Multiple amino acids determine the DNA binding specificity of the Msx-1 homeodomain.

This study investigates the sequence features that contribute to the differential DNA binding properties of two divergent homeodomains, Msx-1 and HoxA3. We show that these homeodomains have overlapping, but nonidentical, DNA binding site preferences. We defined the amino acid residues that contribute to the observed differences in DNA binding specificity by producing a series of mutated polypeptides in which selected residues in Msx-1 were replaced with the corresponding ones in HoxA3. These analyses show that the DNA binding specificity of Msx-1 versus HoxA3 results from the cumulative action of multiple residues in all segments of the homeodomain (i.e., the N-terminal arm and helices I, II, and III). Therefore, substitutions of residues in both helix III and the N-terminal arm (but not in either segment alone) produced an Msx-1 polypeptide whose binding site preference was indistinguishable from that of HoxA3. Residues in helices I and II also influence DNA binding activity; these oppositely charged residues (e.g., lysine 19 and glutamate 30) may mediate ionic interactions between helices I and II which stabilize DNA binding by Msx-1. These findings demonstrate a critical interplay between residues in each homeodomain segment for appropriate conformation of the protein-DNA complex.

Use of fluorescence resonance energy transfer to estimate intramolecular distances in the Msx-1 homeodomain.

We have utilized fluorescence resonance energy transfer (FRET) to investigate the spatial proximities of segments in the Msx-1 homeodomain (Msx). This strategy makes use of a single, invariant tryptophan (Trp-48) in helix III as the donor for FRET. The acceptor molecule, 5-[[[(iodoacetyl)amino]-ethyl]amino]naphthalene-1-sulfonic acid (AEDANS), was incorporated into Msx at positions 6, 10, or 27 which are within the N-terminal arm, and helices I and II since these segments have been implicated in interactions with helix III. Specific incorporation of AEDANS was achieved by using a two-step strategy consisting of site-directed mutagenesis for introducing unique cysteine residues at the selected positions followed by covalent modification of these cysteine residues with AEDANS. Using this approach, we demonstrated energy transfer between Trp-48 and the AEDANS-labeled cysteines at positions 6, 10, and 27 and estimated the distances between the Trp-48 and AEDANS pairs to be 19, 23, and 16 A, respectively. We further demonstrated that FRET provides a strategy for detecting subtle alterations in protein conformation that result from replacement of specific residues in helix III and the N-terminal arm. Together, these findings show that FRET provides a useful approach for estimating intramolecular distances and for examining the conformation of Msx. Moreover, given the fact that Trp-48 is invariant among all homeodomain sequences, we propose that FRET will provide a general approach for facilitating comparative analyses of homeodomain conformations.

Design of a "minimAl" homeodomain: the N-terminal arm modulates DNA binding affinity and stabilizes homeodomain structure.

This report investigates the sequence specificity requirements for homeodomain structure and DNA binding activity by the design and synthesis of a "minimAl" homeodomain (for minimalist design and alanine scanning mutagenesis) which contains the consensus residues and in which all nonconsensus residues have been replaced with alanine. The murine homeodomain Msx served as the prototype for the minimAl homeodomain, Ala-Msx. We show that Ala-Msx binds to DNA specifically, albeit with lower affinity than Msx. A derivative of the minimAl homeodomain, Ala-Msx(NT), which contains a native rather than an alanine-substituted N-terminal arm, has similar DNA binding affinity as Msx. We show that the native N-terminal arm stabilizes the tertiary structure of the minimAl homeodomain. Although Ala-Msx resembles a molten-globule protein, the structure of Ala-Msx(NT) is similar to Msx. The requirement for an intact N-terminal arm is not unique to the minimAl homeodomain, since the N-terminal arm also promotes high-affinity binding activity and appropriate tertiary structure of Msx. Therefore, the homeodomain "scaffold" consists of consensus residues, which are sufficient for DNA recognition, and nonconsensus residues in the N-terminal arm, which are required for optimal DNA binding affinity and appropriate tertiary structure. MinimAl design provides a powerful strategy to probe homeodomain structure and function. This approach should be of general utility to study the sequence specificity requirements for structure and function of other DNA-binding domains.