ENGRAILED (GLN50-]LYS) HOMEODOMAIN-DNA COMPLEX AT 1.9 ANGSTROM RESOLUTION - STRUCTURAL BASIS FOR ENHANCED AFFINITY AND ALTERED SPECIFICITY

Background: The homeodomain is one of the key DNA-binding motifs used in eukaryotic gene regulation, and homeodomain proteins play critical roles in development. The residue at position 50 of many homeodomains appears to determine the differential DNA-binding specificity, helping to distinguish among binding sites of the form TAATNN. However, the p recise role(s) of residue 50 in the differential recognition of altern ative sites has not been clear. None of the previously determined stru ctures of homeodomain-DNA complexes has shown evidence for a stable hy drogen bond between residue 50 and a base, and there has been much dis cussion, based in part on NMR studies, about the potential importance of water-mediated contacts. This study was initiated to help clarify s ome of these issues. Results: The crystal structure of a complex conta ining the engrailed Gln50-->Lys variant (QK50) with its optimal bindin g site TAATCC (versus TAATTA for the wild-type protein) has been deter mined at 1.9 Angstrom resolution. The overall structure of the QK50 va riant is very similar to that of the wild-type complex, but the sidech ain of Lys50 projects directly into the major groove and makes several hydrogen bonds to the O6 and N7 atoms of the guanines at base pairs 5 and 6. Lys50 also makes an additional water-mediated contact with the guanine at base pair 5 and has an alternative conformation that allow s a hydrogen bond with the O4 of the thymine at base pair 4. Conclusio ns: The structural context provided by the folding and docking of the engrailed homeodomain allows Lys50 to make remarkably favorable contac ts with the guanines at base pairs 5 and 6 of the binding site. Althou gh many different residues occur at position 50 in different homeodoma ins, and although numerous position 50 Variants have been constructed, the most striking examples of altered specificity usually involve int roducing or removing a lysine sidechain from position 50. This high-re solution structure also confirms the critical role of Asn51 in homeodo main-DNA recognition and further clarifies the roles of water molecule s near residues 50 and 51.