Helix-Loop-Helix Motif in HIV-1 Rev

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Abstract
Circular dichroism (CD) spectra of C-terminal deletion mutants of the HIV-1 Rev protein, Rev M9 Delta 14 (missing aa 68-112) and Rev M11 Delta 14 (lacking aa 92-112), indicated that Rev contains 46-49 residues in alpha-helical conformation within the N-terminal 71 or 95 amino acids of the 116 residue protein. Complexation with a 40-nucleotide fragment of the Rev responsive element, RRE, (G(39) to C-78), containing the minimal element for Rev binding, induced an A to B form structural transition in the RRE fragment, whereas the percentage of alpha-helical conformation in the protein stays constant on substrate binding. When complexed to the RNA, neither mutant protein showed structural changes upon raising the temperature to 40 degrees C, as determined by the lack of decrease of the signal intensity at 222 nm, indicative for alpha-helical conformation. In contrast, Rev M9 Delta 14, which is shorter than Rev M11 Delta 14 by 24 amino acids, in the absence of RNA, lost about 60% of the spectral minima at 222 nm at the same temperature. The Rev M11 Delta 14 mutant, in the absence of RNA, showed a decrease of 20% in spectral intensity upon heating to 40 degrees C. Free and RNA-bound mutant proteins showed reversible transitions upon heating to 80 degrees C and subsequent cooling down to 10 degrees C overnight. The Rev peptide Cys 75-93, spanning the Rev transactivation domain, showed secondary structure in 40% and 60% hexafluoropropanol (HFP) solutions. The CD spectrum of the peptide in the presence of HFP showed little change upon heating to 60 degrees C. The CD spectrum of Rev 8-26 at a concentration of 1 mM in aqueous solution contained 28% alpha-helix, whereas no alpha-helix could be detected in a peptide solution of 0.2 mM. However, at this concentration helical conformation was induced as a function of HFP concentration with a maximum negative band intensity at 222 nm at 60% HFP. These data together with an analysis of Rev primary sequence based on general structural arguments and secondary structural calculations led to the identification of a potential helix(1)-loop-helix(2) motif within the Rev N-terminus. This model proposes that between 70% and 100% of all residues in the RNA-binding and nucleolar localization domain of Rev are included in helical conformation. Three distinct hydrophobic motifs within the two helices suggest the formation of a hydrophobic helix(1)-helix(2) contact between an ''isoleucine motif'' of helix(2) (I 52, I 55, and I 59) and one of the two hydrophobic motifs between residues 12 and 22 in helix(1).