Poster Presentation The 47th Lorne Conference on Protein Structure and Function 2022

Engineering of Evasins by Exchanging their N-termini Alters Chemokine Binding Selectivity (#329)

Pramod Aryal 1 2 , Shankar Raj Devkota 1 2 , Ram Prasad Bhusal 1 2 , Martin J. Stone 1 2
  1. Infection and Immunity Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia
  2. Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia

Chemokines are critical mediators in the inflammatory cascade. Binding of chemokines to their receptors, expressed on the surface of leukocytes, results in leukocyte migration towards injured or infected tissues.  Evasins are tick salivary proteins that bind to chemokines and prevent activation of chemokine receptors to subvert the host immune response. To understand the principles governing the CC chemokine selectivity of class A evasins, we have characterised the novel evasins EVA-P974, from Amblyomma cajennense, and EVA-RPU-02, from Rhipicephalus pulchellus, and compared them to the archetypal class A evasins, EVA-1 and EVA-4. Our crystal structure of EVA-P974 complexed to two different chemokines (CCL7 and CCL17) together with extensive mutational analysis, revealed the role of the evasin N-terminus in chemokine binding affinity and selectivity. To test the hypothesis that swapping the N-terminus between evasins would alter their chemokine selectivity, we designed chimeric evasins by interchanging the N-terminal regions between EVA-1, EVA-4, EVA-P974 and EVA-RPU802. We expressed and purified these chimeric evasins and examined their chemokine binding properties. In line with our hypothesis, we found that the chemokine binding selectivity and affinity of these chimeric evasins differed from those of the wild type evasins, but not in the predicted manner. The chimeric evasin consisting of the N-terminus of EVA-4 and the core region of EVA-RPU802 was absolutely selective for the chemokine CCL8 (Kd = 64.4 ± 0.1 nM). Likewise, another chimeric evasin, which contains the N-terminus of EVA-1 and core region of EVA-RPU802, was able to bind to CCL14 selectively. We found that different evasins achieve binding to similar group of chemokines by utilising different residues.  Overall, our study showed that the chemokine binding specificity and affinity of evasins can be altered by interchanging their N-terminal regions. This study provides proof of principle that evasins can be semi-rationally engineered to target specific chemokines.