Structure-guided engineering of tick evasins for targeting chemokines in inflammatory diseases

As natural chemokine inhibitors, evasin proteins produced in tick saliva are potential therapeutic agents for numerous inflammatory diseases. Engineering evasins to block the desired chemokines and avoid off-target side effects requires structural understanding of their target selectivity. Structures of the class A evasin EVA-P974 bound to human CC chemokine ligands 7 and 17 (CCL7 and CCL17) and to a CCL8-CCL7 chimera reveal that the specificity of class A evasins for chemokines of the CC subfamily is defined by conserved, rigid backbone–backbone interactions, whereas the preference for a subset of CC chemokines is controlled by side-chain interactions at four hotspots in flexible structural elements. Hotspot mutations alter target preference, enabling inhibition of selected chemokines. The structure of an engineered EVA-P974 bound to CCL2 reveals an underlying molecular mechanism of EVA-P974 target preference. These results provide a structure-based framework for engineering evasins as targeted antiinflammatory therapeutics.

While some proteins exhibit absolute specificity for a unique binding partner, many others display “multispecificity,” whereby they interact with several, but not all, members of a partner protein family (1, 2). Understanding how proteins achieve such selectivity provides a basis for rational engineering to regulate alternative targets. In this study, we investigated the structural basis for multispecific recognition of human proinflammatory chemokines by tick evasin proteins.Chemokines are the master regulators of leukocyte-trafficking, the unifying feature of immune homeostasis and all inflammatory diseases (3). Chemokines stimulate leukocyte migration via activation of chemokine receptors, G protein–coupled receptors expressed on the surfaces of leukocytes. Chemokines are classified into two major families (CCL and CXCL) and two minor families (XCL and CX₃CL) based on the arrangement of conserved cysteine residues near the N termini of their amino acid sequences. Chemokine receptors are classified (CCR, CXCR, XCR, and CX₃CR) based on their chemokine selectivity. The types of leukocytes recruited to specific tissues depend on the array of chemokines expressed in those tissues and the selectivity of those chemokines for the receptors expressed on different leukocyte subsets. For example, in vascular inflammation associated with hypertension, elevated levels of the chemokines CCL2, CCL7, and CCL8 act via the receptor CCR2 (and possibly also CCR1) to stimulate migration of monocytes into the blood vessel wall (4).To suppress leukocyte recruitment in inflammatory diseases, numerous antagonists of specific chemokine receptors have been evaluated in clinical trials. However, these trials have not yielded any new antiinflammatory therapeutics (5), in part because most leukocytes can utilize multiple chemokine receptors, thus circumventing the specific antagonists. The alternative approach of targeting chemokines has not generally been favored, because it would require agents that bind with high affinity to multiple chemokines. However, the natural chemokine-binding proteins of ticks, helminths, and viruses (6–8) display multispecificity for mammalian chemokines, suggesting that they could potentially be deployed as antiinflammatory therapeutics.Evasins are two families of chemokine-binding, antiinflammatory proteins from tick saliva (6). Class A evasins each inhibit multiple CC chemokines of their mammalian hosts but none of the closely related CXC chemokines. Conversely, class B evasins are specific for CXC over CC chemokines but exhibit variable selectivity among CXC chemokines. Typically, each tick species secretes a mixture of evasins, thereby accomplishing broad-spectrum suppression of the host inflammatory response, presumably enabling the tick to feed on the host for extended periods.The in vivo antiinflammatory activity of tick evasins has been demonstrated using a variety of animal models of inflammatory diseases, including lung fibrosis, skin inflammation, arthritis, colitis, pancreatitis, ischemic reperfusion injury, postinfarction myocardial injury, and Leishmania major infection (9–13). However, deploying evasins as effective antiinflammatory therapeutics in humans would require engineering the natural evasins to selectively target the relevant array of chemokines for any given indication while minimizing off-target inhibition (14). Such engineering requires understanding both the specificity of evasins for a single chemokine subfamily and their target preference among chemokines within that subfamily.Previously, only a single structure has been reported for an evasin:chemokine complex, class A evasin EVA-1 bound to CCL3 (15), so it has not been possible to identify the conserved and variable features of the interactions. Nevertheless, the structure revealed that EVA-1 binds to several receptor recognition elements of CCL3, explaining its inhibitory activity. Moreover, limited mutational data (15, 16) have confirmed that residues in the N- and C-terminal regions of EVA-1 and the homologous EVA-4, respectively, contribute to binding affinity, raising the question of whether the structural basis of CC chemokine recognition varies across the class A evasin family.To establish a structure-based platform for engineering the chemokine selectivity of class A evasins, we now report the structures of EVA-P974 (previously called ACA-01) (17, 18), from the Cayenne tick (Amblyomma cajennense), bound to each of two wild-type chemokines and one chimeric CC chemokine. Structural comparisons and extensive evasin and chemokine mutational data revealed the structural basis for CC chemokine specificity and identified several “hotspots” that define target preference among CC chemokines. These insights enabled EVA-P974 to be engineered to modify its target preference. We further verified the molecular basis of the modified selectivity by solving the chemokine-bound structure of the engineered evasin. Finally, by inhibiting a chemokine mixture, we provide proof of principle for applying engineered evasins as multichemokine inhibitors.