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Is EIAV Tat Protein a Homeodomain?

Recently two publications (1) reported RNA-binding capacity of the Drosophila homeotic bicoid protein bcd (2). We would like to point out that the reverse discovery, namely that a protein turned out to be a homeodomain-like structure after it was discovered as an RNA binding factor, happened in our laboratory. The HIV-1 transactivator protein (hltat) is considered a tat response region (TAR) RNA binding protein, and the equine infectious anemia virus (EIAV) tat protein (e-tat) was inferred to play a role similar to the hl-tat protein (3). We recently determined the three-dimensional features of both these proteins by nuclear magnetic resonance (NMR) spectroscopy (4) and found that e-tat contains helical secondary structure as a limit structure. This limit structure was found to be dramatically stabilized by the addition of trifluoroethanol (tfe) (5). The secondary structure of the e-tat protein in tfe showed clearly a helix-loophelix-turn-helix binding motif similar to the homeodomain protein motif (6, 7). Comparison between helix motif proteins sequences, including bcd, and e-tat showed some resemblance, but no striking homologies, as shown by the alignment of the bcd and e-tat sequences as determined with the ClustalV program (8) and corrected for a continuous helix from Ala 28 to Lys 37 in bcd (7) (Fig. 1). Thus, e-tat and bcd are not closely related on the sequence level (12% identity and 44% similarity). On the level of secondary structure, however, the aligned sequences of bcd and e-tat showed unexpected homology (Fig. 1). The recognition helix of the homeodomain proteins, helix III from the NH2-terminus, corresponds to the basic sequence region of e-tat considered to be responsible for TAR recognition. In addition to the similarity of the helix pattems, both proteins show a strictly conserved turn between helices II and III. Unexpectedly, four out of six amino acid residues responsible for contact to DNA in bcd are identical in e-tat (Arg 5-Arg 5, Lys 50-Lys 57, Asn 51-Asn 58, Arg 54-Arg 61), one is nearly identical [Ile 47-Leu 54; the same substitution was observed for the fungal b2 homeotic protein (7)], and only Arg 3 of bcd does not have a direct counterpart in e-tat, although it may well be that Arg 4 of e-tat plays a role similar to Arg 3 of the homeotic proteins. Frequently, in homeodomains, residue 4 is basic, making the homology to e-tat even more striking. In addition to these conserved amino acids, the highly conserved pattern of hydrophobic amino acids in homeotic proteins, 3 1-xxxHHxxHxxxxxxHxxHxx-50, where H denotes a hydrophobic residue and x is either Gly, Cys, or Ala, is also conserved in e-tat (boxed residues in the sequence alignment). This pattem is clearly not matched in hi -tat. Formation of a homeodomain-like rec-