Abstract
EAA26 (VESMNEELKKIIAQVRAQAEHLKTAY) is a better inhibitor of human immunodeficiency virus, type 1, integrase than its parent Lys-159, reproducing the enzyme segment 147-175 with a nonpolar-polar/charged residue periodicity defined by four helical heptads (abcdefg) prone to collapse into a coiled-coil. Circular dichroism, nuclear magnetic resonance, sedimentation equilibrium, and chemical cross-linking were used to analyze EAA26 in various trifluoroethanol/H(2)O mixtures. In pure water the helix content is weak but increases regularly up to 50-60% trifluoroethanol. In contrast the multimerization follows a bell-shaped curve with monomers in pure water, tetramers at 10% trifluoroethanol, and dimers at 40% trifluoroethanol. All suggest that interhelical interactions between apolar side chains are required for the coiled-coil formation of EAA26 and subsist at medium trifluoroethanol concentration. The N(H) temperature coefficients measured by nuclear magnetic resonance show that at low trifluoroethanol concentration the amide groups buried in the hydrophobic interior of four alpha-helix bundles are weakly accessible to trifluoroethanol and are only weakly subject to its hydrogen bond strengthening effect. The increased accessibility of trifluoroethanol to buried amide groups at higher trifluoroethanol concentration entails the reduction of the hydrophobic interactions and the conversion of helix tetramers into helix dimers, the latter displaying a smaller hydrophobic interface. The better inhibitory activity of EAA26 compared with Lys-159 could arise from its better propensity to form a helix bundle structure with the biologically important helical part of the 147-175 segment in integrase.
Highlights
IntroductionWe have reported (13) that Lys-159, a peptide corresponding to the 147–175 segment of HIV-1 IN, inhibited the integration catalyzed by IN, most likely through specific binding to its counterpart in the protein
The well defined conformational properties exhibited by b Recipient of a grant from the Agence Nationale de Recherche sur le SIDA
Concluding Remarks—The inventory of secondary structure predictions, circular dichroism (CD) data, NOE interactions, and CSI data permits the structure of EAA26, which has a periodicity of polar/ charged-nonpolar residues matching the 3.6 repeat of ␣-helices, to be assigned with confidence as predominantly ␣-helical in TFE/H2O mixtures
Summary
We have reported (13) that Lys-159, a peptide corresponding to the 147–175 segment of HIV-1 IN, inhibited the integration catalyzed by IN, most likely through specific binding to its counterpart in the protein Such a specific binding was plausible since in the crystal structure of the catalytic core IN50–212, the major part of the target 147–175 segment (from residue 147 to 166) is stabilized under a characteristic amphipathic helix (helix ␣4) (Fig. 1) with most of its hydrophobic side chains strikingly oriented outward from the molecule (21). The antibodies bind to IN and its catalytic core and exhibit strong inhibitory activity against the enzyme, confirming both the surface exposure of the 147–175 segment and its biological importance This segment has been shown to contain several critical residues for both the binding of IN to the LTR (long terminal repeats) ends of the viral genomic DNA and the DNA end processing (22, 23). Equilibrium sedimentation can be safely used to establish the multimerization of biological molecules (Ref. 52 and references therein)
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