Poster Presentation 14th Australian Peptide Conference 2022

Structural insights into the inhibition of the Chemokine activity by Tick Evasins (#116)

Shankar Devkota 1 , Pramod Aryal 1 , Ram Bhusal 1 , Martin Stone 1
  1. Monash University, Clayton, VIC, Australia

Inflammation is a normal response to any injury or infection. However, sustained inflammation can cause numerous diseases. A hallmark of inflammation is leukocyte recruitment to the affected tissue, regulated by small secretory proteins called chemokines. They bind and activate chemokine receptors expressed on the leukocyte surface (1). As a strategy to suppress detection and  prolong their feeding  time, ticks  produce chemokine-binding proteins called 'Evasins', which bind to host chemokines and block chemokine receptors' activation preventing leukocyte migration. Several studies have shown that Evasins are natural, selective chemokine-inhibitory proteins with excellent potential as therapeutics for inflammatory diseases (2,3). Using bioinformatics, we identified variants of previously characterised Evasins with ten cysteine residues forming five-disulfide bonds. We expressed and purified EVA-ACA1001 using the E. coli expression system and analysis of the purified protein supported the predicted five intramolecular disulfide bonds. Binding data, obtained using a surface plasmon resonance (SPR), showed that EVA-ACA1001 binds to multiple CC chemokines with dissociation equilibrium constants (KD) in the range (0.01-100) nM. Furthermore, cAMP inhibition assay demonstrated the chemokine inhibitory function with IC50 value (1 nM to 100 nM). Truncation of N and C terminus confirmed that the core region confers the chemokine binding and selectivity of EVA-ACA1001, a distinct feature of C10 Evasins. Our crystal structures of a C10 Evasin in complex with the chemokines CCL7 and CCL17 showed extensive hydrogen bonds between Evasin and the CC motif of chemokine, a common feature of Class A Evasins. In contrast to traditional Class A Evasins, a big hydrophobic pocket formed in the core region of C10 Evasin can accommodate at least two amino residues from the N- loop of chemokines, making it a broad binder. Thus, this study describes structure and function of C10 Evasins and pave a pathway for the engineering of a potent anti-inflammatory molecules.

  1. 1. Bhusal RP, Foster SR, Stone MJ. Structural basis of chemokine and receptor interactions: Key regulators of leukocyte recruitment in inflammatory responses. Protein Sci 2019.
  2. 2. Bhusal RP, Eaton JRO, Chowdhury ST, Power CA, Proudfoot AEI, Stone MJ, et al. Evasins: Tick Salivary Proteins that Inhibit Mammalian Chemokines. Trends Biochem Sci 2019.
  3. 3. Hayward J, Sanchez J, Perry A, Huang C, Rodriguez Valle M, Canals M, et al. Ticks from diverse genera encode chemokine-inhibitory Evasin proteins. J Biol Chem. 2017;292(38):15670-80.