title: Molecular interaction studies of HIV-1 matrix protein p17 and heparin: identification of the heparin-binding motif of p17 as a target for the development of multitarget antagonists.
abstract: Once released by HIV(+) cells, p17 binds heparan sulfate proteoglycans (HSPGs) and CXCR1 on leukocytes causing their dysfunction. By exploiting an approach integrating computational modeling, site-directed mutagenesis of p17, chemical desulfation of heparin, and surface plasmon resonance, we characterized the interaction of p17 with heparin, a HSPG structural analog, and CXCR1. p17 binds to heparin with an affinity (K(d) = 190 nm) that is similar to those of other heparin-binding viral proteins. Two stretches of basic amino acids (basic motifs) are present in p17 N and C termini. Neutralization (Arg→Ala substitution) of the N-terminal, but not of the C-terminal basic motif, causes the loss of p17 heparin-binding capacity. The N-terminal heparin-binding motif of p17 partially overlaps the CXCR1-binding domain. Accordingly, its neutralization prevents also p17 binding to the chemochine receptor. Competition experiments demonstrated that free heparin and heparan sulfate (HS), but not selectively 2-O-, 6-O-, and N-O desulfated heparins, prevent p17 binding to substrate-immobilized heparin, indicating that the sulfate groups of the glycosaminoglycan mediate p17 interaction. Evaluation of the p17 antagonist activity of a panel of biotechnological heparins derived by chemical sulfation of the Escherichia coli K5 polysaccharide revealed that the highly N,O-sulfated derivative prevents the binding of p17 to both heparin and CXCR1, thus inhibiting p17-driven chemotactic migration of human monocytes with an efficiency that is higher than those of heparin and HS. Here, we characterized at a molecular level the interaction of p17 with its cellular receptors, laying the basis for the development of heparin-mimicking p17 antagonists.
authors.affiliation: Section of Experimental Oncology and Immunology, School of Medicine, University of Brescia, Brescia 25123, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Bugatti
authors.forename: Cinzia
authors.initials: C
authors.lastname: Giagulli
authors.forename: Chiara
authors.initials: C
authors.lastname: Urbinati
authors.forename: Francesca
authors.initials: F
authors.lastname: Caccuri
authors.forename: Paola
authors.initials: P
authors.lastname: Chiodelli
authors.forename: Pasqua
authors.initials: P
authors.lastname: Oreste
authors.forename: Simona
authors.initials: S
authors.lastname: Fiorentini
authors.forename: Alessandro
authors.initials: A
authors.lastname: Orro
authors.forename: Luciano
authors.initials: L
authors.lastname: Milanesi
authors.forename: Pasqualina
authors.initials: P
authors.lastname: D'Ursi
authors.forename: Arnaldo
authors.initials: A
authors.lastname: Caruso
authors.forename: Marco
authors.initials: M
authors.lastname: Rusnati
mesh_terms: D000595:Amino Acid Sequence; D002634:Chemotaxis, Leukocyte; D015488:HIV Antigens / Q000737:chemistry / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism*
chemical_list: D015488:HIV Antigens; D054301:gag Gene Products, Human Immunodeficiency Virus; C054858:p17 protein, Human Immunodeficiency Virus Type 1; D006493:Heparin