Characterization of the Interactions of vMIP-II, and a Dimeric Variant of vMIP-II, with Glycosaminoglycans

摘要

Chemokines are important immune proteins, carrying out their function by binding tonglycosaminoglycans (GAGs) on the endothelial surface and to cell surface chemokine receptors. A uniquenviral chemokine analogue, viral macrophage inflammatory protein-II (vMIP-II), encoded by humannherpesvirus-8, has garnered interest because of its ability to bind to multiple chemokine receptors, includingnboth HIV coreceptors. In addition, vMIP-II binds to cell surface GAGs much more tightly than most humannchemokines, which may be the key to its anti-inflammatory function in vivo. The goal of this work was tondetermine the mechanism of binding of GAG by vMIP-II. The interaction of vMIP-II with a heparin-derivedndisaccharide was characterized using NMR. Important binding sites were further analyzed by mutagenesisnstudies, in which corresponding vMIP-II mutants were tested for GAG binding ability using heparinnchromatography and NMR.We found that despite having many more basic residues than some chemokines,nvMIP-II shares a characteristic binding site similar to that of its human analogues, utilizing basic residuesnR18, R46, and R48. Interestingly, a particularmutation (Leu13Phe) caused vMIP-II to forma pH-dependentnCC chemokine-type dimer as determined by analytical ultracentrifugation and NMR. To the best of ournknowledge, this is the first example of engineering a naturally predominantly monomeric chemokine into andissociable dimer by a singlemutation. This dimeric vMIP-IImutant binds to heparinmuchmore tightly thannwild-type vMIP-II and provides a new model for studying the relationship between chemokine quaternarynstructure and various aspects of function. Structural differences between monomeric and dimeric vMIP-IInupon GAG binding were characterized by NMR and molecular docking.