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

J Biol Chem. 2013 Jan 11;288(2):1150-61. doi: 10.1074/jbc.M112.400077. Epub 2012 Nov 19.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Chemotaxis, Leukocyte
  • HIV Antigens / chemistry
  • HIV Antigens / metabolism*
  • Heparin / metabolism*
  • Humans
  • Models, Molecular
  • Molecular Docking Simulation
  • Protein Binding
  • Surface Plasmon Resonance
  • gag Gene Products, Human Immunodeficiency Virus / chemistry
  • gag Gene Products, Human Immunodeficiency Virus / metabolism*

Substances

  • HIV Antigens
  • gag Gene Products, Human Immunodeficiency Virus
  • p17 protein, Human Immunodeficiency Virus Type 1
  • Heparin