Structural Investigation of the HIV‐1 Envelope Glycoprotein gp160 Cleavage Site, 2: Relevance of an N‐Terminal Helix

Abstract

Proteolytic activation of the HIV-1 envelope glycoprotein gp160 is selectively performed by the proprotein convertase furin at the C-terminus of the sequence R508-E-K-R511 (site 1), in spite of the presence of another consensus sequence, Lys500-Ala-Lys-Arg503 (site 2). On the basis of the solution structural analysis of the synthetic peptide p498, spanning the gp160 sequence Pro498-Gly516, we previously suggested a possible role of an N-terminal helix in regulating the exposure and accessibility of the gp160 physiological cleavage site, enclosed in a loop. Here we report on the activity and conformation of the 23-residue peptide h-REKR, designed to exhibit a large N-terminal helix, followed by the gp160 native sequence, Arg508-Gly516. h-REKR is digested by furin with high efficiency, comparable to the full native p498. Circular dichroism analyses, in mixtures from pure water to 98 % trifluoroethanol, outline a significant content of helical structure in the peptide conformation. The molecular model obtained from NMR data collected in trifluoroethanol/water, by means of DYANA and AMBER simulations, indeed has helical structure on a large N-terminal segment. Such a long helix does not seem to affect the loop conformation of the C-terminal site 1-containing sequence, which exhibits the same proton chemical shifts already observed for the full native p498.

Structural Investigation of the HIV‐1 Envelope Glycoprotein gp160 Cleavage Site 3: Role of Site‐Specific Mutations

“…As already hypothesized on the basis of homology models for the furin catalytic domain (Siezen and co‐workers23, 24 and our own laboratories8, 11), the furin binding region can be described as a narrow channel, able to accommodate several substrate residues around the multibasic sequence (see also Siezen et al23). The furin catalytic triad (Ser368–His194–Asp153) is located at the bottom of the channel, together with several surface‐exposed acidic residues, making specific electrostatic interactions with the basic amino acids at positions P1, P2, and P4.…”

“… When no mutation occurs in or close to the cleavage site, but different sequences are exhibited at the N terminus (see analogues p498,8 h‐REKR,11 r ‐REKR,14) the efficiency of cleavage seems to be driven by degree of order of the N terminus. In the case of mutations (analogues 2 – 4 ), proline mutation at the P3 position seems to enhance the cleavage efficiency, while proline mutation at P2′ seems not to affect it (or to reduce it if a proline is already present at P3). …”

“…When no mutation occurs in or close to the cleavage site, but different sequences are exhibited at the N terminus (see analogues p498,8 h‐REKR,11 r ‐REKR,14) the efficiency of cleavage seems to be driven by degree of order of the N terminus.…”

“…More recently we reported on the structure and activity of the 23‐residue peptide h‐REKR, the key features of which were the introduction of a large helical sequence replacing that of site 2 and located at the N terminus 11. We showed that the propensity of the peptide to be processed by furin is high and not significantly affected by such modification, in relation to the full‐native analogue p498.…”

C‐terminal tails mimicking bioactive intermediates cause different plasma degradation patterns and kinetics in neuropeptides γ‐MSH, α‐MSH, and neurotensin

“…In It is commonly accepted that the peptide structure around the cleavage site can alter the kinetics of proteolysis. 37,38 We next investigated whether the C-terminal tail was determinant for inducing or disrupting specific conformations, for example, β-turns, important for peptide processing. 38,39 To this aim, γ2-MSH* and γ3-MSH were analyzed by far UV-CD spectroscopy in several means (pH 7.5, pH 2.5, 0.2% SDS).…”