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

Abstract

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...

A role for the Msx-1 homeodomain in transcriptional regulation: residues in the N-terminal arm mediate TATA binding protein interaction and transcriptional repression.

“…4D). This finding was not surprising since many of the alanine-substituted residues mediate DNA-protein contact (e.g., Msx-A and Msx-E) or stabilize the DNA-protein interaction (e.g., Msx-B) (23). However, it is noteworthy that some of the mutants that abolished DNA binding activity did not abolish repression or TBP interaction (Fig.…”

Transcriptional Repression by Msx-1 Does Not Require Homeodomain DNA-Binding Sites

“…There has been ample speculation that the homeodomain also mediates protein-protein interactions, and several functional associations have been described (66,88,90). The contribution of the homeodomain as a structural motif has been less well explored, although we have observed that mutations within the homeodomain of Msx-1 affect both structure and stability (14,78).…”

“…Since our data show that homeodomain DNA-binding sites are not required for repression, it is likely that the Msx-1 homeodomain serves an alternative function. For instance, it may mediate interactions with other protein factors (22,66,88,90), or it may provide a scaffold which maintains appropriate tertiary structure of Msx-1 (78). DNAbinding domains have been shown to serve various functions in transcriptional regulation, particularly as surfaces for proteinprotein interactions.…”

A novel missense mutation in MSX1 underlies autosomal recessive oligodontia with associated dental anomalies in Pakistani families

“…The conserved regions among the MSX proteins are the MSX homology regions I, II and III (MHR1 to -III), the extended homeodomain (EHD), and the homeodomain (Davidson 1995). The homeodomain consisting of 60 amino acids numbered 166-225 is subdivided into the N-terminal arm (NT arm) and helices I, II and III, which contribute to protein stability, DNA binding specificity, transcriptional repression and protein interactions (Shang et al 1994;Isaac et al 1995;Zhang et al 1996Zhang et al , 1997. In the MSX1 homeodomain, there are several conserved residues that constitute a consensus sequence, which promotes tertiary structure and mediates DNA binding activity (Scott et al 1989;Laughon 1991;Billeter 1993).…”

“…In the MSX1 homeodomain, there are several conserved residues that constitute a consensus sequence, which promotes tertiary structure and mediates DNA binding activity (Scott et al 1989;Laughon 1991;Billeter 1993). Consensus residues in helices I-III furnish the hydrophobic core and preserve the amphipathic nature of the alpha helices, whereas other consensus residues in helix III and in the N-terminal arm contact DNA (Shang et al 1994).…”