Recognition of a CXCR4 sulfotyrosine by the chemokine stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12)

Tyrosine sulfation of the chemokine receptor CXCR4 enhances its interaction with the chemokine SDF-1alpha. Given similar post-translational modification of other receptors, including CCR5, CX3CR1 and CCR2b, tyrosine sulfation may be of universal importance in chemokine signaling. N-terminal domains from seven transmembrane chemokine receptors have been employed for structural studies of chemokine-receptor interactions, but never in the context of proper post-translational modifications known to affect function. A CXCR4 peptide modified at position 21 by expressed tyrosylprotein sulfotransferase-1 and unmodified peptide are both disordered in solution, but bind SDF-1alpha with low micromolar affinities. NMR and fluorescence polarization measurements showed that the CXCR4 peptide stabilizes dimeric SDF-1alpha, and that sulfotyrosine 21 binds a specific site on the chemokine that includes arginine 47. We conclude that the SDF-1alpha dimer preferentially interacts with receptor peptide, and residues beyond the extreme N-terminal region of CXCR4, including sulfotyrosine 21, make specific contacts with the chemokine ligand.     

NMR studies of active N-terminal peptides of stromal cell-derived factor-1. Structural basis for receptor binding

Stromal cell-derived factor 1 (SDF-1), a member of the CXC chemokine family, is the only chemokine to bind to the receptor CXCR4. This receptor is also a co-receptor for syncytia-inducing forms of HIV in CD4(+) cells. In addition, SDF-1 is responsible for attracting mature lymphocytes to the bone marrow and can therefore contribute to host versus graft rejection in bone marrow transplantation. Clearly, by manipulating SDF-1 activity, we could find a possible anti-viral AIDS treatment and aid in bone marrow transplantation. SDF-1 binds to CXCR4 primarily via the N terminus, which appears flexible in the recently determined three-dimensional structure of SDF-1. Strikingly, short N-terminal SDF-1 peptides have been shown to have significant SDF-1 activity. By using NMR, we have determined the major conformation of the N terminus of SDF-1 in a 17-mer (residues 1-17 of SDF-1) and a 9-mer dimer (residues 1-9 of SDF-1 linked by a disulfide bond at residue 9). Residues 5-8 and 11-14 form similar structures that can be characterized as a beta-turn of the beta-alphaR type. These structural motifs are likely to be interconverting with other states, but the major conformation may be important for recognition in receptor binding. These results suggest for the first time that there may be a link between structuring of short N-terminal chemokine peptides and their ability to activate their receptor. These studies will act as a starting point for synthesizing non-peptide analogs that act as CXCR4 antagonists.     

Identification of CXCR4 domains that support coreceptor and chemokine receptor functions

The interaction of the chemokine stromal cell-derived factor 1 (SDF-1) with its receptor CXCR4 is vital for cell trafficking during development, is capable of inhibiting human immunodeficiency virus type 1 (HIV-1) utilization of CXCR4 as a coreceptor, and has been implicated in delaying disease progression to AIDS in vivo. Because of the importance of this chemokine-chemokine receptor pair to both development and disease, we investigated the molecular basis of the interaction between CXCR4 and its ligands SDF-1 and HIV-1 envelope. Using CXCR4 chimeras and mutants, we determined that SDF-1 requires the CXCR4 amino terminus for binding and activates downstream signaling pathways by interacting with the second extracellular loop of CXCR4. SDF-1-mediated activation of CXCR4 required the Asp-Arg-Tyr motif in the second intracellular loop of CXCR4, was pertussis toxin sensitive, and did not require the distal C-terminal tail of CXCR4. Several CXCR4 mutants that were not capable of binding SDF-1 or signaling still supported HIV-1 infection, indicating that the ability of CXCR4 to function as a coreceptor is independent of its ability to signal. Direct binding studies using the X4 gp120s HXB, BH8, and MN demonstrated the ability of HIV-1 gp120 to bind directly and specifically to the chemokine receptor CXCR4 in a CD4-dependent manner, using a conformationally complex structure on CXCR4. Several CXCR4 variants that did not support binding of soluble gp120 could still function as viral coreceptors, indicating that detectable binding of monomeric gp120 is not always predictive of coreceptor function.     

Functional mimetic of the G-protein coupled receptor CXCR4 on a soluble antibody scaffold

G-protein coupled receptors (GPCRs) constitute major drug targets due to their involvement in critical biological functions and pathophysiological disorders. The leading challenge in their structural and functional characterization has been the need for a lipid environment to accommodate their hydrophobic cores. Here, we report an antibody scaffold mimetic (ASM) platform where we have recapitulated the extracellular functional domains of the GPCR, C-X-C chemokine receptor 4 (CXCR4) on a soluble antibody framework. The engineered ASM molecule can accommodate the N-terminal loop and all three extracellular loops of CXCR4. These extracellular features are important players in ligand recruitment and interaction for allostery and signal transduction. Our study shows that ASM^CXCR4 can be recognized by the anti-CXCR4 antibodies, MEDI3185, 2B11, and 12G5, and that ASM^CXCR4 can bind the HIV-1 glycoprotein ligand gp120, and the natural chemokine ligand SDF-1α. Further, we show that ASM^CXCR4 can competitively inhibit the SDF-1α signaling pathway, and be used as an immunogen to generate CXCR4-specific antibodies. This platform will be useful in the study of GPCR biology in a soluble receptor context for evaluating its extracellular ligand interactions.     

Identification of residues of CXCR4 critical for human immunodeficiency virus coreceptor and chemokine receptor activities

CXCR4 is a G-coupled receptor for the stromal cell-derived factor (SDF-1) chemokine, and a CD4-associated human immunodeficiency virus type 1 (HIV-1) coreceptor. These functions were studied in a panel of CXCR4 mutants bearing deletions in the NH(2)-terminal extracellular domain (NT) or substitutions in the NT, the extracellular loops (ECL), or the transmembrane domains (TMs). The coreceptor activity of CXCR4 was markedly impaired by mutations of two Tyr residues in NT (Y7A/Y12A) or at a single Asp residue in ECL2 (D193A), ECL3 (D262A), or TMII (D97N). These acidic residues could engage electrostatical interactions with basic residues of the HIV-1 envelope protein gp120, known to contribute to the selectivity for CXCR4. The ability of CXCR4 mutants to bind SDF-1 and mediate cell signal was consistent with the two-site model of chemokine-receptor interaction. Site I involved in SDF-1 binding but not signaling was located in NT with particular importance of Glu(14) and/or Glu(15) and Tyr(21). Residues required for both SDF-1 binding and signaling, and thus probably part of site II, were identified in ECL2 (Asp(187)), TMII (Asp(97)), and TMVII (Glu(288)). The first residues () of NT also seem required for SDF-1 binding and signaling. A deletion in the third intracellular loop abolished signaling, probably by disrupting the coupling with G proteins. The identification of CXCR4 residues involved in the interaction with both SDF-1 and HIV-1 may account for the signaling activity of gp120 and has implications for the development of antiviral compounds.     

The CXC chemokine receptor 4 ligands ubiquitin and stromal cell-derived factor-1α function through distinct receptor interactions

Recently, we identified extracellular ubiquitin as an endogenous CXC chemokine receptor (CXCR) 4 agonist. However, the receptor selectivity and molecular basis of the CXCR4 agonist activity of ubiquitin are unknown, and functional consequences of CXCR4 activation with ubiquitin are poorly defined. Here, we provide evidence that ubiquitin and the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1α do not share CXCR7 as a receptor. We further demonstrate that ubiquitin does not utilize the typical two-site binding mechanism of chemokine-receptor interactions, in which the receptor N terminus is important for ligand binding. CXCR4 activation with ubiquitin and SDF-1α lead to similar Gα(i)-responses and to a comparable magnitude of phosphorylation of ERK-1/2, p90 ribosomal S6 kinase-l and Akt, although phosphorylations occur more transiently after activation with ubiquitin. Despite the similarity of signal transduction events after activation of CXCR4 with both ligands, ubiquitin possesses weaker chemotactic activity than SDF-lα in cell migration assays and does not interfere with productive entry of HIV-1 into P4.R5 multinuclear activation of galactosidase indicator cells. Unlike SDF-1α, ubiquitin lacks interactions with an N-terminal CXCR4 peptide in NMR spectroscopy experiments. Binding and signaling studies in the presence of antibodies against the N terminus and extracellular loops 2/3 of CXCR4 confirm that the ubiquitin CXCR4 interaction is independent of the N-terminal receptor domain, whereas blockade of extracellular loops 2/3 prevents receptor binding and activation. Our findings define ubiquitin as a CXCR4 agonist, which does not interfere with productive cellular entry of HIV-1, and provide new mechanistic insights into interactions between CXCR4 and its natural ligands.     

Structural and functional characterization of human CXCR4 as a chemokine receptor and HIV-1 co-receptor by mutagenesis and molecular modeling studies

The human CXC chemokine receptor 4 (CXCR4) is a receptor for the chemokine stromal cell-derived factor (SDF-1alpha) and a co-receptor for the entry of specific strains of human immunodeficiency virus type I (HIV-1). CXCR4 is also recognized by an antagonistic chemokine, the viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus type VIII. SDF-1alpha or vMIP-II binding to CXCR4 can inhibit HIV-1 entry via this co-receptor. An approach combining protein structural modeling and site-directed mutagenesis was used to probe the structure-function relationship of CXCR4, and interactions with its ligands SDF-1alpha and vMIP-II and HIV-1 envelope protein gp120. Hypothetical three-dimensional structures were proposed by molecular modeling studies of the CXCR4.SDF-1alpha complex, which rationalize extensive biological information on the role of CXCR4 in its interactions with HIV-1 envelope protein gp120. With site-directed mutagenesis, we have identified that the amino acid residues Asp (D20A) and Tyr (Y21A) in the N-terminal domain and the residue Glu (E268A) in extracellular loop 3 (ECL3) are involved in ligand binding, whereas the mutation Y190A in extracellular loop 2 (ECL2) impairs the signaling mediated by SDF-1alpha. As an HIV-1 co-receptor, we found that the N-terminal domain, ECL2, and ECL3 of CXCR4 are involved in HIV-1 entry. These structural and mutational studies provide valuable information regarding the structural basis for CXCR4 activity in chemokine binding and HIV-1 viral entry, and could guide the design of novel targeted inhibitors.     

Identification of the cytoplasmic domains of CXCR4 involved in Jak2 and STAT3 phosphorylation

The chemokine SDF-1alpha transduces G(i)-dependent and -independent signals through CXCR4. Activation of Jak2/STAT3, a G(i)-independent signaling pathway, which plays a major role in survival signals, is known to be activated after SDF-1alpha binding to CXCR4 but the domains of CXCR4 involved in this signaling remain unexplored. Using human embryonic kidney HEK-293 cells stably expressing wild-type or mutated forms of CXCR4, we demonstrated that STAT3 phosphorylation requires the N-terminal part of the third intracellular loop (ICL3) and the tyrosine 157 present at the end of the second intracellular loop (ICL2) of CXCR4. In contrast, neither the conserved Tyr(135) in the DRY motif at the N terminus of ICL2 nor the Tyr(65) and Tyr(76) in the first intracellular loop (ICL1) are involved in this activation. ICL3, which does not contain any tyrosine residues, is needed to activate Jak2. These results demonstrate that two separate domains of CXCR4 are involved in Jak2/STAT3 signaling. The N-terminal part of ICL3 is needed to activate Jak2 after SDF-1alpha binding to CXCR4, leading to phosphorylation of only one cytoplasmic Tyr, present at the C terminus of ICL2, which triggers STAT3 activation. This work has profound implications for the understanding of CXCR4-transduced signaling.     

Optimal inhibition of X4 HIV isolates by the CXC chemokine stromal cell-derived factor 1 alpha requires interaction with cell surface heparan sulfate proteoglycans

The chemokine stromal cell-derived factor 1 (SDF-1) is the natural ligand for CXC chemokine receptor 4 (CXCR4). SDF-1 inhibits infection of CD4+ cells by X4 (CXCR4-dependent) human immunodeficiency virus (HIV) strains. We previously showed that SDF-1 alpha interacts specifically with heparin or heparan sulfates (HSs). Herein, we delimited the boundaries of the HS-binding domain located in the first beta-strand of SDF-1 alpha as the critical residues. We also provide evidence that binding to cell surface heparan sulfate proteoglycans (HSPGs) determines the capacity of SDF-1 alpha to prevent the fusogenic activity of HIV-1 X4 isolates in leukocytes. Indeed, SDF-1 alpha mutants lacking the capacity to interact with HSPGs showed a substantially reduced capacity to prevent cell-to-cell fusion mediated by X4 HIV envelope glycoproteins. Moreover, the enzymatic removal of cell surface HS diminishes the HIV-inhibitory capacity of the chemokine to the levels shown by the HS-binding-disabled mutant counterparts. The mechanisms underlying the optimal HIV-inhibitory activity of SDF-1 alpha when attached to HSPGs were investigated. Combining fluorescence resonance energy transfer and laser confocal microscopy, we demonstrate the concomitant binding of SDF-1 alpha to CXCR4 and HSPGs at the cell membrane. Using FRET between a Texas Red-labeled SDF-1 alpha and an enhanced green fluorescent protein-tagged CXCR4, we show that binding of SDF-1 alpha to cell surface HSPGs modifies neither the kinetics of occupancy nor activation in real time of CXCR4 by the chemokine. Moreover, attachment to HSPGs does not modify the potency of the chemokine to promote internalization of CXCR4. Attachment to cellular HSPGs may co-operate in the optimal anti-HIV activity of SDF-1 alpha by increasing the local concentration of the chemokine in the surrounding environment of CXCR4, thus facilitating sustained occupancy and down-regulation of the HIV coreceptor.     

Unique ligand binding sites on CXCR4 probed by a chemical biology approach: implications for the design of selective human immunodeficiency virus type 1 inhibitors

The chemokine receptor CXCR4 plays an important role as the receptor for the normal physiological function of stromal cell-derived factor 1alpha (SDF-1alpha) and the coreceptor for the entry of human immunodeficiency virus type 1 (HIV-1) into the cell. In a recent work (S. Tian et al., J. Virol. 79:12667-12673, 2005), we found that many residues throughout CXCR4 transmembrane (TM) and extracellular loop 2 domains are specifically involved in interaction with HIV-1 gp120, as most of these sites did not play a role in either SDF-1alpha binding or signaling. These results provided direct experimental evidence for the distinct functional sites on CXCR4 for HIV-1 and the normal ligand SDF-1alpha. To further understand the CXCR4-ligand interaction and to develop new CXCR4 inhibitors to block HIV-1 entry, we have recently generated a new family of unnatural chemokines, termed synthetically and modularly modified (SMM) chemokines, derived from the native sequence of SDF-1alpha or viral macrophage inflammatory protein II (vMIP-II). These SMM chemokines contain various de novo-designed sequence replacements and substitutions by d-amino acids and display more enhanced CXCR4 selectivity, binding affinities, and/or anti-HIV activities than natural chemokines. Using these novel CXCR4-targeting SMM chemokines as receptor probes, we conducted ligand binding site mapping experiments on a panel of site-directed mutants of CXCR4. Here, we provide the first experimental evidence demonstrating that SMM chemokines interact with many residues on CXCR4 TM and extracellular domains that are important for HIV-1 entry, but not SDF-1alpha binding or signaling. The preferential overlapping in the CXCR4 binding residues of SMM chemokines with HIV-1 over SDF-1alpha illustrates a mechanism for the potent HIV-1 inhibition by these SMM chemokines. The discovery of distinct functional sites or conformational states influenced by these receptor sites mediating different functions of the natural ligand versus the viral or synthetic ligands has important implications for drug discovery, since the sites shared by SMM chemokines and HIV-1 but not by SDF-1alpha can be targeted for the development of selective HIV-1 inhibitors devoid of interference with normal SDF-1alpha function.     

Inhibition of CXCR4 by LY2624587, a Fully Humanized Anti-CXCR4 Antibody Induces Apoptosis of Hematologic Malignancies

SDF-1 and CXCR4 are a chemokine and chemokine receptor pair playing critical roles in tumorigenesis. Overexpression of CXCR4 is a hallmark of many hematological malignancies including acute myeloid leukemia, chronic lymphocytic leukemia and non-Hodgkin’s lymphoma, and generally correlates with a poor prognosis. In this study, we developed a humanized anti-CXCR4 monoclonal antibody, LY2624587 as a potent CXCR4 antagonist that was advanced into clinical study for cancer. LY2624587 blocked SDF-1 binding to CXCR4 with an IC₅₀ of 0.26 nM, and inhibited SDF-1-induced GTP binding with a K_b of 0.66 nM. In human lymphoma U937 and leukemia CCRF-CEM cells expressing endogenous CXCR4, LY2624587 inhibited SDF-1-induced cell migration with IC₅₀ values of 3.7 and 0.26 nM, respectively. This antibody also inhibited CXCR4 and SDF-1 mediated cell signaling including activation of MAPK and AKT in tumor cells expressing CXCR4. Bifocal microscopic and flow cytometry analyses revealed that LY2624587 mediated receptor internalization and caused CXCR4 down-regulation on the cell surface. In human hematologic cancer cells, LY2624587 caused dose dependent apoptosis in vitro and in vivo. In mouse xenograft models developed with human leukemia and lymphoma cells expressing high levels of CXCR4, LY2624587 exhibited dose-dependent tumor growth inhibition and provided significant survival benefit in a disseminated lymphoma model. Collectively, we have demonstrated that CXCR4 inhibition by LY2624587 has the potential for the treatment of human hematological malignancies.     

CD4-Chemokine Receptor Hybrids in Human Immunodeficiency Virus Type 1 Infection

Most human immunodeficiency virus (HIV) strains require both CD4 and a chemokine receptor for entry into a host cell. In order to analyze how the HIV-1 envelope glycoprotein interacts with these cellular molecules, we constructed single-molecule hybrids of CD4 and chemokine receptors and expressed these constructs in the mink cell line Mv-1-lu. The two N-terminal (2D) or all four (4D) extracellular domains of CD4 were linked to the N terminus of the chemokine receptor CXCR4. The CD4(2D)CXCR4 hybrid mediated infection by HIV-1(LAI) to nearly the same extent as the wild-type molecules, whereas CD4(4D)CXCR4 was less efficient. Recombinant SU(LAI) protein competed more efficiently with the CXCR4-specific monoclonal antibody 12G5 for binding to CD4(2D)CXCR4 than for binding to CD4(4D)CXCR4. Stromal cell-derived factor 1 (SDF-1) blocked HIV-1(LAI) infection of cells expressing CD4(2D)CXCR4 less efficiently than for cells expressing wild-type CXCR4 and CD4, whereas down-modulation of CXCR4 by SDF-1 was similar for hybrids and wild-type CXCR4. In contrast, the bicyclam AMD3100, a nonpeptide CXCR4 ligand that did not down-modulate the hybrids, blocked hybrid-mediated infection at least as potently as for wild-type CXCR4. Thus SDF-1, but not the smaller molecule AMD3100, may interfere at multiple points with the binding of the surface unit (SU)-CD4 complex to CXCR4, a mechanism that the covalent linkage of CD4 to CXCR4 impedes. Although the CD4-CXCR4 hybrids yielded enhanced SU interactions with the chemokine receptor moiety, this did not overcome the specific coreceptor requirement of different HIV-1 strains: the X4 virus HIV-1(LAI) and the X4R5 virus HIV-1(89. 6), unlike the R5 strain HIV-1(SF162), infected Mv-1-lu cells expressing the CD4(2D)CXCR4 hybrid, but none could use hybrids of CD4 and the chemokine receptor CCR2b, CCR5, or CXCR2. Thus single-molecule hybrid constructs that mimic receptor-coreceptor complexes can be used to dissect coreceptor function and its inhibition.     

HIV-1 envelope is a neutral antagonist to CXCR4 in T-cells

The chemokine receptor CXCR4 is the principal coreceptor for X4 strains of HIV-1. We show that gp120 is unable to induce interactions between CXCR4 and G-protein in T-cells, but antagonized the agonist effect of SDF-1alpha, the natural ligand for CXCR4. Gp120 had ten times lower affinity for CXCR4 than CD4, implying that a substantial role for cellular CD4 may be to facilitate binding of the viral envelope to CXCR4. Binding of gp120 to CXCR4 was neither regulated by guanine nucleotides, nor affected by divalent cations, was temperature independent and bound to a homogenous population of CXCR4, which is characteristic for an antagonist to a G-protein coupled receptor. In contrast, SDF-1alpha binds to two affinity states of CXCR4 in T-cell membranes, which are modulated by guanine nucleotides. Binding of SDF-1alpha to CXCR4 was highly temperature dependent. Thus, the interaction of CXCR4 with HIV-1 viral envelope and chemokine exhibits fundamental differences.     

A syndecan-4/CXCR4 complex expressed on human primary lymphocytes and macrophages and HeLa cell line binds the CXC chemokine stromal cell-derived factor-1 (SDF-1)

The stromal cell-derived factor-1 (SDF-1) is a CXC chemokine, which plays critical roles in migration, proliferation, and differentiation of leukocytes. SDF-1 is the only known ligand of CXCR4, the coreceptor of X4 HIV strains. We show that SDF-1 binds to high- and low-affinity sites on HeLa cells. Coimmunoprecipitation studies demonstrate that glycanated and oligomerized syndecan-4 but neither syndecan-1, syndecan-2, betaglycan, nor CD44 forms complexes with SDF-1 and CXCR4 on these cells as well as on primary lymphocytes or macrophages. Moreover, biotinylated SDF-1 directly binds in a glycosaminoglycans (GAGs)-dependent manner to electroblotted syndecan-4, and colocalization of SDF-1 with syndecan-4 was visualized by confocal microscopy. Glycosaminidases pretreatment of the HeLa cells or the macrophages decreases the binding of syndecan-4 to the complex formed by it and SDF-1. In addition, this treatment also decreases the binding of the chemokine to CXCR4 on the primary macrophages but not on the HeLa cells. Therefore GAGs-dependent binding of SDF-1 to the cells facilitates SDF-1 binding to CXCR4 on primary macrophages but not on HeLa cell line. Finally, an SDF-1-independent heteromeric complex between syndecan-4 and CXCR4 was visualized on HeLa cells by confocal microscopy as well as by electron microscopy. Moreover, syndecan-4 from lymphocytes, monocyte derived-macrophages, and HeLa cells coimmunoprecipitated with CXCR4. This syndecan-4/CXCR4 complex is likely a functional unit involved in SDF-1 binding. The role of these interactions in the pathophysiology of SDF-1 deserves further study.     

A simplified model of the binding interaction between stromal cell-derived factor-1 chemokine and CXC chemokine receptor 4

We have synthesised the CXC chemokine receptor 4 (29-39) peptide, CXCR4[29-39]. This peptide is located in the N-terminal region of the receptor and is likely to be involved in the docking step of the receptor interaction with its natural ligand stromal cell-derived factor-1 chemokine, SDF-1. Preliminary experiments, performed in the presence of micellar detergents to model a membrane-like environment, show that the (1-17) segment of SDF-1 binds to CXCR4[29-39].     

Replacement of the V3 region of gp120 with SDF-1 preserves the infectivity of T-cell line-tropic human immunodeficiency virus type 1

Interaction between the human immunodeficiency virus type 1 (HIV-1) envelope and the relevant chemokine receptors is crucial for subsequent membrane fusion and viral entry. Although the V3 region of gp120 is known to determine the cell tropism as well as the coreceptor usage, the significance of the binding of the V3 region to the chemokine receptor has not been fully understood. To address this issue, we adopted the pseudotyped virus infection assay in which the V3 region of the T-cell line-tropic (T-tropic) NL4-3 envelope was replaced with a portion of stromal cell-derived factor 1 (SDF-1), the ligand of CXCR4. The V3 region of the NL4-3 envelope expression vector was replaced with three different stretches of SDF-1 cDNA. Expression of each chimeric envelope protein was confirmed by immunoprecipitation and Western blotting. Luciferase reporter viruses were prepared by cotransfection of the pNL4-3.Luc.E(-)R(-) vector and each chimeric envelope expression vector, and the infection assay was then carried out. We showed that pseudotyped viruses with one of the chimeric envelopes, NL4-3/SDF1-51, could infect U87.CD4.CXCR4 but not U87.CD4 or U87.CXCR4 cells and that this infection was inhibited by the ligand of CXCR4, SDF-1beta, by anti-human SDF-1 antibody, or by an anti-CD4 antibody, Leu3a, in a dose-dependent manner. Furthermore, chimeric NL4-3/SDF1-51 gp120 significantly inhibited binding of labeled SDF-1 to CXCR4. It was suggested that replacement of the V3 region of the NL4-3 envelope with SDF-1 preserved the CD4-dependent infectivity of T-tropic HIV-1. These results indicate that binding between the V3 region and the relevant coreceptor is important for viral entry, whether its amino acid sequence is indigenous to the virus or not.     

Mapping the binding of the N-terminal extracellular tail of the CXCR4 receptor to stromal cell-derived factor-1alpha

The solution structure of monomeric stromal cell-derived factor-1alpha (SDF-1alpha), the natural ligand for the CXCR4 G-coupled receptor, has been solved by multidimensional heteronuclear NMR spectroscopy. The structure has a characteristic chemokine fold and is in excellent agreement with the individual subunits observed in the crystal structures of dimeric SDF-1alpha. Using various peptides derived from the N-terminal extracellular tail of the CXCR4 receptor, we show that the principal determinants of binding reside in the N-terminal 17 residues of CXCR4, with a major contribution from the first six residues. From 15N/1HN chemical shift pertubation studies we show that the interaction surface on SDF-1alpha is formed by the undersurface of the three-stranded antiparallel beta-sheet bounded by the N-terminal loop on one side and the C-terminal helix on the other. This surface overlaps with but is not identical to that mapped on several other chemokines for the binding of equivalent peptides derived from their respective receptors.     

Attenuation of osteoarthritis via blockade of the SDF-1/CXCR4 signaling pathway

Introduction

This study was performed to evaluate the attenuation of osteoarthritic (OA) pathogenesis via disruption of the stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) signaling with AMD3100 in a guinea pig OA model.

Methods

OA chondrocytes and cartilage explants were incubated with SDF-1, siRNA CXCR4, or anti-CXCR4 antibody before treatment with SDF-1. Matrix metalloproteases (MMPs) mRNA and protein levels were measured with real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The 35 9-month-old male Hartley guinea pigs (0.88 kg ± 0.21 kg) were divided into three groups: AMD-treated group (n = 13); OA group (n = 11); and sham group (n = 11). At 3 months after treatment, knee joints, synovial fluid, and serum were collected for histologic and biochemical analysis. The severity of cartilage damage was assessed by using the modified Mankin score. The levels of SDF-1, glycosaminoglycans (GAGs), MMP-1, MMP-13, and interleukin-1 (IL-1β) were quantified with ELISA.

Results

SDF-1 infiltrated cartilage and decreased proteoglycan staining. Increased glycosaminoglycans and MMP-13 activity were found in the culture media in response to SDF-1 treatment. Disrupting the interaction between SDF-1 and CXCR4 with siRNA CXCR4 or CXCR4 antibody attenuated the effect of SDF-1. Safranin-O staining revealed less cartilage damage in the AMD3100-treated animals with the lowest Mankin score compared with the control animals. The levels of SDF-1, GAG, MMP1, MMP-13, and IL-1β were much lower in the synovial fluid of the AMD3100 group than in that of control group.

Conclusions

The binding of SDF-1 to CXCR4 induces OA cartilage degeneration. The catabolic processes can be disrupted by pharmacologic blockade of SDF-1/CXCR4 signaling. Together, these findings raise the possibility that disruption of the SDF-1/CXCR4 signaling can be used as a therapeutic approach to attenuate cartilage degeneration.     

Distinct functional sites for human immunodeficiency virus type 1 and stromal cell-derived factor 1alpha on CXCR4 transmembrane helical domains

The entry of human immunodeficiency virus type 1 (HIV-1) into the cell is initiated by the interaction of the viral surface envelope protein with two cell surface components of the target cell, CD4 and a chemokine coreceptor, usually CXCR4 or CCR5. The natural ligand of CXCR4 is stromal cell-derived factor 1alpha (SDF-1alpha). Whereas the overlap between HIV-1 and SDF-1alpha functional sites on the extracellular domains of CXCR4 has been well documented, it has yet to be determined whether there are sites in the transmembrane (TM) helices of CXCR4 important for HIV-1 and/or SDF-1alpha functions, and if such sites do exist, whether they are overlapping or distinctive for the separate functions of CXCR4. For this study, by employing alanine-scanning mutagenesis, (125)I-SDF-1alpha competition binding, Ca(2+) mobilization, and cell-cell fusion assays, we found that the mutation of many CXCR4 TM residues, including Tyr(45), His(79), Asp(97), Pro(163), Trp(252), Tyr(255), Asp(262), Glu(288), His(294), and Asn(298), could selectively decrease HIV-1-mediated cell fusion but not the binding activity of SDF-1alpha. Phe(87) and Phe(292), which were involved in SDF-1alpha binding, did not play a significant role in the coreceptor activity of CXCR4, further demonstrating the disconnection between physiological and pathological activities of CXCR4 TM domains. Our data also show that four mutations of the second extracellular loop, D182A, D187A, F189A, and P191A, could reduce HIV-1 entry without impairing either ligand binding or signaling. Taken together, our first detailed characterization of the different functional roles of CXCR4 TM domains may suggest a mechanistic basis for the discovery of new selective anti-HIV agents.     

Molecular dynamics simulations on SDF-1alpha: binding with CXCR4 receptor

Insights into the interacting mode of CXCR4 with SDF-1alpha are crucial in understanding the structural and functional characteristics of CXCR4 receptor. In this paper a computational pipeline, integrating protein structure prediction, molecular dynamics simulations, automated molecular docking, and Brownian dynamics simulations were employed to investigate the dynamic and energetic aspects of CXCR4 associating with SDF-1alpha. The entire simulation revealed the surface distribution feature of electrostatic potentials and conformational "open-close" process of the receptor. The possible binding conformation of CXCR4 was identified, and the CXCR4-SDF-1alpha binding complex was generated. Arg188-Glu277 salt bridge plays an important role for both the extracellular domain conformational change and SDF-1alpha binding. Two binding sites were mapped at the extracellular domain (Site 1) and inside the transmembrane domain (Site 2), which are composed of conserved residues. Sites 1 and 2 contribute approximately 60% and 40% to the binding affinity with SDF-1alpha, respectively. The binding model is in agreement with most of the experimental data. Transmembrane VI has more significant motion in the harmonious conformational transition of CXCR4 during SDF-1alpha binding, which may be possibly associated with signal transduction. Based on the modeling and simulation, a binding mechanism hypothesis between CXCR4 and SDF-1alpha and its relationship to the signal transduction has been proposed.