Abstract
Chemokines, a sub-family of cytokines, are small, secreted proteins that mediate a variety of biological processes. Various chemokines, despite the low degree of sequence homology, adopt remarkable conserved tertiary structure comprising an anti-parallel beta-sheet core domain followed by a C-terminal helix that packs onto the beta-sheet despite the low degree of sequence homology. The conserved structural feature allows chemokines to bind cell surface glycosaminoglycans (GAGs), crucially for recruitment of chemokines to a specific anatomical location. The recently isolated variant, CXCL4L1, is a homologue of CXCL4 chemokine (or platelet factor 4, PF-4) with potent anti-angiogenic activity and differed only in three amino acid residues of P58L, K66E and L67H. In this study, we show by X-ray structural determination that CXCL4L1 adopts a novel structure at its C-terminus. The orientation of the C-terminal helix protrudes into the aqueous space to expose the entire helix. The alternative helix orientation modifies the overall chemokine shape and surface properties. This change results in the decrease of its GAG-binding properties. A combined NMR and SPR investigation on a set of intrinsic mutants further shows that L67H is mainly responsible for the swing-out effect of the helix and the reduced GAGs binding activities, while mutations of P58L and K66E act secondarily. Our results demonstrate that the structural reorganization of the C-terminal helix mediates their anti-angiogenic effects and diffusibility. The data suggest that based on just three mutations in the primary sequence, chemokine enables to adapt an alternate C-terminal helix conformation to alter the cell surface recognition.