Swapping N-terminal regions among tick evasins reveals cooperative interactions influencing chemokine binding and selectivity

Abstract

Class A tick evasins are natural chemokine-binding proteins that block the signaling of multiple chemokines from the CC subfamily through their cognate receptors, thus suppressing leukocyte recruitment and inflammation. Development of tick evasins as chemokine-targeted anti-inflammatory therapeutics requires an understanding of the factors controlling their chemokine recognition and selectivity. To investigate the role of the evasin N-terminal region for chemokine recognition, we prepared chimeric evasins by interchanging the N-terminal regions of four class A evasins, including a newly identified evasin, EVA-RPU02. We show through chemokine binding analysis of the parental and chimeric evasins that the N-terminal region is critical for chemokine binding affinity and selectivity. Notably, we found some chimeras were unable to bind certain cognate chemokine ligands of both parental evasins. Moreover, unlike any natural evasins characterized to date, some chimeras exhibited specific binding to a single chemokine. These results indicate that the evasin N terminus interacts cooperatively with the “body” of the evasin to enable optimum chemokine recognition. Furthermore, the altered chemokine selectivity of the chimeras validates the approach of engineering the N termini of evasins to yield unique chemokine recognition profiles. Class A tick evasins are natural chemokine-binding proteins that block the signaling of multiple chemokines from the CC subfamily through their cognate receptors, thus suppressing leukocyte recruitment and inflammation. Development of tick evasins as chemokine-targeted anti-inflammatory therapeutics requires an understanding of the factors controlling their chemokine recognition and selectivity. To investigate the role of the evasin N-terminal region for chemokine recognition, we prepared chimeric evasins by interchanging the N-terminal regions of four class A evasins, including a newly identified evasin, EVA-RPU02. We show through chemokine binding analysis of the parental and chimeric evasins that the N-terminal region is critical for chemokine binding affinity and selectivity. Notably, we found some chimeras were unable to bind certain cognate chemokine ligands of both parental evasins. Moreover, unlike any natural evasins characterized to date, some chimeras exhibited specific binding to a single chemokine. These results indicate that the evasin N terminus interacts cooperatively with the “body” of the evasin to enable optimum chemokine recognition. Furthermore, the altered chemokine selectivity of the chimeras validates the approach of engineering the N termini of evasins to yield unique chemokine recognition profiles. The central hallmark of inflammation is the recruitment of leukocytes from the bloodstream to the affected tissues. This process is regulated by chemokines, a family of proteins expressed in response to tissue injury or infection. Chemokines bind and activate chemokine receptors expressed on the surface of leukocytes, resulting in leukocyte chemotaxis to the affected tissues and a variety of downstream responses, including innate and adaptive immunity and tissue repair (1Keane M.P. Strieter R.M. Chemokine signaling in inflammation.Crit. Care Med. 2000; 28: N13-N26Crossref PubMed Scopus (80) Google Scholar, 2Zhang H. Chen K. Tan Q. Shao Q. Han S. Zhang C. et al.Structural basis for chemokine recognition and receptor activation of chemokine receptor CCR5.Nat. Commun. 2021; 12: 4151Crossref PubMed Scopus (36) Google Scholar). There are ∼50 chemokines in humans that are classified as CCL, CXCL, CX3CL, or XCL (L indicates ligand) based on the arrangement of the first two of four conserved cysteine residues in their sequences (3Kufareva I. Salanga C.L. Handel T.M. Chemokine and chemokine receptor structure and interactions: Implications for therapeutic strategies.Immunol. Cell Biol. 2015; 93: 372-383Crossref PubMed Scopus (147) Google Scholar). Chemokine receptors are G protein–coupled receptors, classified as CCR, CXCR, CX3CR, or XCR (R indicates receptor), named according to the chemokines to which they respond. However, the chemokine–chemokine receptor network exhibits bidirectional promiscuity, whereby each receptor typically responds to several chemokines, and each chemokine typically activates multiple receptors. This complexity is likely to be one factor contributing to the failure of receptor-specific antagonists in clinical trials (4Zhao S. Wu B. Stevens R.C. Advancing chemokine GPCR structure based drug discovery.Structure. 2019; 27: 405-408Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Thus, it is attractive to consider alternative approaches for the development of anti-inflammatories, such as simultaneously suppressing the activity of several chemokines or receptors. The simultaneous inhibition of several chemokines is used naturally by viruses, helminths, and ticks, which all secrete armories of molecules, including chemokine-binding proteins, to subvert the host immune response (5González-Motos V. Kropp K.A. Viejo-Borbolla A. Chemokine binding proteins: an immunomodulatory strategy going viral.Cytokine Growth Factor Rev. 2016; 30: 71-80Crossref PubMed Scopus (41) Google Scholar). In particular, ticks are hematophagous ectoparasites that can remain undetected by their hosts for several days to weeks. Ticks from several genera secrete salivary proteins, called evasins (designated by the prefix ‘EVA’), that each bind to multiple chemokines, preventing the activation of the cognate chemokine receptors and suppressing the inflammatory response to the tick bite during acquisition of a blood meal (6Bhattacharya S. Nuttall P.A. Phylogenetic analysis indicates that evasin-like proteins of ixodid ticks fall into three distinct classes.Front. Cell Infect. Microbiol. 2021; 11: 769542Crossref PubMed Scopus (5) Google Scholar, 7Bhusal R.P. Eaton J.R.O. Chowdhury S.T. Power C.A. Proudfoot A.E.I. Stone M.J. et al.Evasins: tick salivary proteins that inhibit mammalian chemokines.Trends Biochem. Sci. 2020; 45: 108-122Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The earliest-discovered evasins, EVA-1, EVA-3, and EVA-4, have all exhibited promising anti-inflammatory activities in murine models of various inflammatory diseases (8Bonvin P. Power C.A. Proudfoot A.E.I. Evasins: therapeutic potential of a new family of chemokine-binding proteins from ticks.Front. Immunol. 2016; 7: 208Crossref PubMed Scopus (26) Google Scholar, 9Déruaz M. Bonvin P. Severin I.C. Johnson Z. Krohn S. Power C.A. et al.Evasin-4, a tick-derived chemokine-binding protein with broad selectivity can be modified for use in preclinical disease models.FEBS J. 2013; 280: 4876-4887Crossref PubMed Scopus (32) Google Scholar, 10Montecucco F. Mach F. Lenglet S. Vonlaufen A. Gomes Quinderé A.L. Pelli G. et al.Treatment with Evasin-3 abrogates neutrophil-mediated inflammation in mouse acute pancreatitis.Eur. J. Clin. Invest. 2014; 44: 940-950Crossref PubMed Scopus (39) Google Scholar, 11Russo R.C. Alessandri A.L. Garcia C.C. Cordeiro B.F. Pinho V. Cassali G.D. et al.Therapeutic effects of evasin-1, a chemokine binding protein, in bleomycin-induced pulmonary fibrosis.Am. J. Respir. Cell Mol. Biol. 2011; 45: 72-80Crossref PubMed Scopus (47) Google Scholar, V. F. Pelli G. K. Proudfoot A. et al.Treatment with the CC chemokine-binding protein injury and in 2013; PubMed Scopus (47) Google Scholar). evasins that bind to the chemokines in specific the of or it is to natural evasins with the selectivity they or to natural evasins to the of distinct of evasins are Class A evasins and conserved cysteine which four and bind to CC Class evasins conserved three and bind to chemokines R.P. Eaton J.R.O. Chowdhury S.T. Power C.A. Proudfoot A.E.I. Stone M.J. et al.Evasins: tick salivary proteins that inhibit mammalian chemokines.Trends Biochem. Sci. 2020; 45: 108-122Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). each each evasin exhibits a unique of chemokine binding from selectivity chemokine to broad activity chemokine A. Power C.A. M. et and of a chemokine-binding protein from the tick Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, M. C. G. K. et the of tick Biol. 2019; Full Text Full Text PDF PubMed Scopus Google Scholar, K. G. Eaton J.R.O. A. S. surface a family of evasins from ticks with CC chemokine-binding 7: PubMed Scopus Google Scholar). we on class A evasins, for which the basis of chemokine recognition is to be R.P. P. M.J. et engineering of tick evasins for chemokines in inflammatory Sci. S. A. PubMed Scopus Google Scholar, M. et al.Structural of activity of the tick salivary protein Biol. 2020; Full Text Full Text PDF PubMed Scopus Google Scholar, C. M. Power C.A. Proudfoot A.E.I. basis of chemokine by a tick chemokine binding the structure of the and Scholar). of to C. M. Power C.A. Proudfoot A.E.I. basis of chemokine by a tick chemokine binding the structure of the and and to and a by R.P. P. M.J. et engineering of tick evasins for chemokines in inflammatory Sci. S. A. PubMed Scopus Google that the of class A evasins for CC chemokines chemokine is by In binding CC chemokines is by the of in the and regions of the Thus, of the regions enable engineering of evasins to chemokines of In we found that a new class A evasin, from tick or exhibits chemokine selectivity to that of EVA-1, from To investigate the of the N terminus on chemokine we characterized several chimeric evasins. The results that the chemokine binding affinity and selectivity can be altered by interchanging the N-terminal regions the evasins. However, they for in chemokine binding the evasin N terminus and the in engineering to chemokines of In trials of several evasins identified in we found that to as to in and a by However, it as a broad to by and and in the to by which is the of with to of the as K. G. Eaton J.R.O. A. S. surface a family of evasins from ticks with CC chemokine-binding 7: PubMed Scopus Google Scholar, K. G. Eaton J.R.O. P. A. et evasins a to of chemokine PubMed Scopus Google Scholar, J.R.O. K. G. B. et N-terminal of a tick evasin is critical for chemokine binding and and specific binding activity to Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, C. J. Chowdhury S. M. R.P. et of an evasin from tick a critical role of for chemokine binding and Sci. S. A. 2020; PubMed Scopus Google results that the expressed protein which is with the of to potential in the protein and of class A evasins and of EVA-4, EVA-1, and of class A evasins conserved cysteine and potential residues the residues in that a and regions of parental and chimeric evasins, the evasins from which the N-terminal region and of each of and chimeric evasins. expressed and evasins and chimeras were to and with and of parental and chimeric evasins by We the binding of to all chemokines and surface a single chemokine for class A evasins, we binding of to any of the or chemokines the CC chemokines that and multiple that the from to and and with and the Notably, the and the binding chemokines chemokine binding and and the of to chemokines The are as from three in a new The and and the of to chemokines The are as from three We the of to inhibit the of chemokines in a activation by chemokines, chemokine receptors to inhibition of which can be in the chemokine receptor and a In the chemokines and activation of their receptor This by in a with their binding for inhibition of to show that a class A evasin, to chemokines their We the chemokine-binding selectivity of to of three class A evasins. to bind to three of CC chemokines and to as have to bind to and CC chemokines, R.P. P. M.J. et engineering of tick evasins for chemokines in inflammatory Sci. S. A. PubMed Scopus Google Scholar, C. M. Power C.A. Proudfoot A.E.I. basis of chemokine by a tick chemokine binding the structure of the and Scholar, P. F. J. Power C.A. et of the of the CC chemokine-binding proteins and Biol. 2014; Full Text Full Text PDF PubMed Scopus (22) Google Scholar). We expressed evasins and their chemokine binding selectivity and Notably, in we of the N-terminal to the evasin to the in a specific have used with evasins M. Bonvin P. Severin I.C. Johnson Z. Krohn S. Power C.A. et al.Evasin-4, a tick-derived chemokine-binding protein with broad selectivity can be modified for use in preclinical disease models.FEBS J. 2013; 280: 4876-4887Crossref PubMed Scopus (32) Google Scholar, A. Power C.A. M. et and of a chemokine-binding protein from the tick Biol. Full Text Full Text PDF PubMed Scopus Google or K. G. Eaton J.R.O. A. S. surface a family of evasins from ticks with CC chemokine-binding 7: PubMed Scopus Google which is from T.M. for of and 2014; Scholar). the chemokine binding and were in with with to the of the used M. Bonvin P. Severin I.C. Johnson Z. Krohn S. Power C.A. et al.Evasin-4, a tick-derived chemokine-binding protein with broad selectivity can be modified for use in preclinical disease models.FEBS J. 2013; 280: 4876-4887Crossref PubMed Scopus (32) Google Scholar, A. Power C.A. M. et and of a chemokine-binding protein from the tick Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, K. G. Eaton J.R.O. A. S. surface a family of evasins from ticks with CC chemokine-binding 7: PubMed Scopus Google binding of class A evasins EVA-4, and of of four class A evasins for their cognate A indicates binding The are as from three in a new The of four class A evasins for their cognate A indicates binding The are as from three To the of evasins that to their in chemokine we their sequences four evasins the conserved cysteine and two conserved which the evasin In they to potential with in to chemokine binding A. Power C.A. M. et and of a chemokine-binding protein from the tick Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, K. G. Eaton J.R.O. A. S. surface a family of evasins from ticks with CC chemokine-binding 7: PubMed Scopus Google Scholar, M. A. Alessandri A.L. R.C. et chemokine binding proteins with Med. PubMed Scopus Google Scholar). have that residues the N-terminal regions the first conserved of and in chemokine binding R.P. P. M.J. et engineering of tick evasins for chemokines in inflammatory Sci. S. A. PubMed Scopus Google Scholar, C. M. Power C.A. Proudfoot A.E.I. basis of chemokine by a tick chemokine binding the structure of the and Scholar). The N-terminal regions of all four evasins in the one or two In the of to chemokine binding affinity C. J. Chowdhury S. M. R.P. et of an evasin from tick a critical role of for chemokine binding and Sci. S. A. 2020; PubMed Scopus Google Scholar). However, the N-terminal regions in and as as the the and the first conserved We that the the N-terminal regions of evasins are in for their distinct chemokine recognition profiles. To the that the N-terminal regions of class A evasins to their chemokine recognition we prepared a of chimeric proteins in which the N-terminal regions were evasins we the N-terminal region of with of and and the N-terminal regions of and evasins were named the in which the evasin from which the N-terminal region and the evasin from which the of the “body” of the of the N terminus of and the of chimeric evasin expressed and from each the and by were to that of the parental evasin from which the of the This is with of the in the of each evasin that the N-terminal regions of evasins are likely to be the of of the chimeric evasins is to to by the of the To we the of each and each evasin The evasins exhibited in the to with of and in of the of all the chimeric evasins exhibited and to the evasin from which the of the exhibits to exhibits to a a and to that be of chimeric the of the of the of the of of parental and chimeric evasins from The are as from three in a new of parental and chimeric evasins from The are as from three The results that of the chimeras are to their chemokine binding to the of the N-terminal region and the of each We used to the binding of each to all all the chimeras to bind to or chemokines, of the chimeras to of CC We for binding of each chimeric evasin to each chemokine and binding of chimeric of chimeric are the from three A indicates binding in a new are the from three A indicates binding a broad of chemokine recognition, a of Thus, N-terminal chimeras of two evasins the role of the N terminus in chemokine recognition. binding to any CC chemokines, it is to with affinity bind to any CC chemokines selectivity for CC chemokines and with or and To is the first an evasin to bind and inhibit a single chemokine. The selectivity of from of both the parental evasins, that it from the chemokine binding of the evasin and N-terminal chemokine recognition to two regions of the evasins that to all the chemokines as as as a of chemokines, we that the chimeras and a bind to the chemokines as However, we found that the binding of two chimeras were distinct from of the parental evasins. exhibited binding to and chemokine to both of the parental evasins three of the chemokines as and and three chemokines and bind to which is a to both parental evasins to and to These indicate that the N-terminal region and of evasins cooperatively to cognate and have chemokine recognition profiles. a chemokine recognition to EVA-1, for of binding to This that the of binding to and binding to requires some of the N-terminal In exhibited selectivity to all chemokines and This is to the that exhibited for a role for chemokine-binding by the and N-terminal region of chimeric that the regions of and for each in chimeras of two evasins, we that regions of two evasins into chemokine recognition In of we found that the chemokine recognition of from that of This an role for the evasin in chemokine selectivity. The exhibited the chemokine recognition of all the chimeras binding to of the chemokines as with affinity evasins are natural chemokine that inhibit receptor signaling and have the potential to be modified for use as clinical anti-inflammatory However, in to tick-derived we an understanding of the factors that the affinity and selectivity of evasins for In the we characterized the evasin for the first and chimeras and three evasins EVA-4, and to investigate the role of the evasin N-terminal region in chemokine recognition. the results a role for the evasin N they show that the N terminus cooperatively with the of the evasin to chemokine binding selectivity. The that the N-terminal a on the chemokine selectivity of class A evasins. the N terminus of with that of a that to of the chemokine ligands of The of the N terminus is with several Bonvin et P. F. J. Power C.A. et of the of the CC chemokine-binding proteins and Biol. 2014; Full Text Full Text PDF PubMed Scopus (22) Google that two residues in the N-terminal region of and were critical for binding to Eaton et J.R.O. K. G. B. et N-terminal of a tick evasin is critical for chemokine binding and and specific binding activity to Biol. Full Text Full Text PDF PubMed Scopus Google that the N terminus and the first of with of a in chemokine selectivity. we have that and in the N-terminal region of the chemokine selectivity of evasin R.P. P. M.J. et engineering of tick evasins for chemokines in inflammatory Sci. S. A. PubMed Scopus Google Scholar). results the of the evasin N-terminal they indicate that the N-terminal region is to the chemokine selectivity of each evasin, that regions of the structure to binding affinity and selectivity. and which have the N-terminal exhibited selectivity to with or affinity to and which the N-terminal region as to bind to binding to and a single structure of an the structure of to C. M. Power C.A. Proudfoot A.E.I. basis of chemokine by a tick chemokine binding the structure of the and Scholar). However, we have the of to each of two chemokines and and a chimeric chemokine which the N-terminal and regions of were by of as as the structure of an to R.P. P. M.J. et engineering of tick evasins for chemokines in inflammatory Sci. S. A. PubMed Scopus Google Scholar). These the role of the evasin N-terminal region indicate two regions that to recognition of several residues from the first two a that to the first of the chemokine a conserved The residues the four evasins in contributing to their chemokine binding selectivity. the of the evasin is in to the N-terminal of the chemokine. In regions are and the residues show However, the structure of to the a and surface the N-terminal regions of R.P. P. M.J. et engineering of tick evasins for chemokines in inflammatory Sci. S. A. PubMed Scopus Google Scholar). that the residues to affinity binding of The regions of the four evasins in the both in their and sequences in that the of regions with the chemokine N termini the chemokine binding and of evasins. the results that the N-terminal and regions of the evasins are in chemokine recognition, they that the of are the for from the This is from of and of parental evasins bind to the chemokines and The is and of chemokine binding However, it bind to any of the chemokines that bind to both parental evasins Thus, binding to chemokines is by the N-terminal region of in the of the of or by the N-terminal region of in the of the of it is by the N-terminal region of in the of the of This indicates that the regions of the evasin the chemokines in a we that the regions of the evasins such we can based on the structure of to R.P. P. M.J. et engineering of tick evasins for chemokines in inflammatory Sci. S. A. PubMed Scopus Google Scholar). The structure that the chemokine interacts with both the and the N terminus of the Moreover, that to the binding the first of the chemokine in interacts with a in the of the evasin surface in and several chemokine with residues in the a surface surface in that interacts with evasin N-terminal residues and However, the structure and several the N-terminal region and the of These to the N-terminal region to bind to the chemokine Thus, of the N terminus the of two evasins the of the N-terminal region to with the chemokine and a for the the evasin and N-terminal is the class A evasin from the tick chemokine binding characterized K. G. Eaton J.R.O. A. S. surface a family of evasins from ticks with CC chemokine-binding 7: PubMed Scopus Google Scholar, K. G. Eaton J.R.O. P. A. et evasins a to of chemokine PubMed Scopus Google Scholar, J.R.O. K. G. B. et N-terminal of a tick evasin is critical for chemokine binding and and specific binding activity to Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. A. C. M. M. et from genera evasin Biol. Full Text Full Text PDF PubMed Scopus (41) Google Scholar, J. H. J. et of host immunity by tick 2015; PubMed Scopus Google Scholar, Alessandri A.L. et al.Treatment with a chemokine-binding protein or from J. Full Text Full Text PDF PubMed Scopus (80) Google Scholar). We found that to CC chemokines, evasins and have to bind to CC chemokines, on the of an affinity K. G. Eaton J.R.O. A. S. surface a family of evasins from ticks with CC chemokine-binding 7: PubMed Scopus Google Scholar, J.R.O. K. G. B. et N-terminal of a tick evasin is critical for chemokine binding and and specific binding activity to Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the chemokines by are a of by two evasins, which bind to of This the of is some to the tick of all three evasins. the analysis of class A evasins to be from and it likely that some of evasins bind to of CC which is to of a variety of including and as as some J. M. J. of in tick in 2013; PubMed Scopus Google Scholar, S. Ticks with and of the Med. PubMed Google Scholar, in an in Med. Sci. 2013; Google and can and is to that by a of salivary evasins that inhibit chemokines, both on a host and host on an understanding of chemokine recognition by class A evasins and of evasin we that the N-terminal region evasins yield with altered selectivity for results show that the selectivity in an In particular, as a of the chemokine of the N-terminal region and regions of the evasins, the N-terminal regions can to chimeras with chemokine selectivity selectivity for a single chemokine. Thus, it be to evasin with chemokine results that an approach to such be the of of evasin parental and chimeric evasins were expressed in The parental and chimeric evasins, a an and a on the N were into the and and into the were to a of in of and were with of for and to the with The were in a and for to The for and the the protein were and The were a with The with the and the protein with the The proteins were to an and by with and a of The the proteins were and used or and for The and of parental evasins and chimeras were by and of protein a with with The protein with of with of protein a and with a of The prepared by protein the In all the proteins were by the and the with for protein expressed and of with and by the for The protein and with and F. The for The protein by and by with The of parental and chimeric evasins were with of protein and to an from to a of The in with an of and of and The of to and in a to yield the and the of three parental and chimeric evasins were to of the by evasins with and in the of for in proteins were from the by as The the proteins were for chemokine binding chemokines and were as R.P. P. M.J. et engineering of tick evasins for chemokines in inflammatory Sci. S. A. PubMed Scopus Google with and were from The chemokine binding analysis of evasins by a and a with evasins in were a of to an of to response the binding of each chemokine or from in the through the a of for by of to binding and the chemokines that exhibited binding on were through an and The and binding were by the with binding three inhibition of receptor signaling by evasins J. et of a to a of by the Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). chemokine receptor were in with and in a with of with of and and the and for into a four M. Stone M.J. of chemokine signaling chemokine receptors and J. Mol. Sci. 2021; PubMed Scopus (5) Google Scholar). the were and to a for were and with for with for To chemokine were by of or for by the of a the by and were a of to and to a one in a by the of to on the were with or or or with of evasins, and the as The in is expressed as a of inhibition of the chemokine in the of are to be to the This The that they have of with the of We of for with and of for with P. P. J. and M. J. S. P. S. and P. B. P. S. P. and M. J. S. P. P. and M. J. S. P. S. P. J. P. and M. J. S. and P. J. P. and M. J. S. P. A. and P. B. P. and M. J. S. J. P. and M. J. S. P. B. and M. J. S. This by and J. S. and J. and J. and J. by a from P. and by a from P. B. and M. J.

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