From Proteopedia

Hox Proteins Specifically Recognize the Sequence-Dependent Shape of the Minor Groove

Biological Role of Hox Proteins

Figure 1: Crystal structure of Exd-Scr-DNA ternary complex; PDB ID# 2R5Z^[1]. The Hox protein Scr (yellow) and its cofactor Exd (blue) bind to its specific fkh20 site.

Figure 2: Hox proteins require a cofactor to achieve high binding specificity in order to execute their distinct functions in developing various parts of the fly embryo ^[2]. Elsevier/Cell Press has provided permission for usage of this figure.

Hox proteins are transcription factors that play a key role in the embryonic development across species by activating and repressing genes. Eight Drosophila Hox proteins are responsible for the development of different body segments of the fly, for example its antennae, wings, or legs. Hox proteins execute their distinct functions through binding to closely related but different in vivo binding sites. This page discusses molecular mechanisms through which Hox proteins recognize their DNA targets with very high binding specificity.

The crystal structure of a Hox-DNA complex (Figure 1) shows for the Hox protein Sex combs reduced (Scr) that it binds its specific in vivo site with the help of a cofactor, Extradenticle (Exd). Hox proteins bind DNA as monomers but their biniding specificity of enhanced when the co-factor is present, a principle that is called latent specificity (Figure 2). In Drosophila for instance, eight Hox proteins bind as heterodimer with their cofactor Exd to similar but distinct target sites.

Hox proteins are expressed along the anterior-posterior axis of an embryo, thus determining the localization for the development of different body segments (Figure 2). This spatial order of expression from anterior to posterior is congruent with the location of the respective Hox binding sites at the chromosome, a fact knowm as collinearity.

Hox mutants can lead to malformations, and studying the molecular basis of how Hox proteins execute distinct in vivo functions, therefore, remains an important field of biomedical research.

Structural Description of Hox-DNA Complex

spinqualitylabelsall models Figure 3: 3D-Representation of Exd-Scr-DNA ternary complex with Scr specific site; PDB ID# 2R5Z. Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Homeodomain Architecture

Hox proteins and their cofactors belong to the family of homeodomain proteins, which are encoded by homeoboxes. comprised of three alpha helices (Figure 3). Both Scr (yellow) and Exd (blue) are homeodomain proteins. The interface residues of both proteins are (dark purple vs. cyan).

Their third alpha helix of the Scr and Exd homeodomains, the so-called , inserts into the major groove where hydrogen bonds are formed between protein side chains and base pairs. An N-terminal tail forms contacts with the minor groove.

Hox Protein-Cofactor Interactions

Scr interacts with its cofactor Exd through hydrophobic interactions via a located at its N-terminal tail. This interaction spans Scr's flexible N-terminal linker across the minor groove of its binding site. In the absence of the YPWM motif, Scr and Exd would not form a heterodimer.

Major Groove Base Readout

Ho proteins achieve a large fraction of their binding specificity through . This form of protein-DNA recognition in the major groove is characterized as base readout since hydrogen bonds in the major groove can be used to distinguish between all four possible base pairs.

Minor Groove Shape Readout

This level of accuracy, however, cannot be achieved in the minor groove. Hydrogen bonds between protein side chains and functional groups of the bases are unable to distinguish A/T and T/A, or C/G and G/C base pairs due to the overlapping location of hydrogen bond donors and acceptors. On the other hand, it has been shown that minor groove contacts are essential for achieving specificity. Three side chains, of the Scr in vivo site fkh250.

The mechanism through which these three residues recognize the DNA minor groove is called shape readout as they do not form base-specific hydrogen bonds but rather recognize the sequence-specific narrowing of the minor groove. AT-rich regions can be characterized through an intrinsically narrow minor groove, leading to enhanced negative electrostatic potential, which in turn attracts basic side chains. This shape readout mechanisms was found to be broadly employed by for arginine residues ^[3].

Caption ^[1].

spinqualitylabelsall models Figure 3: 3D-Representation of Exd-Scr-DNA ternary complex with Hox consensus site; PDB ID# 2R5Y. Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image ' /> In vitro experiments have shown that His-12 and Arg3 mutations have a large effect when exposed to the Scr specific site whereas the effect is small when exposed to a Hox consensus site. Upon mutations to alanine and ectopic expression in a fly embryo, the biological importance of both side chains in vivo becomes apparent. Whereas there is only a residual expression detected in the thorax region of the double mutant when the Scr specific site is tested, there is no apparent effect in the presence of a Hox consensus site. This observation can be explained based on a second Scr-Exd-DNA ternary complex where the Hox-Exd hetrodimer is bound to a Hox consensus site, which is not specific to Scr. In this structure it is apparent that only is disordered (Figure 5). Figure 4: Comparison of intrinsic DNA shape of Scr specific in vivo site (left panel) vs. Hox consensus site (right panel)[1]. Elsevier/Cell Press has provided permission for usage of this figure. Based on the comparison of the two crystal structures of Scr-Exd-DNA ternary complexes (Figure 4), it was found that hree N-terminal residues contact the minor groove of the Scr specific site fkh250 (A) compared to only Arg5 binding the Hox consensus site fkh250con (B). Based on crystal structures of protein-DNA complexes, the shape of both sites is distinct (dark gray, concave; green, convex surfaces). Minor groove width (green plots) and electrostatic potential (pink plots) correlate and form two binding pockets in fkh250 (C) and only a binding site for Arg5 in fkh250con (D). The distinct shapes of the two DNA binding sites are already present when the protein is not bound to the DNA, with two minima in fkh250 (E) vs. one minimum in fkh250con (F), as inferred by Monte Carlo simulations (red plots) and high-throughput predictions (blue plots). Image:Slattery-etal-Figure6.jpg Caption [2]. References ↑ 1.0 1.1 1.2 Joshi R, Passner JM, Rohs R, Jain R, Sosinsky A, Crickmore MA, Jacob V, Aggarwal AK, Honig B, Mann RS. Functional specificity of a Hox protein mediated by the recognition of minor groove structure. Cell. 2007;131(3):530-43. ↑ 2.0 2.1 Slattery M, Riley T, Liu P, Abe N, Gomez-Alcala P, Dror I, Zhou T, Rohs R, Honig B, Bussemaker HJ, Mann RS. Cofactor binding evokes latent differences in DNA binding specificity between Hox proteins. Cell. 2011;147(6):1270-82. ↑ Rohs R, West SM, Sosinsky A, Liu P, Mann RS, Honig B. The role of DNA shape in protein-DNA recognition. Nature. 2009;461(7268):1248-53.