DNA-binding specificity of the homeodomain-leucine zipper domain.
Homeodomain-leucine zipper (HD-Zip) proteins are putative transcription factors identified only in plants. The study of the DNA-binding properties of the ATHB-1 and -2 HD-Zip (HD-Zip-1 and -2) domains showed that they interact with DNA as homodimers and recognize two distinct 9 bp pseudopalindromic sequences, CAAT(A/T)ATTG (BS-1) and CAAT(G/C)ATTG (BS-2), respectively, as determined by selecting high-affinity binding sites from random-sequence DNA. Here, we report a mutational analysis of the HD-Zip-2 domain. We determined that conserved amino acid residues of helix 3, Val47 and Asn51, and Arg55 are essential for the DNA-binding activity of the HD-Zip-2 domain. We demonstrated that the preferential recognition of a G/C base-pair at the central position by the HD-Zip-2 domain is abolished either by the replacement of Arg55 with lysine or by the substitution of Glu46 and Thr56 with the corresponding residues of the HD-Zip-1 domain (alanine and tryptophan, respectively). In contrast, substitution of Arg55 with lysine in the HD-Zip-1 domain significantly reduced DNA-binding activity without changing the specificity of recognition. Finally, we determined that differences in residues outside helix 3 further contribute to the DNA-binding specificity of the HD-Zip domain. Taken together, the data strongly suggest that the preferential recognition of BS-2 and -1 by the HD-Zip-2 and -1 domains, respectively, may be attributable to a distinct orientation of the side-chain of Arg55 in these two domains.
Sessa
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G
Dipartimento di Genetica e Biologia Molecolare, Rome, Italy.
Morelli
G
G
Ruberti
I
I
D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D004586:Electrophoresis; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism
D029681:Arabidopsis Proteins; D004268:DNA-Binding Proteins; C069386:HB-2 protein, Arabidopsis; D018398:Homeodomain Proteins; D010940:Plant Proteins; D004247:DNA