Molecular Interaction Studies of HIV-1 Matrix Protein p17 and\ud Heparin: IDENTIFICATION OF THE HEPARIN-BINDING MOTIF OF p17 AS A TARGET FOR THE DEVELOPMENT OF MULTITARGET ANTAGONISTS

摘要

Once released by HIV cells, p17 binds heparan sulfate proteoglycans\ud(HSPGs) and CXCR1 on leukocytes causing their\uddysfunction. By exploiting an approach integrating computational\udmodeling, site-directed mutagenesis of p17, chemical\uddesulfation of heparin, and surface plasmon resonance, we characterized\udthe interaction of p17 with heparin, a HSPG structural\udanalog, and CXCR1. p17 binds to heparin with an affinity (Kd\ud190 nM) that is similar to those of other heparin-binding viral\udproteins. Two stretches of basic amino acids (basic motifs) are\udpresent in p17 N and C termini. Neutralization (Arg3Ala substitution)\udof the N-terminal, but not of the C-terminal basic\udmotif, causes the loss of p17 heparin-binding capacity. The\udN-terminal heparin-binding motif of p17 partially overlaps the\udCXCR1-binding domain. Accordingly, its neutralization prevents\udalso p17 binding to the chemochine receptor. Competition\udexperiments demonstrated that free heparin and heparan\udsulfate (HS), but not selectively 2-O-, 6-O-, and N-O desulfated\udheparins, prevent p17 binding to substrate-immobilized heparin,\udindicating that the sulfate groups of the glycosaminoglycan\udmediate p17 interaction. Evaluation of the p17 antagonist activity\udof a panel of biotechnological heparins derived by chemical\udsulfation of the Escherichia coli K5 polysaccharide revealed that\udthe highlyN,O-sulfated derivative prevents the binding of p17 to\udboth heparin and CXCR1, thus inhibiting p17-driven chemotactic\udmigration of human monocytes with an efficiency that is\udhigher than those of heparin and HS. Here, we characterized at a\udmolecular level the interaction of p17 with its cellular receptors,\udlaying the basis for the development of heparin-mimicking p17\udantagonists.