This report investigates the sequence specificity requirements for homeodomain structure and DNA binding activity by the design and synthesis of a "minimAl" homeodomain (for mimalist design and alanine sning mutagenesis) which contain the consensus residues and in which all nonconsensus residues have been replaced with alanin. 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 Msn. A derivative of the minimAl homeodomain, AlaMsx(NT), which contain a native rather than an alaninesubstituted N-terminal arm, has similar DNA binding affinity as Mnx. We show that the native N-terminal arm sbiizes the tertiary structure of the minimAl homeodomain. Although Ala-Mn resembles a molten-globule protein, the structure of Ala-Mnx(NT) is similar to Mn. The requirement for an intact N-terminal arm is not unique to the minimA homeodomain, since the N-ternl arm also promotes hih-affinity binding activit and appropriate tertiary structure of Mn. Therefore, the homeodomain "scaffold" consists of consensus residues, which are s t for DNA recognition, and nonconsensus residues in the N-terminal arm, which are required for optimal DNA binding affinity and appropriate tertiary structure. MinImAM design provides a powerful strategy to probe homeodomain structure and function. This approach should be of general utility to study the sequence specfcit requirements for structure and function of other DNA-bining domains.The homeodomain, a conserved DNA-binding domain, was originally described as a sequence motif shared among Drosophila homeotic proteins and was subsequently shown to be ubiquitous among developmental regulatory proteins from nematodes to humans (1-5). A high degree of conservation is evident in primary sequence (3, 5), tertiary structure (5-7), and mode of interaction with DNA (5-7). The sequence corresponds to 60 amino acid residues that assemble into three a-helices and an extended N-terminal arm (Fig. 1). Several residues are invariant or highly conserved among a majority of homeodomain proteins (Fig. 2). These constitute a consensus sequence that promotes tertiary structure and mediates DNA binding activity (3,5,7). Consensus residues in helices I-HI furnish the hydrophobic core and preserve the amphipathic nature of the a-helices, whereas other consensus residues in helix III and in the N-terminal arm contact DNA. Despite extensive conservation, the homeodomain also promotes the selective functions that are characteristic of homeodomain-containing proteins (e.g., refs. 10 and 11).One explanation for this paradox is that the consensus residues form a "scaffold" that provides DNA site recognition, whereas the nonconsensus residues allow functional diversity by promoting selective protein-protein and protein-DNA interactions.To characterize the contribution of consensus and nonconsensus residues toward homeodomain structure and function, we have designed and synthesized a "minimAl" homeodomain which...
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.
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.
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