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J Biol Chem. 2019 Jul 19;294(29):11199-11212. doi: 10.1074/jbc.RA119.008817. Epub 2019 Jun 5.

A knottin scaffold directs the CXC-chemokine-binding specificity of tick evasins.

Author information

1
Radcliffe Department of Medicine Division of Cardiovascular Medicine, University of Oxford, Oxford OX3 7BN, United Kingdom.
2
Serono Pharmaceutical Research Institute, 1228 Geneva, Switzerland.
3
Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom.
4
Serono Pharmaceutical Research Institute, 1228 Geneva, Switzerland jmdias@pfizer.com.
5
Radcliffe Department of Medicine Division of Cardiovascular Medicine, University of Oxford, Oxford OX3 7BN, United Kingdom sbhattac@well.ox.ac.uk.

Abstract

Tick evasins (EVAs) bind either CC- or CXC-chemokines by a poorly understood promiscuous or "one-to-many" mechanism to neutralize inflammation. Because EVAs potently inhibit inflammation in many preclinical models, highlighting their potential as biological therapeutics for inflammatory diseases, we sought to further unravel the CXC-chemokine-EVA interactions. Using yeast surface display, we identified and characterized 27 novel CXC-chemokine-binding evasins homologous to EVA3 and defined two functional classes. The first, which included EVA3, exclusively bound ELR+ CXC-chemokines, whereas the second class bound both ELR+ and ELR- CXC-chemokines, in several cases including CXC-motif chemokine ligand 10 (CXCL10) but, surprisingly, not CXCL8. The X-ray crystal structure of EVA3 at a resolution of 1.79 Å revealed a single antiparallel β-sheet with six conserved cysteine residues forming a disulfide-bonded knottin scaffold that creates a contiguous solvent-accessible surface. Swapping analyses identified distinct knottin scaffold segments necessary for different CXC-chemokine-binding activities, implying that differential ligand positioning, at least in part, plays a role in promiscuous binding. Swapping segments also transferred chemokine-binding activity, resulting in a hybrid EVA with dual CXCL10- and CXCL8-binding activities. The solvent-accessible surfaces of the knottin scaffold segments have distinctive shape and charge, which we suggest drives chemokine-binding specificity. These studies provide structural and mechanistic insight into how CXC-chemokine-binding tick EVAs achieve class specificity but also engage in promiscuous binding.

KEYWORDS:

chemokine; chemotaxis; crystal structure; evasin; host-pathogen interaction; immune response; inflammation; knottin; protein-protein interaction; tick

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