Novel structural features in two ZHX homeodomains derived from a systematic study of single and multiple domains

Abstract

Background: Zhx1 to 3 (zinc-fingers and homeoboxes) form a set of paralogous genes encoding multi-domain proteins. ZHX proteins consist of two zinc fingers followed by five homeodomains. ZHXs have biological roles in cell cycle control by acting as co-repressors of the transcriptional regulator Nuclear Factor Y. As part of a structural genomics project we have expressed single and multi-domain fragments of the different human ZHX genes for use in structure determination.

Results: A total of 30 single and multiple domain ZHX1-3 constructs selected from bioinformatics protocols were screened for soluble expression in E. coli using high throughput methodologies. Two homeodomains were crystallized leading to structures for ZHX1 HD4 and ZHX2 HD2. ZHX1 HD4, although closest matched to homeodomains from 'homez' and 'engrailed', showed structural differences, notably an additional C-terminal helix (helix V) which wrapped over helix I thereby making extensive contacts. Although ZHX2 HD2-3 was successfully expressed and purified, proteolysis occurred during crystallization yielding crystals of just HD2. The structure of ZHX2 HD2 showed an unusual open conformation with helix I undergoing 'domain-swapping' to form a homodimer.

Conclusions: Although multiple-domain constructs of ZHX1 selected by bioinformatics studies could be expressed solubly, only single homeodomains yielded crystals. The crystal structure of ZHX1 HD4 showed additional hydrophobic interactions relative to many known homeodomains via extensive contacts formed by the novel C-terminal helix V with, in particular, helix I. Additionally, the replacement of some charged covariant residues (which are commonly observed to form salt bridges in non-homeotherms such as the Drosophila 'engrailed' homeodomain), by apolar residues further increases hydrophobic contacts within ZHX1 HD4, and potentially stability, relative to engrailed homeodomain. ZHX1 HD4 helix V points away from the normally observed DNA major groove binding site on homeodomains and thus would not obstruct the putative binding of nucleic acid. In contrast, for ZHX2 HD2 the observed altered conformation involving rearrangement of helix I, relative to the canonical homeodomain fold, disrupts the normal DNA binding site, although protein-protein binding is possible as observed in homodimer formation.

Figure 1

Alignment of amino acid sequences for human ZHX paralogs. Positions of zinc finger and homeodomains derived from the alignment are indicated.

Figure 2

Structures of ZHX HD domains. (A) ZHX1 HD4 showing the two molecules in the crystal asymmetric unit (B) ZHX2 HD2 showing the two molecules in the crystal asymmetric unit. The disulfide bridge linking the subunits is shown in light blue and yellow (C) Part of ZHX1 HD4 showing omit electron density for residues 458-462, a region where the switch in helix I conformation occurs in ZHX2 HD2 compared to ZHX1 HD4. The monomer A is coloured in orange and monomer B in cyan. The dotted lines represent hydrogen bonds. The CA trace of a standard HD conformation is shown as thin black sticks.

Figure 3

Comparison of homeodomain structures. (A) ZHX1 HD4 (green) with homez HD (2ECC, red); (B). ZHX1 HD4(green) and ZHX2 HD2 (red).

Figure 4

Interactions formed by helix V in ZHX1 HD4. Packing of ZHX1 HD4 helix V with helices I & III/IV showing extensive hydrophobic contacts involving mainly aromatic residues.

Figure 5

Comparison of a region of engrailed HD and ZHX1 HD4 containing certain co-variant residues. Engrailed HD (blue backbone, orange side-chains) showing the extensive ionic and hydrogen-bonding interactions involving commonly observed co-variant residues. These interactions are absent in ZHX1 HD4 (red backbone, gray side-chains).