Role of Conserved Salt Bridges in Homeodomain Stability and DNA Binding

Mario Torrado,Julia Revuelta,Carlos Gonzalez,Francisco Corzana,Agatha Bastida,Juan Luis Asensio

doi:10.1074/jbc.m109.012054

Abstract

The sequence information available for homeodomains reveals that salt bridges connecting pairs 19/30, 31/42, and 17/52 are frequent, whereas aliphatic residues at these sites are rare and mainly restricted to proteins from homeotherms. We have analyzed the influence of salt and hydrophobic bridges at these sites on the stability and DNA binding properties of human Hesx-1 homeodomain. Regarding the protein stability, our analysis shows that hydrophobic side chains are clearly preferred at positions 19/30 and 31/42. This stabilizing influence results from the more favorable packing of the aliphatic side chains with the protein core, as illustrated by the three-dimensional solution structure of a thermostable variant, herein reported. In contrast only polar side chains seem to be tolerated at positions 17/52. Interestingly, despite the significant influence of pairs 19/30 and 31/42 on the stability of the homeodomain, their effect on DNA binding ranges from modest to negligible. The observed lack of correlation between binding strength and conformational stability in the analyzed variants suggests that salt/hydrophobic bridges at these specific positions might have been employed by evolution to independently modulate both properties.

Highlights

The largest searchable collection of information for the homeodomain protein family is the Homeodomain Resource Data Base (5–7)
We analyze the relative effect of salt and hydrophobic bridges on the homeodomain stability and DNA binding properties, employing the human Hesx-1 DNA binding domain as model system
Salt Versus Hydrophobic Bridges in Stability and DNA Binding ured by NMR spectroscopy to further test the role of conserved salt bridges on the homeodomain stability

Summary

EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis of Hesx-1—Mutations were incorporated into the wild-type Hesx-1 homeobox located on plasmid pT7-7 using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA) and the appropriate mutagenic primers. ⌬G values at a common temperature (55 or 77 °C depending on the particular set of protein variants, see Table 1) were derived from the thermal denaturation data In this procedure, a constant value of Ϫ0.7 kcal/(mol1⁄7K) was used for the change in heat capacity upon folding (⌬Cp). DNA Binding Experiments—Binding studies were performed at 15, 25, and 35 °C, in 150 mM NaCl, 20 mM phosphate, 5 mM MgCl2, pH 6.0, using a VP-ITC titration calorimeter (MicroCal, LLC) with a reaction cell volume of 1.467 ml Both the protein and duplex DNA (5Ј-GTCTAATTGACGCG-3Ј and its complementary 5Ј-CGCGTCAATTAGAC-3Ј) solutions were dialyzed against the same buffer prior to ITC experiments to ensure chemical equilibration. For every single protein variant, thermodynamic parameters were derived from two independent experiments and averaged

RESULTS

21 Wild-type 2 16 17 7 11 15

DISCUSSION