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CC and CXC chemokines are distinct chemokine subfamilies. CC chemokines usually do not bind CXC-chemokine receptors and vice versa. CCR5 and CXCR4 receptors are activated by CCL5 and CXCL12 chemokines, respectively, and are also used as HIV-1 coreceptors. CCL5 contains one conserved binding site for a sulfated tyrosine residue, whereas CXCL12 is unique in having two additional sites for sulfated/nonsulfated tyrosine residues. In this study, N-terminal (Nt) CXCR4 peptides were found to bind CCL5 with somewhat higher affinities in comparison to those of short Nt-CCR5(8-20) peptides with the same number of sulfated tyrosine residues. Similarly, a long Nt-CCR5(1-27)( s Y3, s Y10, s Y14) peptide cross reacts with CXCL12 and with lower K D in comparison to its binding to CCL5. Intermolecular nuclear overhauser effect (NOE) measurements were used to decipher the mechanism of the chemokine/Nt-receptor peptide binding. The Nt-CXCR4 peptides interact with the conserved CCL5 tyrosine sulfate-binding site by an allovalency mechanism like that observed for CCL5 binding of Nt-CCR5 peptides. Nt-CCR5 peptides bind CXCL12 in multiple modes analogous to their binding to HIV-1 gp120 and interact with all three tyrosine/sulfated tyrosine-binding pockets of CXCL12. We suggest that the chemokine-receptors Nt-segments bind promiscuously to cognate and non-cognate chemokines and in a mechanism that is dependent on the number of binding pockets for tyrosine residues found on the chemokine. In conclusion, common features shared among the chemokine-receptors' Nt-segments such as multiple tyrosine residues that are potentially sulfated, and a large number of negatively charged residues are the reason of the cross binding observed in this study., (© 2023 Federation of European Biochemical Societies.)

The large number of pathologies that position CCR5 as a central molecular determinant substantiates the studies aimed at understanding receptor-ligand interactions, as well as the development of compounds that efficiently block this receptor. This perspective focuses on CCR5 antagonism as the preferred landscape for therapeutic intervention, thus the receptor active site occupancy by known antagonists of different origins is overviewed. CCL5 is a natural agonist ligand for CCR5 and an extensively studied scaffold for CCR5 antagonists production through chemokine N-terminus modification. A retrospective 3D modeling analysis on recently developed CCL5 mutants and their contribution to enhanced anti-HIV-1 activity is reported here. These results allow us to prospect the development of conceptually novel amino acid substitutions outside the CCL5 N-terminus hotspot. CCR5 interaction improvement in regions distal to the chemokine N-terminus, as well as the stabilization of the chemokine hydrophobic core are strategies that influence binding affinity and stability beyond the agonist/antagonist dualism. Furthermore, the development of allosteric antagonists topologically remote from the orthosteric site (e.g., intracellular or membrane-embedded) is an intriguing new avenue in GPCR druggability and thus a conceivable novel direction for CCR5 blockade. Ultimately, the three-dimensional structure elucidation of the interaction between various ligands and CCR5 helps illuminate the active site occupancy and mechanism of action., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Amerzhanova and Vangelista.)

The numerous chemokines and their cognate G protein-coupled chemokine receptors on the surface of leukocytes form a complex signaling network, which regulates the immune response and also other key physiological processes. Currently only a very limited number of structures of chemokine•chemokine receptor complexes have been solved. More structures are needed for the understanding of their mechanism of action and the rational design of drugs against these highly relevant therapeutic targets. Recently, we have determined the cryo-EM structure of the human wild-type CCR5 chemokine receptor, which is also the HIV-1 coreceptor, in its active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric G i protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist CCR5 chemokine ligands. In this chapter, we present a detailed protocol for the preparation of the active agonist chemokine•CCR5•G i complex for cryo-EM studies including quality controls and caveats. As such the protocol may serve as starting point for structural and biophysical studies of other chemokine•chemokine receptor complexes., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

GPCR oligomerization plays a critical role in cellular signaling, yet the stoichiometry of the interactions between oligomers and binding ligands in living cells remains a longstanding challenge. Here, by developing a dual-color simultaneous tracking system based on a total internal reflection fluorescence microscope (TIRFM), the CCR5-CCL5 interactions are visualized and quantitatively assessed in real time. Results show that each oligomeric state of CCR5 could bind with CCL5 but with different binding affinities; CCR5 dimers have a 3.5-fold higher binding affinity than the monomers. The dimerization may cause an asymmetric conformational change which makes the first binding pocket have a 3.5-fold higher binding affinity and the second have only a half compared with the monomeric CCR5. This study is the first example to directly scrutinize the CCR5-CCL5 interactions at the single-molecule level on living cell membranes and will offer great potential for the interaction stoichiometry study of diverse surface proteins.

The chemokine receptor CCR5 plays a vital role in immune surveillance and inflammation. However, molecular details that govern its endogenous chemokine recognition and receptor activation remain elusive. Here we report three cryo-electron microscopy structures of G i1 protein-coupled CCR5 in a ligand-free state and in complex with the chemokine MIP-1α or RANTES, as well as the crystal structure of MIP-1α-bound CCR5. These structures reveal distinct binding modes of the two chemokines and a specific accommodate pattern of the chemokine for the distal N terminus of CCR5. Together with functional data, the structures demonstrate that chemokine-induced rearrangement of toggle switch and plasticity of the receptor extracellular region are critical for receptor activation, while a conserved tryptophan residue in helix II acts as a trigger of receptor constitutive activation.

The human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in inflammation and is involved in cancer, HIV, and COVID-19. Despite its importance as a drug target, the molecular activation mechanism of CCR5, i.e., how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric G i protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N terminus of agonist chemokines pushes onto specific structural motifs at the bottom of the orthosteric pocket that activate the canonical GPCR microswitch network. This activation mechanism differs substantially from other CC chemokine receptors that bind chemokines with shorter N termini in a shallow binding mode involving unique sequence signatures and a specialized activation mechanism., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The N-terminal segment of the chemokine receptor Human CC chemokine receptor 5 (CCR5), Nt-CCR5, contains four tyrosine residues, Y3, Y10, Y14, and Y15. Sulfation of at least two of these tyrosine residues was found to be essential for high-affinity binding of CCR5 to its chemokine ligands. Here, we show that among the monosulfated Nt-CCR5(8-20) peptide surrogates (sNt-CCR5) those sulfated at Y15 and Y14 have the highest affinity for the CC chemokine ligand 5 (CCL5) chemokine in comparison with monosulfation at position Y10. Sulfation at Y3 was not investigated. A peptide sulfated at both Y14 and Y15 has the highest affinity for CCL5 by up to a factor of 3, in comparison with the other disulfated (sNt-CCR5) peptides. Chemical shift perturbation analysis and transferred nuclear Overhauser effect measurements indicate that the sulfated tyrosine residues interact with the same CCL5-binding pocket and that each of the sulfated tyrosines at positions 10, 14, and 15 can occupy individually the binding site on CCL5 in a similar manner, although with somewhat different affinity, suggesting the possibility of allovalency in sulfated Nt-CCR5 peptides. The affinity of the disulfated peptides to CCL5 could be increased by this allovalency and by stronger electrostatic interactions., (© 2020 Federation of European Biochemical Societies.)

Respiratory syncytial virus (RSV) is associated with bronchiolitis in infancy and the later development of asthma. Research on RSV in vitro requires preparation of a purified RSV stock. The objective for this work was to develop best methods for RSV purification, while monitoring the samples for potential contaminating proinflammatory mediators. Using polyethylene glycol concentration, and sucrose-gradient ultracentrifugation, we collected samples at each step of purification and measured the values of RSV titer, total protein (µg/mL), and proinflammatory cytokines (ELISA). We analyzed the efficacy of each step in the purification procedure. In so doing, we also determined that despite optimal purification methods, a well-known chemokine in the field of allergic disease, CCL5 (RANTES), persisted within the virus preparations, whereas other cytokines did not. We suggest that researchers should be aware that CCL5 appears to co-purify with RSV. Despite reasonable purification methods, a significant level of CCL5 (RANTES) persists in the virus preparation. This is relevant to the study of RSV-induced allergic disease., (©2020 Society for Leukocyte Biology.)

CC-type chemokine ligand 5 (CCL5) has been known to regulate immune responses by mediating the chemotaxis of leukocytes. Depending on the environment, CCL5 forms different orders of oligomers to interact with targets and create functional diversity. A recent CCL5 trimer structure revealed that the N-terminal conversed F12-A13-Y14 ( 12 FAY 14 ) sequence is involved in CCL5 aggregation. The CCL5- 12 AAA 14 mutant with two mutations had a deficiency in the formation of high-order oligomers. In the study, we clarify the respective roles of F12 and Y14 through NMR analysis and structural determination of the CCL5- 12 AAA 14 mutant where F12 is involved in the dimer assembly and Y14 is involved in aggregation. The CCL5- 12 AAA 14 structure contains a unique dimer packing. The backbone pairing shifts for one-residue in the N-terminal interface, when compared to the native CCL5 dimer. This difference creates a new structural orientation and leads to the conclusion that F12 confines the native CCL5 dimer configuration. Without F12 anchoring in the position, the interfacial backbone pairing is permitted to slide. Structural plasticity occurs in the N-terminal interaction. This is the first case to report this structural rearrangement through mutagenesis. The study provides a new idea for chemokine engineering and complements the understanding of CCL5 oligomerization and the role of the 12 FAY 14 sequence.

CC-type chemokine ligand 5 (CCL5) is involved in the pathogenesis of many inflammatory conditions. Under physiological conditions, CCL5 oligomerization and aggregation are considered to be responsible for its inflammatory properties. The structural basis of CCL5 oligomerization remains controversial because the current oligomer models contain no consensus interactions. In this study, NMR and biophysical analyses proposed evidence that the CC-type CCL5 dimer acts as the basic unit to constitute the oligomer and that CCL5 oligomerizes alternatively through E66-K25 and E66-R44/K45 interactions. In addition, a newly determined trimer structure, constituted by CCL5 and the E66S mutant, reported an interfacial interaction through the N-terminal 12 FAY 14 sequence. The interaction contributes to CCL5 aggregation and precipitation but not to oligomerization. In accordance with the observations, an integrative model explains the CCL5 oligomerization and aggregation mechanism in which CCL5 assembly consists of two types of dimer-dimer interactions and one aggregation mechanism. For full-length CCL5, the molecular accumulation triggers oligomerization through the E66-K25 and E66-R44/K45 interactions, and the 12 FAY 14 interaction acts as a secondary effect to derive aggregation and precipitation. In contrast, the E66-R44/K45 interaction might dominate in CCL5 N-terminal truncations, and the interaction would lead to the filament-like formation in solution., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

HIV-1 envelope glycoprotein (Env), which consists of trimeric (gp160) 3 cleaved to (gp120 and gp41) 3 , interacts with the primary receptor CD4 and a coreceptor (such as chemokine receptor CCR5) to fuse viral and target-cell membranes. The gp120-coreceptor interaction has previously been proposed as the most crucial trigger for unleashing the fusogenic potential of gp41. Here we report a cryo-electron microscopy structure of a full-length gp120 in complex with soluble CD4 and unmodified human CCR5, at 3.9 Å resolution. The V3 loop of gp120 inserts into the chemokine-binding pocket formed by seven transmembrane helices of CCR5, and the N terminus of CCR5 contacts the CD4-induced bridging sheet of gp120. CCR5 induces no obvious allosteric changes in gp120 that can propagate to gp41; it does bring the Env trimer close to the target membrane. The N terminus of gp120, which is gripped by gp41 in the pre-fusion or CD4-bound Env, flips back in the CCR5-bound conformation and may irreversibly destabilize gp41 to initiate fusion. The coreceptor probably functions by stabilizing and anchoring the CD4-induced conformation of Env near the cell membrane. These results advance our understanding of HIV-1 entry into host cells and may guide the development of vaccines and therapeutic agents.

Peptides represent a promising source of new medicines, but improved technologies are needed to facilitate discovery and optimization campaigns. In particular, longer peptides with multiple disulfide bridges are challenging to produce, and producing large numbers of structurally related variants is dissuasively costly and time-consuming. The principal cost and time drivers are the multiple column chromatography purification steps that are used during the multistep chemical synthesis procedure, which involves both ligation and oxidative refolding steps. In this study, we developed a method for multiplex parallel synthesis of complex peptide analogs in which the structurally variant region of the molecule is produced as a small peptide on a 384-well synthesizer with subsequent ligation to the longer, structurally invariant region and oxidative refolding carried out in-well without any column purification steps. To test the method, we used a panel of 96 analogs of the chemokine RANTES (regulated on activation normal T cell expressed and secreted)/CCL5 (69 residues, two disulfide bridges), which had been synthesized using standard approaches and characterized pharmacologically in an earlier study. Although, as expected, the multiplex method generated chemokine analogs of lower purity than those produced in the original study, it was nonetheless possible to closely match the pharmacological attributes (anti-HIV potency, capacity to elicit G protein signaling, and capacity to elicit intracellular receptor sequestration) of each chemokine analog to reference data from the earlier study. This rapid, low-cost approach has the potential to support discovery and optimization campaigns based on analogs of other chemokines as well as those of other complex peptide and small protein targets of a similar size., (© 2018 Paolini-Bertrand et al.)

The inflammatory chemokine CCL5, which binds the chemokine receptor CCR5 in a two-step mechanism so as to activate signaling pathways in hematopoetic cells, plays an important role in immune surveillance, inflammation, and development as well as in several immune system pathologies. The recently published crystal structure of CCR5 bound to a high-affinity variant of CCL5 lacks the N-terminal segment of the receptor that is post-translationally sulfated and is known to be important for high-affinity binding. Here, we report the NMR solution structure of monomeric CCL5 bound to a synthetic doubly sulfated peptide corresponding to the missing first 27 residues of CCR5. Our structures show that two sulfated tyrosine residues, sY10 and sY14, as well as the unsulfated Y15 form a network of strong interactions with a groove on a surface of CCL5 that is formed from evolutionarily conserved basic and hydrophobic amino acids. We then use our NMR structures, in combination with available crystal data, to create an atomic model of full-length wild-type CCR5:CCL5. Our findings reveal the structural determinants involved in the recognition of CCL5 by the CCR5 N terminus. These findings, together with existing structural data, provide a complete structural framework with which to understand the specificity of receptor:chemokine interactions., Database: Structural data are available in the PDB under the accession number 6FGP., (© 2018 Federation of European Biochemical Societies.)

The chemokine CCL5 is considered to be a potential therapeutic target because of its ability to recruit immune cells to inflammatory sites. CCL5 aggregates under physiological conditions, and high-order oligomer formation is considered to be significant for cell migration, immune-cell activation and HIV cell entry. The structure of the high-order oligomer is unknown and the mechanism by which the oligomer is derived has yet to be established. Here, a CCL5 mutant (CCL5-E66S) which is deficient in oligomer formation was mixed with native CCL5 to prepare a protein trimer. At an optimized ratio the trimeric CCL5 crystallized, and the crystal belonged to the tetragonal space group P4 1 2 1 2, with unit-cell parameters a = 56.6, b = 56.6, c = 154.1 Å. The Matthews coefficient (V M ) of the crystal is 2.58 Å 3  Da -1 (three molecules in the asymmetric unit), with a solvent content of 52.32%. Diffraction data were collected to a resolution of 1.87 Å and the statistics indicated satisfactory data quality. The new structure will reveal the interfaces in the CCL5 oligomer, therefore assisting in understanding the mechanism of CCL5 oligomerization.

Many human proteins have the potential to be developed as therapeutic agents. However, side effects caused by direct administration of natural proteins have significantly slowed expansion of protein therapeutics into the clinic. Post-translational modifications (PTMs) can improve protein properties, but because of significant knowledge gaps, we are considerably limited in our ability to apply PTMs to generate better protein therapeutics. Here, we seek to fill the gaps by studying the PTMs of a small representative chemotactic cytokine, RANTES. RANTES can inhibit HIV-1 infection by competing with it for binding to receptor CCR5 and stimulating CCR5 endocytosis. Unfortunately, RANTES can induce strong signaling, leading to severe inflammatory side effects. We apply a chemical biology approach to explore the potential of post-translationally modified RANTES as safe inhibitors of HIV-1 infection. We synthesized and systematically tested a library of RANTES isoforms for their ability to inhibit inflammatory signaling and prevent HIV-1 infection of primary human cells. Through this research, we revealed that most of the glycosylated variants have decreased inflammation-associated properties and identified one particular glyco variant, a truncated RANTES containing a Galβ1-3GalNAc disaccharide α-linked to Ser4, which stands out as having the best overall properties: relatively high HIV-1 inhibition potency but also weak inflammatory properties. Moreover, our results provided a structural basis for the observed changes in the properties of RANTES. Taken together, this work highlights the potential importance of glycosylation as an alternative strategy for developing CCR5 inhibitors to treat HIV-1 infection and, more generally, for reducing or eliminating unwanted properties of therapeutic proteins.

CCR5 is the primary chemokine receptor utilized by HIV to infect leukocytes, whereas CCR5 ligands inhibit infection by blocking CCR5 engagement with HIV gp120. To guide the design of improved therapeutics, we solved the structure of CCR5 in complex with chemokine antagonist [5P7]CCL5. Several structural features appeared to contribute to the anti-HIV potency of [5P7]CCL5, including the distinct chemokine orientation relative to the receptor, the near-complete occupancy of the receptor binding pocket, the dense network of intermolecular hydrogen bonds, and the similarity of binding determinants with the FDA-approved HIV inhibitor Maraviroc. Molecular modeling indicated that HIV gp120 mimicked the chemokine interaction with CCR5, providing an explanation for the ability of CCR5 to recognize diverse ligands and gp120 variants. Our findings reveal that structural plasticity facilitates receptor-chemokine specificity and enables exploitation by HIV, and provide insight into the design of small molecule and protein inhibitors for HIV and other CCR5-mediated diseases., (Copyright © 2017 Elsevier Inc. All rights reserved.)

NMR is a powerful tool for studying structural details of protein/peptide complexes exhibiting weak to medium binding (K D > 10 μm). However, it has been assumed that intermolecular nuclear Overhauser effect (NOE) interactions are difficult to observe in such complexes. We demonstrate that intermolecular NOEs can be revealed by combining the 13 C-edited/ 13 C-filtered experiment with the transferred NOE effect (TRNOE). Due to the TRNOE phenomenon, intermolecular NOE cross peaks are characterized by both the chemical shifts (CSs) of the protein protons and the average CSs of the peptide protons, which are dominated by the CSs of the protons of the free peptide. Previously, the TRNOE phenomenon was used almost exclusively to investigate the conformation of small ligands bound to large biomolecules. Here, we demonstrate that TRNOE can be extended to enable the study of intermolecular interactions in small- and medium-sized protein complexes. We used the 13 C-edited/ 13 C-filtered TRNOE experiment to study the interactions of the chemokine regulated upon activation, normal T cell, expressed and secreted (RANTES) with a 27-residue peptide, containing two sulfotyrosine residues, representing the N-terminal segment of the CCR5 receptor ((Nt-CCR5(1-27). The TRNOE phenomenon led to more than doubling of the signal-to-noise ratios (SNRs) for the intermolecular NOEs observed in the 13 C-edited/ 13 C-filtered experiment for the 11.5-kDa monomeric RANTES/Nt-CCR5(1-27) complex. An even better improvement in the SNR was achieved with dimeric Nt-CCR5(1-27)/RANTES (23 kDa), especially in comparison with the spectra measured with a 1 : 1 protein to peptide ratio. In principle, the isotope-edited/isotope-filtered TRNOE spectrum can discern all intermolecular interactions involving nonexchangeable protons in the complex., (© 2017 Federation of European Biochemical Societies.)

CC chemokine ligand 5 (CCL5) and CCL3 are critical for immune surveillance and inflammation. Consequently, they are linked to the pathogenesis of many inflammatory conditions and are therapeutic targets. Oligomerization and glycosaminoglycan (GAG) binding of CCL5 and CCL3 are vital for the functions of these chemokines. Our structural and biophysical analyses of human CCL5 reveal that CCL5 oligomerization is a polymerization process in which CCL5 forms rod-shaped, double-helical oligomers. This CCL5 structure explains mutational data and offers a unified mechanism for CCL3, CCL4, and CCL5 assembly into high-molecular-weight, polydisperse oligomers. A conserved, positively charged BBXB motif is key for the binding of CC chemokines to GAG. However, this motif is partially buried when CCL3, CCL4, and CCL5 are oligomerized; thus, the mechanism by which GAG binds these chemokine oligomers has been elusive. Our structures of GAG-bound CCL5 and CCL3 oligomers reveal that these chemokine oligomers have distinct GAG-binding mechanisms. The CCL5 oligomer uses another positively charged and fully exposed motif, KKWVR, in GAG binding. However, residues from two partially buried BBXB motifs along with other residues combine to form a GAG-binding groove in the CCL3 oligomer. The N termini of CC chemokines are shown to be involved in receptor binding and oligomerization. We also report an alternative CCL3 oligomer structure that reveals how conformational changes in CCL3 N termini profoundly alter its surface properties and dimer-dimer interactions to affect GAG binding and oligomerization. Such complexity in oligomerization and GAG binding enables intricate, physiologically relevant regulation of CC chemokine functions.

Chemical reaction rates are generally decreased at lower temperatures. Here, we report that an oxime ligation reaction in water at neutral pH is accelerated by freezing. The freezing method and its rate effect on oxime ligation are systematically studied on a peptide model system, and applied to a larger chemokine protein, containing a single acetyl butyrate group, which is conjugated to an aminooxy-labeled ligand. Our improved ligation protocol now makes it possible to efficiently introduce oxime-bond coupled ligands into proteins under aqueous conditions at low concentrations and neutral pH.

Interactions between chemokines such as CCL5 and glycosaminoglycans (GAGs) are essential for creating haptotactic gradients to guide the migration of leukocytes into inflammatory sites, and the GAGs that interact with CCL5 with the highest affinity are heparan sulfates/heparin. The interaction between CCL5 and its receptor on monocytes, CCR1, is mediated through residues Arg-17 and -47 in CCL5, which overlap with the GAG-binding (44)RKNR(47) "BBXB" motifs. Here we report that heparin and tetrasaccharide fragments of heparin are able to inhibit CCL5-CCR1 binding, with IC50 values showing strong dependence on the pattern and extent of sulfation. Modeling of the CCL5-tetrasaccharide complexes suggested that interactions between specific sulfate and carboxylate groups of heparin and residues Arg-17 and -47 of the protein are essential for strong inhibition; tetrasaccharides lacking the specific sulfation pattern were found to preferentially bind CCL5 in positions less favorable for inhibition of the interaction with CCR1. Simulations of a 12-mer heparin fragment bound to CCL5 indicated that the oligosaccharide preferred to interact simultaneously with both (44)RKNR(47) motifs in the CCL5 homodimer and engaged residues Arg-47 and -17 from both chains. Direct engagement of these residues by the longer heparin oligosaccharide provides a rationalization for its effectiveness as an inhibitor of CCL5-CCR1 interaction. In this mode, histidine (His-23) may contribute to CCL5-GAG interactions when the pH drops just below neutral, as occurs during inflammation. Additionally, an examination of the contribution of pH to modulating CCL5-heparin interactions suggested a need for careful interpretation of experimental results when experiments are performed under non-physiological conditions., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Interactions of the chemokine CCL5 (RANTES) with glycosaminoglycans (GAGs) are crucial to the CCL5-mediated inflammation process. However, structural information on interactions between CCL5 and longer GAG fragments is lacking. In this study, the interactions between oligosaccharides derived from chondroitin sulfate and a dimeric variant of CCL5 were investigated using solution nuclear magnetic resonance. The data indicate that, in addition to the BBXB motif in the 40s loop, GAGs also contact residues in the N loop in a manner similar to interactions between chemokine and the receptor N terminus, leading to possible stabilization of the dimer. Using 2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl-tagged hexasaccharides, the binding orientation of the hexasaccharides was shown to be highly dependent on the sulfation pattern of the N-acetyl galactosamine groups. Finally, a model of the CCL5 dimer complexed to chondroitin sulfate hexasaccharides was constructed using paramagnetic relaxation enhancement and intra- and intermolecular nuclear Overhauser effect constraints., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

To elucidate the ligand-binding surface of the CC chemokine-binding proteins Evasin-1 and Evasin-4, produced by the tick Rhipicephalus sanguineus, we sought to identify the key determinants responsible for their different chemokine selectivities by expressing Evasin mutants using phage display. We first designed alanine mutants based on the Evasin-1·CCL3 complex structure and an in silico model of Evasin-4 bound to CCL3. The mutants were displayed on M13 phage particles, and binding to chemokine was assessed by ELISA. Selected variants were then produced as purified proteins and characterized by surface plasmon resonance analysis and inhibition of chemotaxis. The method was validated by confirming the importance of Phe-14 and Trp-89 to the inhibitory properties of Evasin-1 and led to the identification of a third crucial residue, Asn-88. Two amino acids, Glu-16 and Tyr-19, were identified as key residues for binding and inhibition of Evasin-4. In a parallel approach, we identified one clone (Y28Q/N60D) that showed a clear reduction in binding to CCL3, CCL5, and CCL8. It therefore appears that Evasin-1 and -4 use different pharmacophores to bind CC chemokines, with the principal binding occurring through the C terminus of Evasin-1, but through the N-terminal region of Evasin-4. However, both proteins appear to target chemokine N termini, presumably because these domains are key to receptor signaling. The results also suggest that phage display may offer a useful approach for rapid investigation of the pharmacophores of small inhibitory binding proteins., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The highly constitutively active G protein-coupled receptor US28 of human cytomegalovirus (HCMV) is thought to camouflage agonism by mediating constitutive endocytosis. With the use of the US28Δ300 mutant, which is largely devoid of constitutive internalization, I have demonstrated that the coupling of the receptor to its downstream signaling partners is responsible for the inverse agonism to agonism efficacy switch in some small-weight ligands of US28., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

G protein-coupled receptor activation and desensitization leads to recruitment of arrestin proteins from cytosolic pools to the cell membrane where they form clusters difficult to characterize due to their small size and further mediate receptor internalization. We quantitatively investigated clustering of arrestin 3 induced by potent anti-HIV analogues of the chemokine RANTES after stimulation of the C-C chemokine receptor 5 using single-molecule localization-based super-resolution microscopy. We determined arrestin 3 cluster sizes and relative fractions of arrestin 3 molecules in each cluster through image-based analysis of the localization data by adapting a method originally developed for co-localization analysis from molecular coordinates. We found that only classical agonists in the set of tested ligands were able to efficiently recruit arrestin 3 to clusters mostly larger than 150 nm in size and compare our results with existing data on arrestin 2 clustering induced by the same chemokine analogues.

CCL5 (RANTES) is an inflammatory chemokine which binds to chemokine receptor CCR5 and induces signaling. The CCL5:CCR5 associated chemotactic signaling is of critical biological importance and is a potential HIV-1 therapeutic axis. Several studies provided growing evidence for the expression of CCL5 and CCR5 in non-hematological malignancies. Therefore, the delineation of the CCL5:CCR5 complex structure can pave the way for novel CCR5-targeted drugs. We employed a computational protocol which is primarily based on free energy calculations and molecular dynamics simulations, and report, what is to our knowledge, the first computationally derived CCL5:CCR5 complex structure which is in excellent agreement with experimental findings and clarifies the functional role of CCL5 and CCR5 residues which are associated with binding and signaling. A wealth of polar and non-polar interactions contributes to the tight CCL5:CCR5 binding. The structure of an HIV-1 gp120 V3 loop in complex with CCR5 has recently been derived through a similar computational protocol. A comparison between the CCL5 : CCR5 and the HIV-1 gp120 V3 loop : CCR5 complex structures depicts that both the chemokine and the virus primarily interact with the same CCR5 residues. The present work provides insights into the blocking mechanism of HIV-1 by CCL5.

Background: A few recent studies have suggested that regulated on activation, normal T cell expressed and secreted (RANTES) polymorphisms (-403 G/A, -28C/G) are associated with asthma. However, there still existed studies which did not confirm these correlations., Objective: The objective of this study was to evaluate the relationship of RANTES and asthma using a meta-analysis., Methods: Pubmed, Embase, and Cochrane library databases were systemically searched. Data were extracted by two independent reviewers and pooled odds ratio (OR) with 95% confidence interval (CI) were calculated., Results: Eighteen studies were enrolled, including a total of 2558 cases and 2630 controls of -403 G/A, as well as 3311 cases and 4031 controls of -28C/G in this meta-analysis. The overall ORs and 95% CIs of -403 G/A were 1.19, 1.06-1.33 (P<0.001) and 1.25, 1.03-1.51 (P = 0.933) in dominant and recessive models, respectively. The overall ORs and 95% CIs of -28G were 1.23, 1.09-1.39 (P = 0.221) and 1.76, 1.32-2.34 (P = 0.356) in dominant and recessive models, respectively. No publication bias among studies was showed., Conclusions: This meta-analysis showed that RANTES -403 G/A polymorphism was a risk factor for asthma, while -28C/G polymorphism were not associated with asthma.

Chemokines play important roles in the immune system, not only recruiting leukocytes to the site of infection and inflammation but also guiding cell homing and cell development. The soluble poxvirus-encoded protein viral CC chemokine inhibitor (vCCI), a CC chemokine inhibitor, can bind to human CC chemokines tightly to impair the host immune defense. This protein has no known homologs in eukaryotes and may represent a potent method to stop inflammation. Previously, our structure of the vCCI·MIP-1β (macrophage inflammatory protein-1β) complex indicated that vCCI uses negatively charged residues in β-sheet II to interact with positively charged residues in the MIP-1β N terminus, 20s region and 40s loop. However, the interactions between vCCI and other CC chemokines have not yet been fully explored. Here, we used NMR and fluorescence anisotropy to study the interaction between vCCI and eotaxin-1 (CCL11), a CC chemokine that is an important factor in the asthma response. NMR results reveal that the binding pattern is very similar to the vCCI·MIP-1β complex and suggest that electrostatic interactions provide a major contribution to binding. Fluorescence anisotropy results on variants of eotaxin-1 further confirm the critical roles of the charged residues in eotaxin-1. In addition, the binding affinity between vCCI and other wild type CC chemokines, MCP-1 (monocyte chemoattractant protein-1), MIP-1β, and RANTES (regulated on activation normal T cell expressed and secreted), were determined as 1.1, 1.2, and 0.22 nm, respectively. To our knowledge, this is the first work quantitatively measuring the binding affinity between vCCI and multiple CC chemokines.

Chemokines are chemotactic cytokines comprised of 70-100 amino acids. The chemokines CXCL12 and CCL5 are the endogenous ligands of the CXCR4 and CCR5 G protein-coupled receptors that are also HIV co-receptors. Biochemical, structural and functional studies of receptors are ligand-consuming and the cost of commercial chemokines hinders their use in such studies. Here, we describe methods for the expression, refolding, purification, and functional characterization of CXCL12 and CCL5 constructs incorporating C-terminal epitope tags. The model tags used were hexahistidines and Strep-Tag for affinity purification, and the double lanthanoid binding tag for fluorescence imaging and crystal structure resolution. The ability of modified and purified chemokines to bind and activate CXCR4 and CCR5 receptors was tested in Xenopus oocytes expressing the receptors, together with a Kir3 G-protein activated K(+) channel that served as a reporter of receptor activation. Results demonstrate that tags greatly influence the biochemical properties of the recombinant chemokines. Besides, despite the absence of any evidence for CXCL12 or CCL5 C-terminus involvement in receptor binding and activation, we demonstrated unpredictable effects of tag insertion on the ligand apparent affinity and efficacy or on the ligand dissociation. These tagged chemokines should constitute useful tools for the selective purification of properly-folded chemokines receptors and the study of their native quaternary structures.

Chemokines constitute a large family of small proteins that regulate leukocyte trafficking to the site of inflammation by binding to specific cell-surface receptors belonging to the G-protein-coupled receptor (GPCR) superfamily. The interactions between N-terminal (Nt-) peptides of these GPCRs and chemokines have been studied extensively using NMR spectroscopy. However, because of the lower affinities of peptides representing the three extracellular loops (ECLs) of chemokine receptors to their respective chemokine ligands, information concerning these interactions is scarce. To overcome the low affinity of ECL peptides to chemokines, we linked two or three CC chemokine receptor 5 (CCR5) extracellular domains using either biosynthesis in Escherichia coli or chemical synthesis. Using such chimeras, CCR5 binding to RANTES was followed using (1)H-(15)N-HSQC spectra to monitor titration of the chemokine with peptides corresponding to the extracellular surface of the receptor. Nt-CCR5 and ECL2 were found to be the major contributors to CCR5 binding to RANTES, creating an almost closed ring around this protein by interacting with opposing faces of the chemokine. A RANTES positively charged surface involved in Nt-CCR5 binding resembles the positively charged surface in HIV-1 gp120 formed by the C4 and the base of the third variable loop of gp120 (V3). The opposing surface on RANTES, composed primarily of β2-β3 hairpin residues, binds ECL2 and was found to be analogous to a surface in the crown of the gp120 V3. The chemical and biosynthetic approaches for linking GPCR surface regions discussed herein should be widely applicable to the investigation of interactions of extracellular segments of chemokine receptors with their respective ligands., (© 2013 The Authors Journal compilation © 2013 FEBS.)

CCR5, the major HIV-1 coreceptor, is a primary target for HIV-1 entry inhibition strategies. CCL5/RANTES, a natural CCR5 ligand, is one of the most potent HIV-1 entry inhibitors and, therefore, an ideal candidate to derive HIV-1 blockers. Peptides spanning the RANTES N-loop/β1-strand region act as specific CCR5 antagonists, with their hydrophobic N- and C termini playing a crucial role in virus blockade. Here, hydrophobic surfaces were enhanced by tryptophan substitution of aromatic residues, highlighting position 27 as a critical hot spot for HIV-1 blockade. In a further molecular evolution step, C-terminal engraftment of RANTES 40' loop produced a peptide with the highest solubility and anti-HIV-1 activity. These modified peptides represent leads for the development of effective HIV-1 inhibitors and microbicides., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Influenza virus is one of the most important causes of acute respiratory disease. Viral infection and viral replication activate multiple cell signalling pathways. Apoptosis of infected cells and immune response against viral replication, which are generally considered to be protective mechanisms, are also probably mediated by viruses, which lead to severe health problems. We previously reported that 3-deoxysappanchalcone (3-DSC), a compound that is isolated from Caesalpinia sappan, exhibited in vitro anti-influenza activity. In the present study, we further identified that 3-DSC inhibited viral genomic replication and transcription only at a relatively high concentration. We then evaluated the effect of 3-DSC on the regulation of virus-induced cellular apoptosis. 3-DSC ameliorated virus-induced DNA fragmentation in a concentration-dependent manner, which tends to be a consequence of its inhibition of upstream caspase activation. 3-DSC also protected host cells against influenza-induced inflammation by suppressing CCL5 and CXCL10 secretions in endothelial cells and reducing the production of IL-6 and IL-1β in monocytes/macrophages. In conclusion, our results demonstrate that anti-influenza virus mechanisms of 3-DSC involved anti-apoptosis and anti-inflammation activities in vitro. Moreover, 3-DSC could be a promising drug candidate for influenza treatment., (Georg Thieme Verlag KG Stuttgart · New York.)

The chemokine CCL5 (RANTES) plays active promalignancy roles in breast malignancy. The secretion of CCL5 by breast tumor cells is an important step in its tumor-promoting activities; therefore, inhibition of CCL5 secretion may have antitumorigenic effects. We demonstrate that, in breast tumor cells, CCL5 secretion necessitated the trafficking of CCL5-containing vesicles on microtubules from the endoplasmic reticulum (ER) to the post-Golgi stage, and CCL5 release was regulated by the rigidity of the actin cytoskeleton. Focusing on the 40s loop of CCL5, we found that the (43)TRKN(46) sequence of CCL5 was indispensable for its inclusion in motile vesicles, and for its secretion. The TRKN-mutated chemokine reached the Golgi, but trafficked along the ER-to-post-Golgi route differently than the wild-type (WT) chemokine. Based on the studies showing that the 40s loop of CCL5 mediates its binding to glycosaminoglycans (GAG), we analyzed the roles of GAG in regulating CCL5 secretion. TRKN-mutated CCL5 had lower propensity for colocalization with GAG in the Golgi compared to the WT chemokine. Secretion of WT CCL5 was significantly reduced in CHO mutant cells deficient in GAG synthesis, and the WT chemokine acquired an ER-like distribution in these cells, similar to that of TRKN-mutated CCL5 in GAG-expressing cells. The release of WT CCL5 was also reduced after inhibition of GAG presence/synthesis by intracellular expression of heparanase, inhibition of GAG sulfation, and sulfate deprivation. The need for a (43)TRKN(46) motif and for a GAG-mediated process in CCL5 secretion may enable the future design of modalities that prevent CCL5 release by breast tumor cells.

Background: The chemokine CCL5 is involved in the recruitment of immune cells and a subsequent activation of hepatic stellate cells (HSC) after liver injury. We here investigate whether inhibition of CCL5 oligomerization and glycosaminoglycan binding by a mutated CCL5 protein ((44)AANA(47)-CCL5) has the potential to ameliorate liver cell injury and fibrosis in vivo., Methodology: Liver injury was induced in C57BL/6 mice by intraperitoneal injection of carbon tetrachloride (CCl(4)) in an acute and a chronic liver injury model. Simultaneously, mice received either (44)AANA(47)-CCL5 or vehicle. Liver cell necrosis and fibrosis was analyzed by histology, and measurement of serum transaminases and hydroxyproline. Intrahepatic mRNA expression of fibrosis and inflammation related genes were determined by quantitative RT-PCR and infiltration of immune cells was assessed by FACS analysis and immunocytochemistry. In vitro, HSC were stimulated with conditioned media of T-cell enriched splenocytes., Principal Findings: (44)AANA(47)-CCL5 treated mice displayed a significantly reduced degree of acute liver injury (liver cell necrosis, transaminases) and fibrosis (Sirus red positive area and hydroxyproline content) compared to vehicle treated mice. Ameliorated fibrosis by (44)AANA(47)-CCL5 was associated with a decreased expression of fibrosis related genes, decreased α-smoth muscle antigen (αSMA) and a reduction of infiltrating immune cells. In the acute model, (44)AANA(47)-CCL5 treated mice displayed a reduced immune cell infiltration and mRNA levels of TNF, IL-1 and CCL3 compared to vehicle treated mice. In vitro, conditioned medium of T-cell enriched splenocytes of (44)AANA(47)-CCL5 treated mice inhibited the chemotaxis and proliferation of HSC., Conclusions: The results provide evidence that inhibition of oligomerization and glycosaminoglycan binding of the chemokine CCL5 is a new therapeutic strategy for the treatment of acute and chronic liver injuries and represents an alternative to chemokine receptor antagonism.

CCL5 (RANTES) is a proinflammatory chemokine known to activate leukocytes through its receptor, CCR5. Although the monomeric form of CCL5 is sufficient to cause cell migration in vitro, CCL5's propensity for aggregation is essential for migration in vivo, T cell activation and apoptosis, and HIV entry into cells. However, there is currently no structural information on CCL5 oligomers larger than the canonical CC chemokine dimer. In this study the solution structure of a CCL5 oligomer was investigated using an integrated approach, including NMR residual dipolar couplings to determine allowed relative orientations of the component monomers, SAXS to restrict overall shape, and hydroxyl radical footprinting and NMR cross-saturation experiments to identify interface residues. The resulting model of the CCL5 oligomer provides a basis for explaining the disaggregating effect of E66 and E26 mutations and suggests mechanisms by which glycosaminoglycan binding may promote oligomer formation and facilitate cell migration in vivo., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The N-terminal region of the chemokine RANTES is critical for its function. A synthesized N-terminally modified analog of RANTES, P2-RANTES, was discovered using a phage display selection against living CCR5-expressing cells, and has been reported to inhibit HIV-1 env-mediated cell-cell fusion at subnanomolar levels (Hartley et al. J Virol 2003;77:6637-6644). In the present study we produced this protein using E. coli overexpression and extensively studied its structure and function. The x-ray crystal structure of P2-RANTES was solved and refined at 1.7 A resolution. This protein was found to be predominantly a monomer in solution by analytical ultracentrifugation, but a tetramer in the crystal. In studies of glycosaminoglycan binding, P2-RANTES was found to be significantly less able to bind heparin than wild type RANTES. We also tested this protein for receptor internalization where it was shown to be functional, in cell-cell fusion assays where recombinant P2-RANTES was a potent fusion inhibitor (IC(50) = 2.4 +/- 0.8 nM), and in single round infection assays where P2-RANTES inhibited at subnanomolar levels. Further, in a modified fusion assay designed to test specificity of inhibition, P2-RANTES was also highly effective, with a 65-fold improvement over the fusion inhibitor C37, which is closely related to the clinically approved inhibitor T-20. These studies provide detailed structural and functional information for this novel N-terminally modified chemokine mutant. This information will be very useful in the development of more potent anti-HIV agents. PDB Accession Number: 2vxw., ((c) 2009 Wiley-Liss, Inc.)

In this article, we introduce the use of a thiaproline-modified lysine side-chain [Lys(Thz)], as an unlockable handle that enables late-stage, site-selective modification of chemically synthesized proteins. The Lys(Thz) residue was incorporated into the murine chemokine RANTES to demonstrate its compatibility with Boc/Bzl solid phase peptide synthesis, native chemical ligation, and disulfide bond formation. After oxidative folding of the protein, the thiol was liberated under mild reaction conditions [0.2 M hydroxylamine (NH2OH) or O-methylhydroxylamine (MeONH2), pH 4] and was subsequently reacted with thiol-selective tags. This side chain protection strategy enables the use of readily available thiol-reactive probes for the modification of internally disulfide bonded proteins., (Copyright (c) 2010 Wiley Periodicals, Inc.)

Development of effective topical microbicides for the prevention of HIV-1 sexual transmission represents a primary goal for the control of the AIDS pandemic. The viral coreceptor CCR5, used by the vast majority of primary HIV-1 isolates, is considered a primary target molecule. RANTES and its derivatives are the most suitable protein-based compounds to fight HIV-1 via CCR5 targeting. Yet, receptor activation should be avoided to prevent pro-inflammatory effects and possibly provide anti-inflammatory properties. C1C5 RANTES is a chemokine mutant that exhibits high anti-HIV-1 potency coupled with CCR5 antagonism. However, the need for the formation of an N-terminal intramolecular disulfide bridge between non-natural cysteine residues at positions 1 and 5 represents a challenge for the correct folding of this protein in recombinant expression systems, a crucial step towards its development as a microbicide against HIV-1. We report here a rare case of superior folding in a prokaryote as compared to an eukaryotic expression system. Production of C1C5 RANTES was highly impaired in CHO cells, with a dramatic yield reduction compared to that of wild type RANTES and secretion of the molecule as disulfide-linked dimer. Conversely, a human vaginal isolate of Lactobacillus jensenii engineered to secrete C1C5 RANTES provided efficient delivery of the monomeric protein. This and other reports on successful secretion of complex proteins indicate that lactic acid bacteria are an excellent system for the expression of therapeutic proteins, which can be used as a platform for the engineering of conceptually novel RANTES mutants with potent anti-HIV-1 activity.

Background: We previously demonstrated that the CC-chemokine Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES)/CCL5 exerts pro-tumoral effects on human hepatoma Huh7 cells through its G protein-coupled receptor, CCR1. Glycosaminoglycans play major roles in these biological events., Methods: In the present study, we explored 1/ the signalling pathways underlying RANTES/CCL5-mediated hepatoma cell migration or invasion by the use of specific pharmacological inhibitors, 2/ the role of RANTES/CCL5 oligomerization in these effects by using a dimeric RANTES/CCL5, 3/ the possible involvement of two membrane heparan sulfate proteoglycans, syndecan-1 (SDC-1) and syndecan-4 (SDC-4) in RANTES/CCL5-induced cell chemotaxis and spreading by pre-incubating cells with specific antibodies or by reducing SDC-1 or -4 expression by RNA interference., Results and Conclusion: The present data suggest that focal adhesion kinase phosphorylation, phosphoinositide 3-kinase-, mitogen-activated protein kinase- and Rho kinase activations are involved in RANTES/CCL5 pro-tumoral effects on Huh7 cells. Interference with oligomerization of the chemokine reduced RANTES/CCL5-mediated cell chemotaxis. This study also indicates that SDC-1 and -4 may be required for HepG2, Hep3B and Huh7 human hepatoma cell migration, invasion or spreading induced by the chemokine. These results also further demonstrate the involvement of glycosaminoglycans as the glycosaminoglycan-binding deficient RANTES/CCL5 variant, in which arginine 47 was replaced by lysine, was devoid of effect., General Significance: The modulation of RANTES/CCL5-mediated cellular effects by targeting the chemokine-syndecan interaction could represent a new therapeutic approach for hepatocellular carcinoma.

Chemokine function in vivo depends on the presentation by structures of the extracellular matrix or on endothelial surfaces. CCL5 contains two clusters of basic amino acid residues ((44)RKNR(47) and (55)KKWVR(59)) implicated in presentation of the protein. While (44)RKNR(47) has been shown to moderate CCL5 binding to glycosaminoglycans (GAGs), no direct role for the basic residues in the so called 50s loop ((55)KKWVR(59)) as a presentation structure has been published to date. In ex vivo studies both regions were found to be necessary for direct tissue binding suggesting a role for (55)KKWVR(59). In vitroT lymphocyte and monocyte induced firm adhesion under flow, as well as leukocyte recruitment to the peritoneal cavity in vivo was reduced in the 50s mutant. The binding of the 50s mutant to endothelial cells was significantly reduced as compared to the wild type protein demonstrated by ELISA. The 50s mutant had little impact on GAG binding in vitro. These data suggest that functional CCL5 presentation is mediated through both the 40s as well as the 50s loop with differential functions of the two loops of clusters of basic residues.

Binding to glycosaminoglycans (GAGs) is a necessary prerequisite for the biological activity of the proinflammatory chemokine RANTES in vivo. We have applied protein engineering methods to modulate equilibrium-binding affinity as well as binding kinetics of RANTES towards its GAG ligand which also altered the chemokine's oligomerization behavior. Out of 10 mutants, A22K and H23K were chosen for further in vitro and in vivo characterization because their stability was comparable with wild-type (wt) RANTES. In chemical cross-linking experiments, A22K gave higher and H23K lower molecular weight aggregates compared with wtRANTES as shown on SDS-PAGE. All mutants contained an N-terminal methionine residue, a well-described G-protein-coupled receptor (GPCR) antagonistic modification, which resulted in the mutants' inability to induce monocyte chemotaxis. In surface plasmon resonance experiments using immobilized heparan sulfate (HS) and physiological buffer conditions, Met-RANTES exhibited a significantly longer residual time on the GAG chip compared with the other RANTES variants. In Scatchard plot analysis, RANTES gave a bi-phasic, bell-shaped curve suggesting 'creation' of ligand-binding sites on the protein during HS interaction. This was not observed in the mutants' Scatchard plots which gave K(d) values of 317.5 and 44.5 nM for the A22K and H23K mutants, respectively. The mutants were subsequently tested for their inhibitory effect in a rat model of autoimmune uveitis where only H23K exhibited a transient improvement of the clinical disease score. H23K is therefore proposed to be a GPCR-inactive GAG antagonist which displaces the wt chemokine from its natural HS-proteoglycan co-receptor. The protein engineering approach presented here opens new ways for the treatment of RANTES-related diseases.

Binding of chemokines to glycosaminoglycans (GAGs) represents a crucial step in leukocyte attraction and activation. Since chemokine oligomerisation was shown to be important for GAG binding, the apparent oligomerisation constant of RANTES was determined to be 225 nM using fluorescence anisotropy. In the presence of heparan sulfate, chemokine oligomerisation was found to be promoted by the glycosaminoglycan as expressed in the increase in cooperativity and a shift towards higher melting temperatures in thermal unfolding experiments. In surface plasmon resonance investigations of RANTES-GAG binding kinetics using a heparan sulfate-coated chip, GAG-induced oligomerisation led to a bell-shaped (bi-phasic) Scatchard plot referring to cooperativity in the chemokine-GAG interaction. This was absent in the oligomerisation deficient RANTES mutants N46R and Q48K. We have further investigated the dependence of RANTES-GAG dissociation constants on oligosaccharide chain length by performing isothermal fluorescence titrations with size-defined heparin and heparan sulfate oligosaccharides as chemokine ligands. Heparin dp18 and heparan sulfate dp14 yielded the highest affinities with Kd values of 31.7 nM and 42.9 nM, respectively. Far-UV CD spectroscopy revealed a significant conformational change of RANTES upon heparan sulfate binding which is suggested to be a pre-requisite for oligomerisation and thus for optimal GPCR activation in vivo. This was shown by the impaired chemotactic activity of the RANTES N46R and Q48K mutants.

New prevention strategies are urgently needed to slow the spread of the HIV/AIDS pandemic, and in the absence of an effective vaccine, there is hope that "microbicides"-HIV inhibitors applied to mucosal surfaces before sexual intercourse-may be able to make an impact. Because developing countries are at the center of the epidemic, affordability and stability during storage are key criteria for candidate microbicides. Furthermore, because formulation strategies that provide long-duration protection after a single dose may enhance acceptability and compliance, stability in the vaginal environment and in the presence of semen should also be considered. PSC-RANTES, a human chemokine analog, has shown promise as a candidate microbicide, but because it contains nonnatural structures that necessitate chemical synthesis steps, it is not suitable for production at a feasible cost and scale for general distribution in developing countries. We have recently developed 2 new fully recombinant chemokine analogs, 5P12-RANTES and 6P4-RANTES, which show equivalent anti-HIV activity to PSC-RANTES. In this study, we tested the stability of these molecules under conditions related to use as microbicides. Our results suggest that stability issues will not present a major obstacle to the further development of these promising molecules as microbicides.

New prevention strategies for use in developing countries are urgently needed to curb the worldwide HIV/AIDS epidemic. The N-terminally modified chemokine PSC-RANTES is a highly potent entry inhibitor against R5-tropic HIV-1 strains, with an inhibitory mechanism involving long-term intracellular sequestration of the HIV coreceptor, CCR5. PSC-RANTES is fully protective when applied topically in a macaque model of vaginal HIV transmission, but it has 2 potential disadvantages related to further development: the requirement for chemical synthesis adds to production costs, and its strong CCR5 agonist activity might induce local inflammation. It would thus be preferable to find a recombinant analogue that retained the high potency of PSC-RANTES but lacked its agonist activity. Using a strategy based on phage display, we set out to discover PSC-RANTES analogs that contain only natural amino acids. We sought molecules that retain the potency and inhibitory mechanism of PSC-RANTES, while trying to reduce CCR5 signaling to as low a level as possible. We identified 3 analogues, all of which exhibit in vitro potency against HIV-1 comparable to that of PSC-RANTES. The first, 6P4-RANTES, resembles PSC-RANTES in that it is a strong agonist that induces prolonged intracellular sequestration of CCR5. The second, 5P12-RANTES, has no detectable G protein-linked signaling activity and does not bring about receptor sequestration. The third, 5P14-RANTES, induces significant levels of CCR5 internalization without detectable G protein-linked signaling activity. These 3 molecules represent promising candidates for further development as topical HIV prevention strategies.

The G protein-coupled receptor CCR5 is a human chemokine receptor involved in the activation and migration of leukocytes. CCR5 is also the major HIV-1 coreceptor that, together with human CD4 and the viral glycoprotein gp120, promotes virus entry into host cells. Thus inhibition of the CCR5-gp120 interaction presents a promising route to prevent HIV infections. Atomic structural details of the interaction between CCR5 and its cognate chemokines or gp120 are presently unknown due to the general difficulties of membrane protein structure determination. Here, we report the high-yield expression of human CCR5 in baculovirus-infected Sf9 insect cells. Highly purified (>90%) CCR5 is obtained in detergent-solubilized form at yields of about 1mg/l cell culture. The conformational integrity of recombinant CCR5 after purification is shown by immunoprecipitation with the conformation-dependent monoclonal antibody 2D7, CD and NMR spectroscopy. The detergent micelles contain CCR5 in monomeric and dimeric forms, which can be separated by size exclusion chromatography and characterized individually. Further functional characterization by isothermal titration calorimetry indicates that the recombinant receptor interacts with its cognate chemokine RANTES. This interaction is strongly suppressed when sulfation of CCR5 is inhibited in the insect cells.

7 transmembrane-spanning (7TM) chemokine receptors having multiple endogenous ligands offer special opportunities to understand the molecular basis for allosteric mechanisms. Thus, CC-chemokine receptor 1 (CCR1) binds CC-chemokine 3 and 5 (CCL3 and CCL5) with K(d) values of 7.3 and 0.16 nm, respectively, as determined in homologous competition binding assays. However, CCL5 appears to have a >10,000-fold lower affinity in competition against (125)I-CCL3. Mutational mapping revealed that CCL3 and CCL5 both are strongly affected by systematic truncations of the N-terminal extension, whereas only CCL5 and not CCL3 activation is affected by substitutions in the main ligand binding pocket including the conserved GluVII:06 anchor point. A series of metal ion chelator complexes were found to act as full agonists on CCR1 and to be critically affected by the same substitutions in the main ligand binding pocket as CCL5 but not by mutations in the extracellular domain. In agreement with the overlapping binding sites, the small non-peptide agonists displaced radiolabeled CCL5 with high affinity. Interestingly, the same compounds acted as allosteric enhancers of the binding of CCL3, with which they did not overlap in binding site, leading to an increased B(max) and affinity of this chemokine mainly due to an increased association rate. It is concluded that a small molecule agonist through binding deep in the main ligand binding pocket can act as an allosteric enhancer for one endogenous chemokine and at the same time as a competitive blocker of the binding of another endogenous chemokine.

RANTES (or regulated upon activation, normal T cell expressed and secreted) belongs to the rapidly growing chemokine family. It is mainly produced by T cells, epithelial cells, monocytes, fibroblasts, and mesanglial cells. Increased RANTES expression has been associated with a wide range of inflammatory disorders and pathologies. Mouse RANTES is the homolog molecule of human RANTES. The two have considerable homology in both sequence and structure. Using hRANTES as immunogen and the technique of rat B lymphocyte hybridoma, we raised two hybridoma cell lines secreting monoclonal antibodies (MAbs) to hRANTES, designated no. 1 and no. 2. Both MAbs can bind the hRANTES in FCM, Western blot analysis, and immunocytochemistry. No. 1 also worked well in immunohistochemistry of rat transplanted intestine, which may recognize the same epitope on human RANTES and rat RANTES. Thus, successful production of rat anti-human RANTES MAbs may provide a useful tool in further exploration of the biological function and pathological significance of RANTES and may provide a new method to judge early rejection after small bowel transplantation.

New HIV prevention methods are needed, and among those currently being explored are "microbicides", substances applied topically to prevent HIV acquisition during sexual intercourse. The chemokine analogue PSC-RANTES (N(alpha)(n-nonanoyl)-des-Ser(1)-[ L-thioprolyl(2), L-cyclohexylglycyl(3)]-RANTES(4-68)) is a highly potent HIV entry inhibitor which has shown promising efficacy in its initial evaluation as a candidate microbicide. However, a way must be found to produce the molecule by cheaper means than total chemical synthesis. Since the only noncoded structures are located at the N-terminus, a possible solution would be to produce a protein fragment representing all but the N-terminal region using low-cost recombinant production methods and then to attach, site specifically, a short synthetic fragment containing the noncoded N-terminal structures. Here, we describe the evaluation of a range of different conjugation chemistries in order to identify those with potential for development as economical routes to production of a PSC-RANTES analogue with antiviral activity as close as possible to that of the parent protein. The strategies tested involved linkage through oxime, hydrazone/hydrazide, and Psi[CH2-NH] bonds, as well as through a peptide bond obtained either by a thiazolidine rearrangement or by direct alpha-amino acylation of a protein fragment in which 4 of the 5 lysine residues of the native sequence were replaced by arginine (the fifth lysine is essential for activity). Where conjugation involved replacement of one or more residues with a linker moiety, the point in the main chain at which the linker was introduced was varied. The resulting panel of 22 PSC-RANTES analogues was evaluated for anti-HIV activity in an entry inhibition assay. The [Arg (25,45,56,57)] PSC-RANTES analogue has comparable potency to PSC-RANTES, and one of the oxime linked analogues, 4L-57, has potency only 5-fold lower, with scope for improvement. Both represent promising leads for development as microbicide compounds that could be produced at low cost via semisynthesis.

A nuclear magnetic resonance (NMR) experiment is described for the direct detection of N-H[...]O=C hydrogen bonds (H-bonds) in 15N and 13C isotope-labeled biomolecules. This quantitative 'long-range' HNCO-COSY (correlation spectroscopy) experiment detects and quantifies electron-mediated scalar couplings across the H-bond (H-bond scalar couplings), which connect the magnetically active (15)N and (13)C nuclei on both sides of the H-bond. Detectable H-bonds comprise the canonical backbone H-bonds in proteins as well as other H-bonds in proteins and nucleic acids with N-H donors and O=C (carbonylic or carboxylic) acceptors. Unlike other NMR observables, which provide only indirect evidence of the presence of H-bonds, the H-bond scalar couplings identify all partners of the H-bond, the donor, the donor proton and the acceptor, in a single experiment. The size of the scalar couplings can be related to H-bond geometries. The time required to detect the N-H[...]O=C H-bonds in small proteins (< or = approximately 10 kDa) is typically on the order of 1 d at millimolar concentrations, whereas H-bond detection for larger proteins (< or = approximately 30 kDa) may be possible within several days depending on concentration, isotope composition, magnetic field strength and molecular weight. The proteins ubiquitin (8.6 kDa), dimeric RANTES (2 x 8.5 kDa) and MAP30 (30 kDa) are used as examples to illustrate this procedure.

Branch-Site Model is a statistical method for detecting molecular adaptation at individual along specific lineages. Not only the model could indicate whether genes in phylogeny under the positive selection or not, but also could forecast positive selection sites in these genes. The sites promote divergence and polymorphism of genes. Chemokines are chemotactic cytokines that can induce immune cells migration, conducting their functions via chemokine receptors. In the test, the molecular adaptation of chemokines and chemokines receptors genes had been analyzed by Branch-Site model. The results showed that only a few of genes, such as RANTES and CCR5, are in the course of positive selection. Several CCR5 positive selection sites are located in a region that involved in binding to receptor and its chemokine.