Structural characterization of the glycoprotein gp160 cleavage site

Cleavage of the HIV envelope glycoprotein gp160 into gp120/gp41 is a prerequisite for virus infectivity. The glycoprotein precursor gp160 undergoes a selective endo-proteolysis at the carboxyl side of a consensus motif consisting of the amino acid sequence Arg508-Glu-Lys510-Arg (site1). The Precursor Convertases (PCs) are proposed enzyme candidates for intracellular processing of the HIV envelope glycoprotein. Furin and PC7, which belong to this endoprotease family, have been shown to correctly cleave gp160. In particular, cleavage occurs selectively at site 1 although other multi-basic sites, which constitute potential substrates for pro-protein convertases, are present. These results suggest that both basic residues and specific secondary structure elements are required for the correct processing of gp160. To determine the role of secondary structure of proteolytic activation, we examined some synthetic peptides reproducing the regions surrounding cleavage site 1 of gp160 HIV-1: 1-19 PTKAKRRVVQREKRAVGIG 505-523 HIV gp160, 1-10 PTKAKRRVVQ 505-514 HIV 1 gp160, 8-19 VVQREKRAVGIG 512-523 HIV-1 gp160. These peptides were analyzed with respect to their ability to be cleaved in vitro by Furin and were structurally characterized by CD and NMR spectroscopy. The kinetic data on cleavage by Furin indicate a clear preference of the enzyme for the larger 1-19 substrate. Furthermore, Furin cleaves this peptide at the processing site 1 REKR-A more efficiently than at the other consensus motif KAKR-R. The experimental data from NMR, here presented, show that in the 1-19 peptide, the cleavage site 1 is likely structured in a loop while the secondary consensus motif is incorporated in a helical structured segment. A model by homology of the three dimensional structure of the catalytic domain of Furin with model substrate 1-19 is here also proposed.