HELIX-LOOP-HELIX MOTIF IN HIV-1 REV

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 Del ta 14 (lacking aa 92-112), indicated that Rev contains 46-49 residues in alpha-helical conformation within the N-terminal 71 or 95 amino aci ds of the 116 residue protein. Complexation with a 40-nucleotide fragm ent of the Rev responsive element, RRE, (G(39) to C-78), containing th e minimal element for Rev binding, induced an A to B form structural t ransition 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 change s upon raising the temperature to 40 degrees C, as determined by the l ack of decrease of the signal intensity at 222 nm, indicative for alph a-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. Th e 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, w hereas no alpha-helix could be detected in a peptide solution of 0.2 m M. 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 pri mary sequence based on general structural arguments and secondary stru ctural calculations led to the identification of a potential helix(1)- loop-helix(2) motif within the Rev N-terminus. This model proposes tha t between 70% and 100% of all residues in the RNA-binding and nucleola r localization domain of Rev are included in helical conformation. Thr ee distinct hydrophobic motifs within the two helices suggest the form ation of a hydrophobic helix(1)-helix(2) contact between an ''isoleuci ne motif'' of helix(2) (I 52, I 55, and I 59) and one of the two hydro phobic motifs between residues 12 and 22 in helix(1).