Crystal Structures of Engrailed Homeodomain Mutants

Abstract

We report the crystal structures and biophysical characterization of two stabilized mutants of the Drosophila Engrailed homeodomain that have been engineered to minimize electrostatic repulsion. Four independent copies of each mutant occupy the crystal lattice, and comparison of these structures illustrates variation that can be partly ascribed to networks of correlated conformational adjustments. Central to one network is leucine 26 (Leu 26 ), which occupies alternatively two side chain rotameric conformations (-gauche and trans) and different positions within the hydrophobic core. Similar sets of conformational substates are observed in other Engrailed structures and in another homeodomain. The pattern of structural adjustments can account for NMR relaxation data and sequence co-variation networks in the wider homeodomain family. It may also explain the dysfunction associated with a P26L mutation in the human ARX homeodomain protein. Finally, we observe a novel dipolar interaction between a conserved tryptophan and a water molecule positioned along the normal to the indole ring. This interaction may explain the distinctive fluorescent properties of the homeodomain family.The homeodomain is a simple fold common to many different DNA-binding proteins from diverse eukaryotes. The domain is found in transcription factors that regulate a variety of genes and so control key processes ranging from early developmental decision to homeostasis and general "housekeeping" (1-3).The homeodomain fold comprises three helices, connected by a flexible loop and a ␤-turn, and has a small hydrophobic core that is remarkably well conserved (4). The side chains in this conserved core appear to be well packed, which is a hallmark of stable folds. However, homeodomain folds generally have poorer stabilities in comparison with other proteins of similar size (5-8). The termini of homeodomains are disordered in solution (9 -11) and structures of free-and DNA-bound forms illustrate that DNA binding is accompanied by a structural condensation of the termini (12). Possibly, the homeodomain core also undergoes subtle structural changes upon DNA binding. Thus, the homeodomain would appear to mold to the surface of specific DNA by induced fit. Despite the wealth of current stereochemical and functional data for homeodomains, the basis for their comparatively low stability and its relationship, if any, to their induced fit remains to be established.Sequence co-variance analysis of homeodomains highlights several strongly correlated residue pairs that form conserved interactions and that may affect stability and DNA binding (4). One pair identified was the surface residues 17 and 52, which forms, in the majority of cases, a salt bridge. However, in the transcriptional repressor Engrailed from Drosophila, two lysines are instead found in these positions. We have substituted lysine 52 for glutamate, to mimic the conserved Glu 17 -Arg 52 salt bridge, and for alanine, to relieve the repulsion of the clustered positive charges. Standard equilibrium ...

Role of Conserved Salt Bridges in Homeodomain Stability and DNA Binding

“…However, it has been shown that the homeodomain core is dynamic leading to the hypothesis that this character is essential for the adaptability of the polypeptide. In addition, it has been suggested that salt bridge 19/30 is part of a co-varying network of interacting residues that plays a role in the induced fit of the protein on complex formation (9). The estimated changes in heat capacity that accompany the specific binding of the selected variants to DNA (⌬Cp, one of the thermodynamic signatures of the induced fit) do not provide support for this hypothesis.…”

“…In some cases, residues 19/30 can establish a cooperative network with positions 15/37, as that observed in the x-ray structure of the Engrailed homeodomain (9,20 From a statistical analysis of homeodomain sequences, Clarke (8) identified a dominating pattern of pairwise co-variation centered on residue 26. Using the co-varying network, homeodomains were divided into two classes.…”

“…One class has branched aliphatic residues at position 26, whereas the second contains proline. Previous analysis of a reduced data set of representative human homeodomain sequences (9) found that the branched aliphatic subgroup usually has a salt bridge connecting residues 19 and 30 (92% of cases). Strikingly, none of the Pro 26 subgroup, with more than 60 members, had this potential interaction.…”

“…Thus, as a first step, the hydrophobic pair Val 19 /Ile 30 , present in the wild-type polypeptide, was replaced by salt bridges of different polarities, either isolated or networked. A previous statistical analysis of the information stored in the homeodomain resource data base (8,9) revealed an intriguing correlation between the nature of pair 19/30 and the loop residue 26 (proline or a branched aliphatic amino acid in all cases). Considering this, both salt bridges and aliphatic residues connecting 19/30 were also considered in the context of either proline or leucine in 26.…”

Understanding ensemble protein folding at atomic detail

“…This study is based on an all-atom model that can accurately fold multiple protein domains using a single potential that includes no knowledge of the native structure beyond the primary amino acid sequence (14). Using this model, we map the complete folding mechanism of the engrailed homeodomain (ENH), which presents an excellent specimen for study due to the wealth of existing experimental data (17)(18)(19)(20)(21)(22)(23). We perform 4,000 independent Ϸ10-s simulations (40 ms of total simulated time), each of which is initiated from a different random coil conformation and is carried out at a fixed temperature (Ϸ25°C).…”