Structurally altered peptides reveal an important role for N-terminal heptad repeat binding and stability in the inhibitory action of HIV-1 peptide DP178.
Human immunodeficiency virus 1 gp41 folds into a six-helix bundle whereby three C-terminal heptad repeat regions pack in an anti-parallel manner against the coiled-coil formed by three N-terminal heptad repeats (NHR). Peptides that inhibit bundle formation contributed significantly to the understanding of the entry mechanism of the virus. DP178, which partially overlaps C-terminal heptad repeats, prevents bundle formation through an undefined mechanism; additionally it has been suggested to bind other ENV regions and arrest fusion in an unknown manner. We used two structurally altered DP178 peptides; in each, two sequential amino acids were substituted into their d configuration, d-SQ in the hydrophilic N-terminal region and d-LW in the hydrophobic C-terminal. Importantly, we generated an elongated NHR peptide, N54, obtaining the full N-helix docking site for DP178. Interestingly, d-LW retained wild type fusion inhibitory activity, whereas d-SQ exhibited significantly reduced activity. In correlation with the inhibitory data, CD spectroscopy and fluorescence studies revealed that all the DP178 peptides interact with N54, albeit with different stabilities of the bundles. We conclude that strong binding of DP178 N-terminal region to the endogenous NHR, without significant contribution of the C-terminal sequence of DP178 to core formation, is vital for DP178 inhibition. The finding that d-amino acid incorporation in the C terminus did not affect activity or membrane binding as revealed by surface plasmon resonance correlates with an additional membrane binding site, or membrane anchoring role, for the C terminus, which works synergistically with the N terminus to inhibit fusion.
Wexler-Cohen
Yael
Y
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.
Johnson
Benitra T
BT
Puri
Anu
A
Blumenthal
Robert
R
Shai
Yechiel
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002459:Cell Fusion; D000077560:Enfuvirtide; D015700:HIV Envelope Protein gp41 / Q000737:chemistry* / Q000502:physiology*; D015497:HIV-1 / Q000472:pathogenicity*; D006367:HeLa Cells; D006801:Humans; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000502:physiology*; D011485:Protein Binding; D017510:Protein Folding; D013329:Structure-Activity Relationship; D014760:Viral Fusion Proteins; D014764:Viral Proteins / Q000737:chemistry* / Q000502:physiology*
D015700:HIV Envelope Protein gp41; D010446:Peptide Fragments; D014760:Viral Fusion Proteins; D014764:Viral Proteins; D000077560:Enfuvirtide