structure to the wild type MCP-1 dimer. Initial cell-based assays indicated that MCP-1(T10C) could activate chemokine receptor CCR2 with potency reduced 1 to 2 orders of magnitude relative to wild type MCP-1. However, analysis of size exclusion chromatography fractions demonstrated that the observed activity was due to a small proportion of MCP-1(T10C) being monomeric and highly potent, whereas the majority dimeric form could neither bind nor activate CCR2 at concentrations up to 1 μm. These observations help to reconcile previous conflicting results and indicate that dimeric CC chemokines do not bind to their receptors with affinities approaching those of the corresponding monomeric chemokines.

      Introduction

      A hallmark of the immune system is its ability to recruit large numbers of leukocytes to sites of infection or injury. This recruitment is mediated by a group of ∼50 small, secreted proteins called chemokines, which act as chemoattractant signals to direct leukocyte migration. Chemokine function is mediated by high affinity interactions with seven-transmembrane G protein-coupled receptors on leukocyte membranes. During inflammatory responses, the array of leukocytes recruited is dependent on the chemokines expressed in the inflamed tissue, the selectivity of those chemokines for chemokine receptors, and the expression of those receptors on various types of leukocytes (
      • Lau E.K.
      • Allen S.
      • Hsu A.R.
      • Handel T.M.
      Chemokine receptor interactions. GPCRs, glycosaminoglycans, and viral chemokine-binding proteins.
      ).
      Chemokines are classified into two major families (CC and CXC) and two minor families (C and CX3C), depending on the spacing of conserved cysteine residues near the protein N terminus. The monomer structure of all chemokines is highly conserved, consisting of a disordered N terminus, an irregularly structured loop (N-loop) ending with a turn of a 310-helix, a three-stranded antiparallel β-sheet, and a C-terminal α-helix (
      • Lodi P.J.
      • Garrett D.S.
      • Kuszewski J.
      • Tsang M.L.
      • Weatherbee J.A.
      • Leonard W.J.
      • Gronenborn A.M.
      • Clore G.M.
      High resolution solution structure of the β-chemokine hMIP-1β by multidimensional NMR.
      ,
      • Handel T.M.
      • Domaille P.J.
      Heteronuclear (1H, 13C, and 15N) NMR assignments and solution structure of the monocyte chemoattractant protein-1 (MCP-1) dimer.
      ,
      • Chung C.W.
      • Cooke R.M.
      • Proudfoot A.E.
      • Wells T.N.
      The three-dimensional solution structure of RANTES.
      ,
      • Clore G.M.
      • Appella E.
      • Yamada M.
      • Matsushima K.
      • Gronenborn A.M.
      Three-dimensional structure of interleukin 8 in solution.
      ). The monomeric unit is sufficient to induce receptor activation in vitro, as demonstrated for constitutively monomeric forms of the CXC chemokine interleukin-8 (IL-8) and the CC chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1β (MIP-1β) (
      • Laurence J.S.
      • Blanpain C.
      • Burgner J.W.
      • Parmentier M.
      • LiWang P.J.
      CC chemokine MIP-1 β can function as a monomer and depends on Phe-13 for receptor binding.
      ,
      • Paavola C.D.
      • Hemmerich S.
      • Grunberger D.
      • Polsky I.
      • Bloom A.
      • Freedman R.
      • Mulkins M.
      • Bhakta S.
      • McCarley D.
      • Wiesent L.
      • Wong B.
      • Jarnagin K.
      • Handel T.M.
      Monomeric monocyte chemoattractant protein-1 (MCP-1) binds and activates the MCP-1 receptor CCR2B.
      ,
      • Rajarathnam K.
      • Sykes B.D.
      • Kay C.M.
      • Dewald B.
      • Geiser T.
      • Baggiolini M.
      • Clark-Lewis I.
      Neutrophil activation by monomeric interleukin-8.
      ). According to the widely accepted two-site model of receptor activation (
      • Wells T.N.
      • Power C.A.
      • Lusti-Narasimhan M.
      • Hoogewerf A.J.
      • Cooke R.M.
      • Chung C.W.
      • Peitsch M.C.
      • Proudfoot A.E.
      Selectivity and antagonism of chemokine receptors.
      ), chemokine monomers activate their receptors in two stages. First, the chemokine uses core (non-N-terminal) residues to bind with high affinity to the receptor N terminus. Subsequently, the chemokine N terminus activates the receptor by binding to its transmembrane helices and/or extracellular loops.
      Although monomeric chemokines are active in vitro, most chemokines are capable of forming dimers or higher order oligomers and can exist in equilibrium between different oligomeric states in solution. Members of the two major chemokine families display different modes of dimerization. CC chemokines use their N-terminal regions to form a new antiparallel β-sheet, resulting in an elongated dimer structure, whereas CXC chemokines dimerize through the existing β1-strands of the monomers, resulting in a more compact structure. Initially, it was suggested that dimeric chemokines are not biologically relevant because the systemic physiological concentrations of chemokines are in the nanomolar range, whereas chemokines typically dimerize with dissociation constants in the micromolar range (
      • Paolini J.F.
      • Willard D.
      • Consler T.
      • Luther M.
      • Krangel M.S.
      The chemokines IL-8, monocyte chemoattractant protein-1, and I-309 are monomers at physiologically relevant concentrations.
      ). However, subsequent studies have shown that the ability of monomeric chemokines to recruit leukocytes in vitro cannot be replicated in vivo. A seminal study by Proudfoot et al. (
      • Proudfoot A.E.
      • Handel T.M.
      • Johnson Z.
      • Lau E.K.
      • LiWang P.
      • Clark-Lewis I.
      • Borlat F.
      • Wells T.N.
      • Kosco-Vilbois M.H.
      Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines.
      ) demonstrated that constitutively monomeric MCP-1, MIP-1β, and RANTES
      The abbreviations used are: RANTES
      regulated on activation, normal T cell expression and secretion
      ΔδNH
      weighted change in NH chemical shift
      CCR
      CC chemokine receptor
      DMEM
      Dulbecco's Modified Eagle Medium
      DSS
      disuccinimidyl suberate
      EC50
      concentration required for 50% maximal signal
      GAG
      glycosaminoglycans
      GPCR
      G protein-coupled receptor
      HSQC
      heteronuclear single quantum coherence spectrum
      IL-8
      interleukin-8
      IPTG
      isopropyl-β-D-thiogalactopyranoside
      LC-MS
      liquid chromatography-mass spectrometry
      MCP-1
      monocyte chemoattractant protein
      MIP
      macrophage inflammatory protein
      NTA
      nitriloacetic acid
      pKi
      negative log of concentration required for 50% binding inhibition
      PMSF
      phenylmethylsulfonyl fluoride
      SEC
      size exclusion chromatography.
      could not recruit leukocytes in vivo when injected into the peritoneal cavity of mice. Furthermore, monomeric IL-8 had weaker leukocyte-recruiting potencies compared with its wild type counterpart (
      • Das S.T.
      • Rajagopalan L.
      • Guerrero-Plata A.
      • Sai J.
      • Richmond A.
      • Garofalo R.P.
      • Rajarathnam K.
      Monomeric and dimeric CXCL8 are both essential for in vivo neutrophil recruitment.
      ). These observations suggested that, although the monomeric state is likely to be important for receptor binding and activation, dimerization is also necessary for physiological function.
      The requirement of oligomerization for the physiological function of chemokines is believed to be due, at least in part, to the ability of chemokine oligomers to bind with high avidity to glycosaminoglycans on endothelial cell surfaces (
      • Lau E.K.
      • Allen S.
      • Hsu A.R.
      • Handel T.M.
      Chemokine receptor interactions. GPCRs, glycosaminoglycans, and viral chemokine-binding proteins.
      ). These interactions are likely to modulate local chemokine concentrations as well as the rates of chemokine clearance from inflammatory loci. However, Rajarathnam and co-workers (
      • Das S.T.
      • Rajagopalan L.
      • Guerrero-Plata A.
      • Sai J.
      • Richmond A.
      • Garofalo R.P.
      • Rajarathnam K.
      Monomeric and dimeric CXCL8 are both essential for in vivo neutrophil recruitment.
      ,
      • Rajarathnam K.
      • Prado G.N.
      • Fernando H.
      • Clark-Lewis I.
      • Navarro J.
      Probing receptor binding activity of interleukin-8 dimer using a disulfide trap.
      ,
      • Nasser M.W.
      • Raghuwanshi S.K.
      • Grant D.J.
      • Jala V.R.
      • Rajarathnam K.
      • Richardson R.M.
      Differential activation and regulation of CXCR1 and CXCR2 by CXCL8 monomer and dimer.
      ,
      • Ravindran A.
      • Joseph P.R.
      • Rajarathnam K.
      Structural basis for differential binding of the interleukin-8 monomer and dimer to the CXCR1 N-domain. Role of coupled interactions and dynamics.
      ) have recently reported that a disulfide-trapped dimer of the CXC chemokine IL-8 can bind and activate the receptors CXCR1 and CXCR2 and elicit neutrophil recruitment in vivo, albeit more weakly than the IL-8 monomer. In contrast, a disulfide-trapped dimer of the CC chemokine MIP-1β is unable to activate the receptor CCR5, and dimerization of the CC chemokine RANTES prevents binding to the N-terminal region of CCR5 (
      • Duma L.
      • Häussinger D.
      • Rogowski M.
      • Lusso P.
      • Grzesiek S.
      Recognition of RANTES by extracellular parts of the CCR5 receptor.
      ,
      • Jin H.
      • Shen X.
      • Baggett B.R.
      • Kong X.
      • LiWang P.J.
      The human CC chemokine MIP-1β dimer is not competent to bind to the CCR5 receptor.
      ). The observations that CXC chemokine dimers are active whereas CC chemokine dimers are not can be rationalized based on the different dimer structures; CXC chemokine dimerization leaves the receptor-binding and activation regions exposed, whereas CC chemokine dimerization involves the N-terminal region, which is critical for receptor activation. In this light, it is surprising that two independent studies of the CC chemokine MCP-1 found that cross-linked dimeric forms of MCP-1 could bind and activate its receptor CCR2 with near wild type potency (
      • Paavola C.D.
      • Hemmerich S.
      • Grunberger D.
      • Polsky I.
      • Bloom A.
      • Freedman R.
      • Mulkins M.
      • Bhakta S.
      • McCarley D.
      • Wiesent L.
      • Wong B.
      • Jarnagin K.
      • Handel T.M.
      Monomeric monocyte chemoattractant protein-1 (MCP-1) binds and activates the MCP-1 receptor CCR2B.
      ,
      • Zhang Y.
      • Rollins B.
      A dominant negative inhibitor indicates that monocyte chemoattractant protein 1 functions as a dimer.
      ). Unfortunately, it is uncertain whether the dimers characterized in those studies resembled the wild type dimer or had alternative structures in which the receptor binding regions were more exposed than in the native dimer. Thus, it remains uncertain whether the wild type MCP-1 dimer acts differently from the other chemokines within the CC chemokine family and whether it is able to bind and activate its receptor.
      In the course of studying the interactions between MCP-1 and an N-terminal peptide derived from CCR2, we observed that both monomeric and dimeric forms of MCP-1 could bind to this fragment of the receptor. To enable detailed characterization of the dimer interactions, we have now designed a new covalent MCP-1 dimer, MCP-1(T10C), which forms an intermolecular disulfide bond in the center of the dimerization interface and therefore should not be capable of dissociating into monomeric units. Biophysical and structural characterization showed that MCP-1(T10C) is a nondissociating disulfide-linked dimer that faithfully mimics the wild type structure. Although initial functional studies suggested that the MCP-1(T10C) dimer could bind and activate CCR2, detailed analysis indicated that the apparent activity was attributable to a low level contaminant of monomer. These results have allowed us to reconcile the previously conflicting data on the functional properties of CC chemokine dimers.

      DISCUSSION

      Previous structural and biophysical studies have shown that many CC chemokines exist in equilibrium between monomeric and dimeric forms in solution. The dimeric forms are required for physiological function as obligate monomers are insufficient for leukocyte recruitment in vivo (
      • Proudfoot A.E.
      • Handel T.M.
      • Johnson Z.
      • Lau E.K.
      • LiWang P.
      • Clark-Lewis I.
      • Borlat F.
      • Wells T.N.
      • Kosco-Vilbois M.H.
      Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines.
      ). Nevertheless, attempts to test the specific roles of the dimers have been impeded by the monomer-dimer equilibrium, which makes it difficult to separate the function of the dimer from that of the monomer. Three previous reports have described the receptor binding and activation properties of cross-linked CC-chemokine dimers. Jin et al. (
      • Jin H.
      • Shen X.
      • Baggett B.R.
      • Kong X.
      • LiWang P.J.
      The human CC chemokine MIP-1β dimer is not competent to bind to the CCR5 receptor.
      ) introduced the A10C mutation into MIP-1β, resulting in a disulfide cross-linked dimer similar to the MCP-1(T10C) mutant described herein. This obligate MIP-1β dimer was unable to activate the MIP-1β receptor CCR5 and did not significantly displace radiolabeled MIP-1β from CCR5, although interestingly, an ∼30% reduction of bound radioligand was observed in the presence of 1 μm MIP-1β(A10C), suggesting potentially weak binding. In contrast, two previous reports (
      • Paavola C.D.
      • Hemmerich S.
      • Grunberger D.
      • Polsky I.
      • Bloom A.
      • Freedman R.
      • Mulkins M.
      • Bhakta S.
      • McCarley D.
      • Wiesent L.
      • Wong B.
      • Jarnagin K.
      • Handel T.M.
      Monomeric monocyte chemoattractant protein-1 (MCP-1) binds and activates the MCP-1 receptor CCR2B.
      ,
      • Zhang Y.
      • Rollins B.
      A dominant negative inhibitor indicates that monocyte chemoattractant protein 1 functions as a dimer.
      ) have described different cross-linked forms of MCP-1, both of which exhibited substantial CCR2 activation ability.
      The data presented here clearly indicate that the MCP-1(T10C) mutant is correctly folded, retains wild type dimer structure, and is unable to bind or activate CCR2 at concentrations up to 1 μm. This mutant is structurally similar to MIP-1β(A10C) (
      • Jin H.
      • Shen X.
      • Baggett B.R.
      • Kong X.
      • LiWang P.J.
      The human CC chemokine MIP-1β dimer is not competent to bind to the CCR5 receptor.
      ). Both of these mutants were purified and then characterized by biophysical methods and both displayed ∼1000-fold or greater reduction in receptor binding and activation relative to the corresponding wild type chemokines. Thus, these two studies suggest that dimeric CC chemokines are indeed unable to bind to their receptors at affinities approaching those of their monomeric counterparts.
      The current conclusion that MCP-1 dimers cannot bind and activate CCR2 is in stark contrast to the previous studies of cross-linked MCP-1 dimers. These differences can be reconciled by considering the different cross-linking methods used. Zhang and Rollins (
      • Zhang Y.
      • Rollins B.
      A dominant negative inhibitor indicates that monocyte chemoattractant protein 1 functions as a dimer.
      ) used the cross-linking reagent disuccinimidyl suberate to chemically cross-link MCP-1 and obtained a mixture of oligomeric species. Disuccinimidyl suberate is a bifunctional cross-linker with amine-reactive functional groups spaced 11.4 Å apart. Although MCP-1 has 10 amine groups per monomer (9 lysine residues and the N terminus), most of them are located distant from the dimer interface, and the closest intermolecular pairs are barely within cross-linking distance; in the Protein Data Bank structure 1DOM, the closest pairs are N terminus to Lys49 (10.8 Å) and Lys38 to Lys49 (12.4 Å). Moreover, it is unlikely that cross-linking of any two amino groups would prevent transient dissociation of the dimerization interface to expose residues that could participate in receptor interactions. In the second study of cross-linked MCP-1 dimers, Paavola et al. (
      • Paavola C.D.
      • Hemmerich S.
      • Grunberger D.
      • Polsky I.
      • Bloom A.
      • Freedman R.
      • Mulkins M.
      • Bhakta S.
      • McCarley D.
      • Wiesent L.
      • Wong B.
      • Jarnagin K.
      • Handel T.M.
      Monomeric monocyte chemoattractant protein-1 (MCP-1) binds and activates the MCP-1 receptor CCR2B.
      ) induced dimer cross-linking by adding a cysteine residue to the C terminus of MCP-1 (mutant designated WT*(Cys77)). Although the resulting obligate dimer had near wild type activity, it is noteworthy that the C termini of the two monomers within the MCP-1 dimer are ∼50 Å apart. Thus, it would not be possible for WT*(Cys77)) to form an intermolecular disulfide bond without completely disrupting the native dimer interface. Based on these considerations of the cross-linking strategies, we suggest that the previous cross-linked dimers displayed activity because the cross-linking approaches did not lock these molecules into native-like dimer structures, whereas the lack of activity for MCP-1(T10C) and MIP-1β(A10C) is a consequence of these mutants being successfully trapped in the native dimer conformation. Alternatively, it is possible that the activity observed for dimers in previous studies may have been due to low concentrations of noncross-linked monomeric contaminants.
      This study was motivated, in part, by our observation that the wild type MCP-1 dimer binds to an N-terminal peptide from CCR2; in a separate study,
      J. H. Y. Tan and M. J. Stone, unpublished results.
      we have found that this interaction is enhanced by sulfation of receptor tyrosine residues, a common post-translational modification of chemokine receptors. Our NMR titration data indicate that the residues of MCP-1(T10C) whose NH groups are sensitive to peptide binding are predominantly located in the N terminus, N-loop, 310-turn, and β3-strand (Fig. 1F and supplemental Fig. S3). These are the same regions that are sensitive to peptide binding in MCP-1(P8A) and also correspond to the chemokine regions found to interact with receptor peptides in previous studies of other chemokines (
      • Ravindran A.
      • Joseph P.R.
      • Rajarathnam K.
      Structural basis for differential binding of the interleukin-8 monomer and dimer to the CXCR1 N-domain. Role of coupled interactions and dynamics.
      ,
      • Clubb R.T.
      • Omichinski J.G.
      • Clore G.M.
      • Gronenborn A.M.
      Mapping the binding surface of interleukin-8 complexed with an N-terminal fragment of the type 1 human interleukin-8 receptor.
      ,
      • Skelton N.J.
      • Quan C.
      • Reilly D.
      • Lowman H.
      Structure of a CXC chemokine-receptor fragment in complex with interleukin-8.
      ,
      • Veldkamp C.T.
      • Seibert C.
      • Peterson F.C.
      • De la Cruz N.B.
      • Haugner 3rd, J.C.
      • Basnet H.
      • Sakmar T.P.
      • Volkman B.F.
      Structural basis of CXCR4 sulfotyrosine recognition by the chemokine SDF-1/CXCL12.
      ,
      • Veldkamp C.T.
      • Seibert C.
      • Peterson F.C.
      • Sakmar T.P.
      • Volkman B.F.
      Recognition of a CXCR4 sulfotyrosine by the chemokine stromal cell-derived factor-1α (SDF-1α/CXCL12).
      ,
      • Mayer K.L.
      • Stone M.J.
      NMR solution structure and receptor peptide binding of the CC chemokine eotaxin-2.
      ,
      • Simpson L.S.
      • Zhu J.Z.
      • Widlanski T.S.
      • Stone M.J.
      Regulation of chemokine recognition by site-specific tyrosine sulfation of receptor peptides.
      ,
      • Ye J.
      • Kohli L.L.
      • Stone M.J.
      Characterization of binding between the chemokine eotaxin and peptides derived from the chemokine receptor CCR3.
      ,
      • Zhu J.Z.
      • Millard C.J.
      • Ludeman J.P.
      • Simpson L.S.
      • Clayton D.J.
      • Payne R.J.
      • Widlanski T.S.
      • Stone M.J.
      Tyrosine sulfation influences the chemokine binding selectivity of peptides derived from chemokine receptor CCR3.
      ). Thus, it appears likely that the observed interactions represent a conserved aspect of chemokine-receptor recognition and could occur in the context of the full-length receptor.
      In light of the peptide binding results, as well as a previous report that the N terminus of CCR2 is necessary and sufficient for MCP-1 binding (
      • Monteclaro F.S.
      • Charo I.F.
      The amino-terminal domain of CCR2 is both necessary and sufficient for high affinity binding of monocyte chemoattractant protein 1. Receptor activation by a pseudo-tethered ligand.
      ), it is therefore surprising that no significant CCR2 binding was observed for MCP-1(T10C) in cell-based assays. There are several possible explanations for this apparent inconsistency. First, the peptide binding is low affinity (Kd values in the micromolar range), whereas the concentrations used in cell-based assays were sub-micromolar. Second, the receptor N terminus might be occluded or conformationally restrained in the cell surface receptor relative to more exposed and flexible peptide models. Third, binding of the dimer, but not the monomer, to the receptor N terminus may be hindered by steric overlap with the receptor extracellular loops. Finally, the signals in the cell-based assays are dependent on the kinetics as well as the thermodynamics of binding, whereas the peptide binding experiments are pure equilibrium assays. Thus, it remains possible that the dimeric chemokine binds transiently to the receptor N terminus, but dissociation is too rapid for the binding to be detected in the calcium mobilization and radioligand binding inhibition assays.
      In summary, we have shown that introduction of a non-native cysteine residue in the center of the MCP-1 dimerization interface gives rise to an obligate covalent dimer whose structure closely resembles that of the native MCP-1 dimer and whose ability to bind or activate CCR2 is dramatically impaired (or completely eliminated) relative to the wild type chemokine. Comparison of these results to those for other cross-linked CC chemokine dimers helps to reconcile previous discrepancies and leads to the overall conclusion that CC-chemokine dimers are unable to bind or activate their receptors at sub-micromolar concentrations. Considering that chemokine dimerization is required for high affinity glycosaminoglycan binding and for in vivo activity, activation of receptors on rolling leukocytes is likely to require dissociation of chemokine dimers from the glycosaminoglycan surface and subsequent dissociation of dimers to their receptor-activating monomeric forms.

      Acknowledgments

      We thank Dr. David Steer for mass spectrometry advice.

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