Structural studies of the engrailed homeodomain

Abstract

The structure of the Drosophila engrailed homeodomain has been solved by molecular replacement and refined to an R-factor of 19.7% at a resolution of 2.1 A. This structure offers a high-resolution view of an important family of DNA-binding proteins and allows comparison to the structure of the same protein bound to DNA. The most significant difference between the current structure and that of the 2.8-A engrailed-DNA complex is the close packing of an extended strand against the rest of the protein in the unbound protein. Structural features of the protein not previously noted include a "herringbone" packing of 4 aromatic residues in the core of the protein and an extensive network of salt bridges that covers much of the helix 1-helix 2 surface. Other features that may play a role in stabilizing the native state include the interaction of buried carbonyl oxygen atoms with the edge of Phe 49 and a bias toward statistically preferred side-chain dihedral angles. There is substantial disorder at both ends of the 61 amino acid protein. A 51-amino acid variant of engrailed (residues 6-56) was synthesized and shown by CD and thermal denaturation studies to be structurally and thermodynamically similar to the full-length domain.Keywords: crystallography; homeodomain; protein stability; protein structure Homeodomains are common eukaryotic DNA-binding domains that consist of a short extended strand followed by 3 helices (Qian et al., 1989;Kissinger et al., 1990;Laughon 1991;Wolberger et al., 1991). The homeodomain itself (conventionally defined as being 60 amino acids in length on the basis of homology) contains much of the DNA-binding specificity and affinity of the much larger proteins in which it is found (Kornberg, 1993). Moderate-resolution crystal structures of 3 protein:DNA complexes that include homeodomains (engrailed, MATa2, and Oct-1) have been determined, as have the NMR structures of Antennapedia (Antp), an Antp:DNA complex, and fushi tarazu (ftz) (Qian et al., 1989(Qian et al., , 1994Kissinger et al., 1990;Wolberger et al., 1991;Billeter et al., 1993;Klemm et al., 1994). There is considerable interest in understanding how these domains recognize DNA.Homeodomains are also of interest because they present an extremely simple model system for studies related to protein folding. They are small and monomeric and do not require disulfide bonds or ligands in order to fold stably. There is considerable sequence diversity among the several hundred homeodomain sequences known, but a conserved pattern of hydrophobic residues and the similarity of the known structures suggests that each of the hundreds of sequences adopts the same overall structure (Scott et al., 1989;Laughon, 1991).We have obtained a high-resolution structure of the engrailed homeodomain in the absence of DNA (Kinemage 1). This structure is useful because (1) it allows direct comparison to the crystallographic structure of a homedomain bound to DNA and (2) it provides a more accurate coordinate set for studies of the sequence-structure relations...

Cross-talk between the Paired Domain and the Homeodomain of Pax3

“…Likewise, DNA binding by the HD also resulted in decreased accessibility of the HD N-terminal linker (Xa216), reflecting a more compact structure of the DNA-bound state of this domain. These results are in agreement with x-ray crystallography and NMR studies of HD proteins that reveal an increased order of the HTH motif, including the N-terminal arm and the recognition helix, of the DNA-bound form of Pbx (61)(62)(63)(64), Antennapedia (65), engrailed (66), and Oct-1 (67) proteins. Increased resistance to protease cleavage of the Xa216 site was more pronounced upon incubation with P2 than with the half site P1/2.…”

Covariation of residues in the homeodomain sequence family

“…Table 2 shows that most of the amino acid pairs found at 19-30 have oppositely charged side chains, and Figure 5 shows that, in the engrailed homeodomain, E19 and R30 form a salt-bridge (Clarke et al, 1994). One other pair in the list of 16 highly correlated residue pairs was also found to be dominated by an amino acid pair of opposite charge, and Figure 6 shows that amino acids Fig.…”

“…5. Stereo drawing ofthe engrailed structure with residues E19 (light) and R30 (dark) indicated (Clarke et al, 1994). This and other structures in this paper were drawn using MOLSCRIPT (Kraulis 1991 ..…”

“…Residue 48 was excluded from the analysis because it is invariably tryptophan. The residues included in this analysis were identified on the basis of solvent accessibility and are residues 8, 16, 20, 26, 34, 38, 40, 45, and 49 (Clarke et al, 1994). The correlation coefficient is 0.43 for an inverse relationship between distance and covariation.…”

“…Homedomains are an interesting example of a large and diverse sequence family that can provide a useful test system for studies of this type. There are now several hundred sequences of this very small and simple motif, and the structures of five homeodomains (Antp, en, ftz, MATa2, and Octl) have been determined (Qian et al, 1989;Kissinger et al, 1990;Wolberger et al, 1991;Billeter et al, 1993;Kappen et al, 1993;Clarke et al, 1994;Klemm et al, 1994;Qian et al, 1994). The structures of the homeodomains are closely related.…”