Benchmarking AlphaFold-Generated Structures of Chemokine-Chemokine Receptor Complexes.

AlphaFold and AlphaFold-Multimer have become two essential tools for the modeling of unknown structures of proteins and protein complexes. In this work, we extensively benchmarked the quality of chemokine-chemokine receptor structures generated by AlphaFold-Multimer against experimentally determined structures. Our analysis considered both the global quality of the model, as well as key structural features for chemokine recognition. To study the effects of template and multiple sequence alignment parameters on the results, a new prediction pipeline called LIT-AlphaFold (https://github.com/LIT-CCM-lab/LIT-AlphaFold) was developed, allowing extensive input customization. AlphaFold-Multimer correctly predicted differences in chemokine binding orientation and accurately reproduced the unique binding orientation of the CXCL12-ACKR3 complex. Further, the predictions of the full receptor N-terminus provided insights into a putative chemokine recognition site 0.5. The accuracy of chemokine N-terminus binding mode prediction varied between complexes, but the confidence score permitted the distinguishing of residues that were very likely well positioned. Finally, we generated a high-confidence model of the unsolved CXCL12-CXCR4 complex, which agreed with experimental mutagenesis and cross-linking data.

Identification of novel human CC chemokine receptor 8 (CCR8) antagonists via the synthesis of naphthalene amide and sulfonamide isosteres.

The human CC chemokine receptor 8 (CCR8) has been extensively pursued as target for the treatment of various inflammatory disorders. More recently, the importance of CCR8 in the tumor microenvironment has been demonstrated, spurring the interest in CCR8 antagonism as therapeutic strategy in immuno-oncology. On a previously described naphthalene sulfonamide with CCR8 antagonistic properties, the concept of isosterism was applied, leading to the discovery of novel CCR8 antagonists with IC50 values in the nM range in both the CCL1 competition binding and CCR8 calcium mobilization assay. The excellent CCR8 antagonistic activity of the most potent congeners was rationalized by homology molecular modeling.

Cholesterol Biases the Conformational Landscape of the Chemokine Receptor CCR3: A MAS SSNMR-Filtered Molecular Dynamics Study.

Cholesterol directs the pathway of ligand-induced G protein-coupled receptor (GPCR) signal transduction. The GPCR C-C motif chemokine receptor 3 (CCR3) is the principal chemotactic receptor for eosinophils, with roles in cancer metastasis and autoinflammatory conditions. Recently, we discovered a direct correlation between bilayer cholesterol and increased agonist-triggered CCR3 signal transduction. However, the allosteric molecular mechanism escalating ligand affinity and G protein coupling is unknown. To study cholesterol-guided CCR3 conformational selection, we implement comparative, objective measurement of protein architectures by scoring shifts (COMPASS) to grade model structures from molecular dynamics simulations. In this workflow, we scored predicted chemical shifts against 2-dimensional solid-state NMR 13C-13C correlation spectra of U-15N,13C-CCR3 samples prepared with and without cholesterol. Our analysis of trajectory model structures uncovers that cholesterol induces site-specific conformational restraint of extracellular loop (ECL) 2 and conserved motion in transmembrane helices and ECL3 not observed in simulations of bilayers with only phosphatidylcholine lipids. PyLipID analysis implicates direct cholesterol agency in CCR3 conformational selection and dynamics. Residue-residue contact scoring shows that cholesterol biases the conformational selection of the orthosteric pocket involving Y411.39, Y1133.32, and E2877.39. Lastly, we observe contact remodeling in activation pathway residues centered on the initial transmission switch, Na+ pocket, and R3.50 in the DRY motif. Our observations have unique implications for understanding of CCR3 ligand recognition and specificity and provide mechanistic insight into how cholesterol functions as an allosteric regulator of CCR3 signal transduction.

Ligand-binding and -scavenging of the chemerin receptor GPR1.

Tight regulation of cytokines is essential for the initiation and resolution of inflammation. Chemerin, a mediator of innate immunity, mainly acts on chemokine-like receptor 1 (CMKLR1) to induce the migration of macrophages and dendritic cells. The role of the second chemerin receptor, G protein-coupled receptor 1 (GPR1), is still unclear. Here we demonstrate that GPR1 shows ligand-induced arrestin3 recruitment and internalization. The chemerin C-terminus triggers this activation by folding into a loop structure, binding to aromatic residues in the extracellular loops of GPR1. While this overall binding mode is shared between GPR1 and CMKLR1, differences in their respective extracellular loop 2 allowed for the design of the first GPR1-selective peptide. However, our results suggest that ligand-induced arrestin recruitment is not the only mode of action of GPR1. This receptor also displays constitutive internalization, which allows GPR1 to internalize inactive peptides efficiently by an activation-independent pathway. Our results demonstrate that GPR1 takes a dual role in regulating chemerin activity: as a signaling receptor for arrestin-based signaling on one hand, and as a scavenging receptor with broader ligand specificity on the other.

UL28 and UL33 homologs of Marek's disease virus terminase complex involved in the regulation of cleavage and packaging of viral DNA are indispensable for replication in cultured cells.

Processing and packaging of herpesvirus genomic DNA is regulated by a packaging-associated terminase complex comprising of viral proteins pUL15, pUL28 and pUL33. Marek's disease virus (MDV) homologs UL28 and UL33 showed conserved functional features with high sequence identity with the corresponding Herpes simplex virus 1 (HSV-1) homologs. As part of the investigations into the role of the UL28 and UL33 homologs of oncogenic MDV for DNA packaging and replication in cultured cells, we generated MDV mutant clones deficient in UL28 or UL33 of full-length MDV genomes. Transfection of UL28- or UL33-deleted BAC DNA into chicken embryo fibroblast (CEF) did not result either in the production of visible virus plaques, or detectable single cell infection after passaging onto fresh CEF cells. However, typical MDV plaques were detectable in CEF transfected with the DNA of revertant mutants where the deleted genes were precisely reinserted. Moreover, the replication defect of the UL28-deficient mutant was completely restored when fragment encoding the full UL28 gene was co-transfected into CEF cells. Viruses recovered from the revertant construct, as well as by the UL28 co-transfection, showed replication ability comparable with parental virus. Furthermore, the transmission electron microscopy study indicated that immature capsids were assembled without the UL28 expression, but with the loss of infectivity. Importantly, predicted three-dimensional structures of UL28 between MDV and HSV-1 suggests conserved function in virus replication. For the first time, these results revealed that both UL28 and UL33 are essential for MDV replication through regulating DNA cleavage and packaging.

FFAT motif phosphorylation controls formation and lipid transfer function of inter-organelle contacts.

Organelles are physically connected in membrane contact sites. The endoplasmic reticulum possesses three major receptors, VAP-A, VAP-B, and MOSPD2, which interact with proteins at the surface of other organelles to build contacts. VAP-A, VAP-B, and MOSPD2 contain an MSP domain, which binds a motif named FFAT (two phenylalanines in an acidic tract). In this study, we identified a non-conventional FFAT motif where a conserved acidic residue is replaced by a serine/threonine. We show that phosphorylation of this serine/threonine is critical for non-conventional FFAT motifs (named Phospho-FFAT) to be recognized by the MSP domain. Moreover, structural analyses of the MSP domain alone or in complex with conventional and Phospho-FFAT peptides revealed new mechanisms of interaction. Based on these new insights, we produced a novel prediction algorithm, which expands the repertoire of candidate proteins with a Phospho-FFAT that are able to create membrane contact sites. Using a prototypical tethering complex made by STARD3 and VAP, we showed that phosphorylation is instrumental for the formation of ER-endosome contacts, and their sterol transfer function. This study reveals that phosphorylation acts as a general switch for inter-organelle contacts.

Atypical chemokine receptor ACKR2-V41A has decreased CCL2 binding, scavenging, and activation, supporting sustained inflammation and increased Alzheimer's disease risk.

A recent genome-wide association study (GWAS) of 59 cerebrospinal fluid (CSF) proteins with a connection to Alzheimer's disease (AD) demonstrated an association between increased levels of chemokine ligand 2 (CCL2) with an atypical chemokine receptor chemokine-binding protein 2 variant V41A (ACKR2-V41A; rs2228467). High levels of CCL2 are associated with increased risk of AD development as well as other inflammatory diseases. In this study we characterized the biological function of the ACKR2-V41A receptor compared to the wild type allele by measuring its ligand binding affinity, CCL2 scavenging efficiency, and cell activation sensitivity. We transfected Chinese hamster ovary cells with plasmids carrying wild type ACKR2 (ACKR2-WT) or the mutant ACKR2-V41A receptor. Binding affinity assays showed that ACKR2-V41A has a lower binding affinity for CCL2 and CCL4 than ACKR2-WT. CCL2 scavenging results aligned with binding affinity assays, with ACKR2-V41A cells scavenging CCL2 with a lower efficiency than ACKR2-WT. Cell activation assays also showed that ACKR2-V41A cells had significantly lower receptor upregulation (ß-Arrestin-dependent signaling pathway) upon stimulation compared to ACKR2-WT cells. These findings provide molecular and biological mechanistic insights into the GWAS association of ACKR2-V41A with increased levels of CCL2 in CSF and possibly other chemokine ligands. Increased CCL2 levels are associated with accelerated cognitive decline and increased risk of AD. Understanding how this atypical chemokine receptor allele increases serum markers of inflammation could lead to novel therapeutic solutions for AD.

Purification of native CCL7 and its functional interaction with selected chemokine receptors.

Chemokine receptors form a major sub-family of G protein-coupled receptors (GPCRs) and they are involved in a number of cellular and physiological processes related to our immune response and regulation. A better structural understanding of ligand-binding, activation, signaling and regulation of chemokine receptors is very important to design potentially therapeutic interventions for human disorders arising from aberrant chemokine signaling. One of the key limitations in probing the structural details of chemokine receptors is the availability of large amounts of purified, homogenous and fully functional chemokine ligands, and the commercially available products, are not affordable for in-depth structural studies. Moreover, production of uniformly isotope-labeled chemokines, for example, suitable for NMR-based structural investigation, also remains challenging. Here, we have designed a streamlined approach to express and purify the human chemokine CCL7 as well as its 15N-, 15N/13C-, 2H/15N/13C- isotope-labeled derivatives, at milligram levels using E. coli expression system. Purified CCL7 not only maintains a well-folded three-dimensional structure as analyzed using circular dichroism and 1H/15N NMR but it also induces coupling of heterotrimeric G-proteins and ß-arrestins for selected chemokine receptors in cellular system. We compared cAMP response induced by histidine tagged CCL7 and native CCL7 and found that modification of the N-terminus of CCL7 compromises its functionality. Our strategy presented here may be applicable to other chemokines and therefore, provide a potentially generic and cost-effective approach to produce chemokines in large amounts for functional and structural studies.

New insights into the structure and function of chemokine receptor:chemokine complexes from an experimental perspective.

Chemokines are small soluble proteins that drive cell migration through the formation of concentration gradients. Chemokine binding to G protein-coupled chemokine receptors in the cell membrane activates intracellular signaling pathways and is a fundamental process involved in numerous physiological and pathophysiological functions. In the past few years, significant experimental developments have made it possible to characterize complexes between chemokine receptors and chemokines at a molecular level. Here, I review these developments from an experimental perspective, focusing on how the ability to express, purify, and stabilize receptor:chemokine complexes have made studies by X-ray crystallography, nuclear magnetic resonance, and other methods possible. I give examples of how these studies have advanced our understanding of the architecture of receptor:chemokine complexes as well as the mechanisms involved in complex formation. Finally, I discuss some of the many remaining questions and challenges that will require studies of more receptors and chemokines as well as further development of experimental methods.

Comparative modeling and docking of chemokine-receptor interactions with Rosetta.

Chemokine receptors are a subset of G protein-coupled receptors defined by the distinct property of binding small protein ligands in the chemokine family. Chemokine receptors recognize their ligands by a mechanism that is distinct from other class A GPCRs that bind peptides or small molecules. For this reason, structural information on other ligand-GPCR interactions are only indirectly relevant to understanding the chemokine receptor interface. Additionally, the experimentally determined structures of chemokine-GPCR complexes represent less than 3% of the known interactions of this complex, multi-ligand/multi-receptor network. To enable predictive modeling of the remaining 97% of interactions, a general in silico protocol was designed to utilize existing chemokine receptor crystal structures, co-crystal structures, and NMR ensembles of chemokines bound to receptor fragments. This protocol was benchmarked on the ability to predict each of the three published co-crystal structures, while being blinded to the target structure. Averaging ensembles selected from the top-ranking models reproduced up to 84% of the intermolecular contacts found in the crystal structure, with the lowest Cα-RMSD of the complex at 3.3 Å. The chemokine receptor N-terminus, unresolved in crystal structures, was included in the modeling and recapitulates contacts with known sulfotyrosine binding pockets seen in structures derived from experimental NMR data. This benchmarking experiment suggests that realistic homology models of chemokine-GPCR complexes can be generated by leveraging current structural data.

In situ crystallography as an emerging method for structure solution of membrane proteins: the case of CCR2A.

The in meso in situ serial X-ray crystallization method (Huang et al., (2015) Acta Crystallogr D Biol Crystallogr 71, 1238) combines lipid cubic phase crystallization, direct freezing of the crystallization droplet without handling of the crystals, and data collection in situ. Recently, this method was used to overcome the mechanical fragility of crystals which enabled the X-ray structure determination of chemokine receptor 2A (Apel et al., (2019) Structure 27, 427) at 2.7 Å resolution. The CCR2 structure provides the structural basis for ligand selectivity of CCR2 against chemokine receptor 5 and provides insights into the residence time of MK-0812 analogs based on molecular dynamics simulations. These findings offer new opportunities for drug discovery targeting chemokine receptors.

Structural basis of chemokine and receptor interactions: Key regulators of leukocyte recruitment in inflammatory responses.

In response to infection or injury, the body mounts an inflammatory immune response in order to neutralize pathogens and promote tissue repair. The key effector cells for these responses are the leukocytes (white blood cells), which are specifically recruited to the site of injury. However, dysregulation of the inflammatory response, characterized by the excessive migration of leukocytes to the affected tissues, can also lead to chronic inflammatory diseases. Leukocyte recruitment is regulated by inflammatory mediators, including an important family of small secreted chemokines and their corresponding G protein-coupled receptors expressed in leukocytes. Unsurprisingly, due to their central role in the leukocyte inflammatory response, chemokines and their receptors have been intensely investigated and represent attractive drug targets. Nonetheless, the full therapeutic potential of chemokine receptors has not been realized, largely due to the complexities in the chemokine system. The determination of chemokine-receptor structures in recent years has dramatically shaped our understanding of the molecular mechanisms that underpin chemokine signaling. In this review, we summarize the contemporary structural view of chemokine-receptor recognition, and describe the various binding modes of peptide and small-molecule ligands to chemokine receptors. We also provide some perspectives on the implications of these data for future research and therapeutic development. IMPORTANCE STATEMENT: Given their central role in the leukocyte inflammatory response, chemokines and their receptors are considered as important regulators of physiology and viable therapeutic targets. In this review, we provide a summary of the current understanding of chemokine: chemokine-receptor interactions that have been gained from structural studies, as well as their implications for future drug discovery efforts.

QTY code designed thermostable and water-soluble chimeric chemokine receptors with tunable ligand affinity.

Chemokine receptors are of great interest as they play a critical role in many immunological and pathological processes. The ability to study chemokine receptors in aqueous solution without detergent would be significant because natural receptors require detergents to become soluble. We previously reported using the QTY code to design detergent-free chemokine receptors. We here report the design of 2 detergent-free chimeric chemokine receptors that were experimentally unattainable in detergent solution. We designed chimeric receptors by switching the N terminus and 3 extracellular (EC) loops between different receptors. Specifically, we replaced the N terminus and 3 EC loops of CCR5QTY with the N terminus and 3 EC loops of CXCR4. The ligand for CXCR4; namely CXCL12, binds to the chimeric receptor CCR5QTY (7TM)-CXCR4 (N terminus+3 EC loops), but with lower affinity compared to CXCR4; the CCL5 ligand of CCR5 binds the chimeric receptor with ∼20× lower affinity. The chimeric design helps to elucidate the mechanism of native receptor-ligand interaction. We also show that all detergent-free QTY-designed chemokine receptors, expressed in Escherichia coli, bind to their respective chemokines with affinities in the nanomolar (nM) range, similar to the affinities of native receptors and SF9-produced QTY variants. These QTY-designed receptors exhibit remarkable thermostability in the presence of arginine and retain ligand-binding activity after heat treatment at 60 °C for 4 h and 24 h, and at 100 °C for 10 min. Our design approach enables affordable scale-up production of detergent-free QTY variant chemokine receptors with tunable functionality for various uses.

Chemokine Receptor Crystal Structures: What Can Be Learned from Them?

Chemokine receptors belong to the class A of G protein-coupled receptors (GPCRs) and are implicated in a wide variety of physiologic functions, mostly related to the homeostasis of the immune system. Chemokine receptors are also involved in multiple pathologic processes, including immune and autoimmune diseases, as well as cancer. Hence, several members of this GPCR subfamily are considered to be very relevant therapeutic targets. Since drug discovery efforts can be significantly reinforced by the availability of crystal structures, substantial efforts in the area of chemokine receptor structural biology could dramatically increase the outcome of drug discovery campaigns. This short review summarizes the available data on chemokine receptor crystal structures, discusses the numerous applications from chemokine receptor structures that can enhance the daily work of molecular pharmacologists, and describes the challenges and pitfalls to consider when relying on crystal structures for further research applications. SIGNIFICANCE STATEMENT: This short review summarizes the available data on chemokine receptor crystal structures, discusses the numerous applications from chemokine receptor structures that can enhance the daily work of molecular pharmacologists, and describes the challenges and pitfalls to consider when relying on crystal structures for further research applications.

QTY code enables design of detergent-free chemokine receptors that retain ligand-binding activities.

Structure and function studies of membrane proteins, particularly G protein-coupled receptors and multipass transmembrane proteins, require detergents. We have devised a simple tool, the QTY code (glutamine, threonine, and tyrosine), for designing hydrophobic domains to become water soluble without detergents. Here we report using the QTY code to systematically replace the hydrophobic amino acids leucine, valine, isoleucine, and phenylalanine in the seven transmembrane α-helices of CCR5, CXCR4, CCR10, and CXCR7. We show that QTY code-designed chemokine receptor variants retain their thermostabilities, α-helical structures, and ligand-binding activities in buffer and 50% human serum. CCR5QTY, CXCR4QTY, and CXCR7QTY also bind to HIV coat protein gp41-120. Despite substantial transmembrane domain changes, the detergent-free QTY variants maintain stable structures and retain their ligand-binding activities. We believe the QTY code will be useful for designing water-soluble variants of membrane proteins and other water-insoluble aggregated proteins.

Viral GPCR US28 can signal in response to chemokine agonists of nearly unlimited structural degeneracy.

Human cytomegalovirus has hijacked and evolved a human G-protein-coupled receptor into US28, which functions as a promiscuous chemokine 'sink' to facilitate evasion of host immune responses. To probe the molecular basis of US28's unique ligand cross-reactivity, we deep-sequenced CX3CL1 chemokine libraries selected on 'molecular casts' of the US28 active-state and find that US28 can engage thousands of distinct chemokine sequences, many of which elicit diverse signaling outcomes. The structure of a G-protein-biased CX3CL1-variant in complex with US28 revealed an entirely unique chemokine amino terminal peptide conformation and remodeled constellation of receptor-ligand interactions. Receptor signaling, however, is remarkably robust to mutational disruption of these interactions. Thus, US28 accommodates and functionally discriminates amongst highly degenerate chemokine sequences by sensing the steric bulk of the ligands, which distort both receptor extracellular loops and the walls of the ligand binding pocket to varying degrees, rather than requiring sequence-specific bonding chemistries for recognition and signaling.

Remodeling our concept of chemokine receptor function: From monomers to oligomers.

The chemokines direct leukocyte recruitment in both homeostatic and inflammatory conditions, and are therefore critical for immune reactions. By binding to members of the class A G protein-coupled receptors, the chemokines play an essential role in numerous physiological and pathological processes. In the last quarter century, the field has accumulated much information regarding the implications of these molecules in different immune processes, as well as mechanistic insight into the signaling events activated through their binding to their receptors. Here, we will focus on chemokine receptors and how new methodological approaches have underscored the role of their conformations in chemokine functions. Advances in biophysical-based techniques show that chemokines and their receptors act in very complex networks and therefore should not be considered isolated entities. In this regard, the chemokine receptors can form homo- and heterodimers as well as oligomers at the cell surface. These findings are changing our view as to how chemokines influence cell biology, identify partners that regulate chemokine function, and open new avenues for therapeutic intervention.

Maximal Unbiased Benchmarking Data Sets for Human Chemokine Receptors and Comparative Analysis.

Chemokine receptors (CRs) have long been druggable targets for the treatment of inflammatory diseases and HIV-1 infection. As a powerful technique, virtual screening (VS) has been widely applied to identifying small molecule leads for modern drug targets including CRs. For rational selection of a wide variety of VS approaches, ligand enrichment assessment based on a benchmarking data set has become an indispensable practice. However, the lack of versatile benchmarking sets for the whole CRs family that are able to unbiasedly evaluate every single approach including both structure- and ligand-based VS somewhat hinders modern drug discovery efforts. To address this issue, we constructed Maximal Unbiased Benchmarking Data sets for human Chemokine Receptors (MUBD-hCRs) using our recently developed tools of MUBD-DecoyMaker. The MUBD-hCRs encompasses 13 subtypes out of 20 chemokine receptors, composed of 404 ligands and 15756 decoys so far and is readily expandable in the future. It had been thoroughly validated that MUBD-hCRs ligands are chemically diverse while its decoys are maximal unbiased in terms of "artificial enrichment", "analogue bias". In addition, we studied the performance of MUBD-hCRs, in particular CXCR4 and CCR5 data sets, in ligand enrichment assessments of both structure- and ligand-based VS approaches in comparison with other benchmarking data sets available in the public domain and demonstrated that MUBD-hCRs is very capable of designating the optimal VS approach. MUBD-hCRs is a unique and maximal unbiased benchmarking set that covers major CRs subtypes so far.

Closely related, yet unique: Distinct homo- and heterodimerization patterns of G protein coupled chemokine receptors and their fine-tuning by cholesterol.

Chemokine receptors, a subclass of G protein coupled receptors (GPCRs), play essential roles in the human immune system, they are involved in cancer metastasis as well as in HIV-infection. A plethora of studies show that homo- and heterodimers or even higher order oligomers of the chemokine receptors CXCR4, CCR5, and CCR2 modulate receptor function. In addition, membrane cholesterol affects chemokine receptor activity. However, structural information about homo- and heterodimers formed by chemokine receptors and their interplay with cholesterol is limited. Here, we report homo- and heterodimer configurations of the chemokine receptors CXCR4, CCR5, and CCR2 at atomistic detail, as obtained from thousands of molecular dynamics simulations. The observed homodimerization patterns were similar for the closely related CC chemokine receptors, yet they differed significantly between the CC receptors and CXCR4. Despite their high sequence identity, cholesterol modulated the CC homodimer interfaces in a subtype-specific manner. Chemokine receptor heterodimers display distinct dimerization patterns for CXCR4/CCR5 and CXCR4/CCR2. Furthermore, associations between CXCR4 and CCR5 reveal an increased cholesterol-sensitivity as compared to CXCR4/CCR2 heterodimerization patterns. This work provides a first comprehensive structural overview over the complex interaction network between chemokine receptors and indicates how heterodimerization and the interaction with the membrane environment diversifies the function of closely related GPCRs.

The N-terminal domain of a tick evasin is critical for chemokine binding and neutralization and confers specific binding activity to other evasins.

Tick chemokine-binding proteins (evasins) are an emerging class of biologicals that target multiple chemokines and show anti-inflammatory activities in preclinical disease models. Using yeast surface display, we identified a CCL8-binding evasin, P672, from the tick Rhipicephalus pulchellus We found that P672 binds CCL8 and eight other CC-class chemokines with a Kd < 10 nm and four other CC chemokines with a Kd between 10 and 100 nm and neutralizes CCL3, CCL3L1, and CCL8 with an IC50 < 10 nm The CC chemokine-binding profile was distinct from that of evasin 1 (EVA1), which does not bind CCL8. We also show that P672's binding activity can be markedly modulated by the location of a StrepII-His purification tag. Combining native MS and bottom-up proteomics, we further demonstrated that P672 is glycosylated and forms a 1:1 complex with CCL8, disrupting CCL8 homodimerization. Homology modeling of P672 using the crystal structure of the EVA1 and CCL3 complex as template suggested that 44 N-terminal residues of P672 form most of the contacts with CCL8. Replacing the 29 N-terminal residues of EVA1 with the 44 N-terminal residues of P672 enabled this hybrid evasin to bind and neutralize CCL8, indicating that the CCL8-binding properties of P672 reside, in part, in its N-terminal residues. This study shows that the function of certain tick evasins can be manipulated simply by adding a tag. We conclude that homology modeling helps identify regions with transportable chemokine-binding functions within evasins, which can be used to construct hybrid evasins with altered properties.