Integration of the human immunodeficiency virus (HIV‐1) DNA into the host genome is catalysed by a virus‐encoded protein integrase. Here, we report some of the structural and functional properties of two synthetic peptides: integrase‐(147–175)‐peptide reproducing the residues 147–175 (SQGVVESMNKELK159KIIGQVRDQAEHLKTAY) of the HIV‐1 integrase, and [Pro159] integrase‐(147–175)‐peptide where the lysine 159 is substituted for a proline. Circular dichroism revealed that both peptides are mostly under unordered conformation in aqueous solution, contrasting with the α‐helix exhibited by residues 147–175 in the protein crystal structure. In a weak α‐helix‐promoting environment, integrase‐(147–175)‐peptide self‐associated into stable coiled‐coil oligomers, while [Pro159] integrase‐(147–175)‐peptide did not. This property was further confirmed by cross‐linking experiments. In our in vitro experiments, only integrase‐(147–175)‐peptide was able to reduce the integration activity of the enzyme. We propose that the inhibitory activity shown by integrase‐(147–175)‐peptide is dependent on its ability to bind to its counterpart in integrase through a peptide‐protein coiled‐coil structure disturbing the catalytic properties of the enzyme.

Results are presented on a peptide fragment (1013-1056) from human DNA topoisomerase II alpha. This was selected using the procedure of Lupas et al. (Lupas, A., Van Dyke, M., and Stock, J. (1991) Science 252, 1162-1164) for its potential to adopt a stable coiled-coil structure. The same theoretical treatment rejected the segment 994-1021 proposed by Zwelling and Perry (Zwelling, L. A., and Perry, W. M. (1989) Mol. Endocrinol. 3, 603-604) as a possible core for leucine-zipper formation. Our experimental studies combine cross-linking and CD analysis. Cross-linking establishes that the 1013-1056 fragment forms a stable homodimer in solution. Effects of increasing peptide concentration on CD spectra confirm that only the 1013-1056 fragment can undergo a coiled-coil stabilization from an isolated alpha-helix. Unfolding experiments further show that the coiled-coil is more stable in guanidium chloride than in urea. Values of -6.8 and -7.4 kcal/mol for the dimerization free energy are determined by thermal and urea unfolding, respectively. These are strikingly similar to the value recently found for the dissociation/reassociation of the entire yeast topoisomerase II from sedimentation equilibrium experiments (Lamhasni, S., Larsen, A. K., Barray, M., Monnot, M., Delain, E., and Fermandjian, S. (1995) Biochemistry 34, 3632-3639), although their significance relatively to topoisomerase II undoubtedly requires further analysis.

Human immunodeficiency virus type 1 integrase (HIV‐1 IN) which catalyzes viral DNA integration into the host genome of infected cells represents an attractive target for AIDS therapy. We have previously demonstrated the ability of the IN‐(147−175)‐peptide derived from the catalytic core domain of HIV‐1 IN to inhibit the enzyme activity in vitro. IN‐(147−175)‐peptide contains four heptad repeats and displays a high propensity for coiled‐coil formation while its [P159]IN‐(147−175)‐peptide analog (Lys159←Pro in the protein, Lys13←Pro in the peptide) is unable to form a stable coiled‐coil and is devoid of inhibitory activity [Sourgen, F., Maroun, R. G., Frère, V., Bouziane, M., Auclair, C., Troalen, F. & Fermandjian, S. (1996) Eur. J. Biochem. 240, 765−773]. Now, we report results from an NMR study on IN‐(147−175)‐peptide and [P159]IN‐(147−175)‐peptide as well as on an optimized [E156, A163, A167]IN‐(147−175)‐peptide that is a better inhibitor of IN than IN‐(147−175)‐peptide. While in aqueous solution, IN‐(147−175)‐peptide and [P159]IN‐(147−175)‐peptide display only nascent helical features, [E156, A163, A167]IN‐(147−175)‐peptide exhibits 20 % of helical content. In 20 % trifluoroethanol/80 % H2O, the helix content is the highest for [E156, A163, A167]IN‐(147−175)‐peptide (≈70 %) and the lowest for [P159]IN‐(147−175)‐peptide (≈40 %), due to a local helix break caused by the Pro residue. The NHs of residues in the two central helical heptads (a−g) of IN‐(147−175)‐peptide and [E156, A163, A167]IN‐(147−175)‐peptide display a regular periodic variation of their temperature coefficients in 20 % trifluoroethanol. The b, c and f residues on the hydrophilic face of the amphipathic helix show high coefficients reflecting hydrogen bonded NHs, while the a and d residues on the hydrophobic face exhibit low coefficients, near random‐coil values. The particular arrangement of the hydrophobic side‐chains of a and d residues at the coiled‐coil interface reduces the access of trifluoroethanol molecules to their amide groups. The inability of trifluoroethanol molecules to create interactions with the amide C=O groups, these being required to strengthen the intrahelical C=O    H‐N hydrogen bonds, is the main cause for observation of heptadic a and d residues with low NH temperature coefficients. Such effects concern mostly the two central helical heptads of IN‐(147−175)‐peptide and [E156, A163, A167]IN‐(147−175)‐peptide implying that these ones are engaged in stable parallel coiled coils. Our results provide a link between the propensity of peptides for helix formation, their coiled‐coil properties and their efficiency to inhibit IN.

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