Role of Conserved Salt Bridges in Homeodomain Stability and DNA Binding

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.Homeodomain proteins are transcription factors present in all eukaryotes and play key roles in cellular differentiation during development (1, 2). The homeodomain encodes a 60-residue DNA binding domain composed of a disordered N-terminal arm, three helical segments (herein referred as I, II, and III) connected by short loops, and a disordered C-terminal region. The secondary and tertiary structures are stabilized by the formation of a well defined hydrophobic core, which results from the packing of the three helices (3, 4).Presently, the largest searchable collection of information for the homeodomain protein family is the Homeodomain Resource Data Base (5-7). It contains around 1056 full-length protein sequences isolated from 112 different species. Inspection of these data shows that helices I/II, I/III, and II/III can be connected by three salt bridges involving pairs 19/30, 31/42, and 17/52, respectively, which in addition can exhibit different polarities. In contrast, hydrophobic bridges at these sites are present only in a minor fraction of sequences (and mainly restricted to homeodomains from warm-blooded animals). This predominance of salt versus hydrophobic bridges suggests a role for the former in homeodomain function and/or stability.Here 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. Our approach involves the extensive use of sitedirected mutagenesis experiments together with CD, NMR, and isothermal titration microcalorimetry (ITC) 4 measurements. 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 sta...

Dlx5 Homeodomain:DNA Complex: Structure, Binding and Effect of Mutations Related to Split Hand and Foot Malformation Syndrome

“…Q186 is located in the middle of helix α3 of the homeodomain structure within the protein-DNA binding interface (Figure 3C). Q186, which corresponds to Q50 in standard homeodomain nomenclature [35], is preserved in all six Dlx proteins and highly conserved in other homeodomains. Only a few residues types can be tolerated in position 50 and, while glutamine is most common, some homeodomain structures contain lysine, serine or cysteine instead [36].…”

“…The second mutation linked to SHFM-1 involves residue Q178, which is preserved across all six Dlx homeodomains and is highly conserved within DLX5 genes from different species [23]. Q178 corresponds to Q42 in the standard homeodomain nomenclature, and is positioned too far from the DNA and is typically occupied by a glutamic acid involved in a salt bridge with R167 (R31), which is one of three highly conserved salt bridges that are believed to play a role in stabilizing the homeodomain structure [35]. Mutations of residues in positions 31/42 induce stabilizing or destabilizing effects on the protein.…”

Dynamic Local Polymorphisms in the Gbx1 Homeodomain Induced by DNA Binding

“…Others have shown for nine different homeodomains that replacement of either Glu17 or Arg52 (residue numbering according to Torrado et al, 2009; in the Gbx1 constructs used in our work, these positions are shifted to Glu23 and Arg58, but to facilitate correlations with other homeodomains we also use the numbering in Torrado et al, 2009; see Fig. 1a) by a neutral amino acid caused decreased protein stability, resulting in protein aggregation during purification (Chi et al, 2005).…”

“…The residues Glu17 and Arg52 (residue numbering of Torrado et al, 2009) are highly conserved among homeodomains, and crystal structures (Grant et al, 2000; Jauch et al, 2008; Hovde et al, 2001) and molecular dynamics studies (Flader et al, 2003) of homeodomain–DNA complexes suggested that these two residues form a salt bridge in the free homeodomains which is preserved in their DNA complexes. In apparent contrast, interactions between Arg52 and the DNA backbone had either been directly observed in or inferred by NMR structure determinations of different homeodomain–DNA complexes (Baird-Titus et al, 2006; Billeter et al, 1993; Chaney et al, 2005).…”

“…Furthermore, several amino acid pairs have been identified that contribute to protein stability through the formation of salt bridges between the different α-helices. The most highly conserved salt bridge in the homeodomain family links Glu17 and Arg52 in helices 1 and 3, and salt bridges between residues Glu19 and Arg30, and between Arg31 and Glu42 are also quite common (Torrado et al, 2009). Comparison of the different individual molecules in the crystallographic asymmetric unit (Longo et al, 2007) and molecular dynamics studies (Babin et al, 2013; Flader et al, 2003) identified small rearrangements of the Glu17 and Arg52 salt bridge and the recognition helix upon binding to DNA, whereas NMR solution structures showed larger conformational changes, which may include disruption of the Glu17 interaction with Arg52 (Billeter et al, 1993; Torrado et al, 2009; Baird-Titus et al, 2006; Chaney et al, 2005).…”