Hox protein: Difference between revisions

''This is a joint project of students at La Cañada High School, La Cañada Flintridge, California USA, and students at the University of Southern California, Los Angeles, California USA, mentored by [[User:Remo Rohs|Professor Remo Rohs]].''

[[Image:Cell.jpg|thumb|right|300px|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. Elsevier/Cell Press has provided permission for usage of this figure<ref name="slattery">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. [http://www.ncbi.nlm.nih.gov/pubmed/22153072 PMID:22153072]</ref>.]]

The crystal structure of a Hox-DNA complex (Figure 1) shows that the Hox protein ''Sex combs reduced'' (Scr) binds its specific ''in vivo'' site with the help of cofactors, ''Extradenticle (Exd)/Pbx proteins''. Hox proteins can bind DNA as monomers but their binding specificity is 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 heterodimers with their cofactor Exd to similar but distinct target sites.<br/>

==Structural Description of Hox-DNA Complex==

Both <span style="background-color: black; color: yellow;">'''&nbsp;Scr&nbsp;'''</span> and <font color="blue">'''Exd'''</font> belong to the family of homeodomain proteins, which are encoded by homeoboxes. Homeodomains are helix-turn-helix motifs (<scene name='Sandbox_Reserved_169/Complex/1'>restore initial scene</scene>) comprised of three alpha helices (Figure 3). The <font color="#e06800">'''DNA'''</font>-binding interface residues of both proteins are <scene name='Sandbox_Reserved_169/Complex/2'> evolutionarily most conserved</scene>. {{Template:ColorKey_ConSurf_NoYellow_NoGray}}

[[Image:Joshi-etal-Figure7.jpg‎ |thumb|left|300px|Figure 4: Expression patterns of Scr in presence of Scr specific site (left panel) vs. Hox consensus site (right panel). Elsevier/Cell Press has provided permission for usage of this figure<ref name="joshi"/>.]]

In vitro binding studies have shown that His-12 and Arg-3 mutations have a large effect when exposed to the Scr specific site, whereas the effect is small when exposed to a Hox consensus site. The biological importance of both side chains becomes apparent in ''in vivo'' experiments. Upon mutations of His-12 and Arg-3 to alanine, Scr expression in a fly embryo is dramatically affected (Figure 4). In comparison to  wild type Scr (A) and based on ectopic expression (B), there is only residual expression detected in  

the thorax region of the double mutant when the Scr specific site is tested (C), whereas there is no apparent effect on expression in the presence of a Hox consensus site (D-F).<br/>

==Recognition of Scr Specific vs. Hox Consensus Site==

This observation can be explained based on a second crystal structure of an <span style="background-color: black; color: yellow;">'''&nbsp;Scr&nbsp;'''</span>-<font color="blue">'''Exd'''</font>-<font color="#e06800">'''DNA'''</font> ternary complex where the Hox-Exd hetrodimer is bound to a Hox consensus site, which is not specific to Scr (<scene name='Sandbox_Reserved_169/Con/3'>restore initial scene</scene>). In this structure it is apparent that only <scene name='Sandbox_Reserved_169/Con/2'>Arg5 binds the minor groove</scene> and the remainder of the N-terminal linker is disordered (Figure 5).<br/>

[[Image:Cell2007-Fig4.jpg‎ |thumb|left|300px|Figure 6: Comparison of DNA shape of Scr specific in vivo site (left panel) vs. Hox consensus site (right panel). Elsevier/Cell Press has provided permission for usage of this figure<ref name="joshi"/>.]]

Based on the comparison of the two crystal structures of a Scr-Exd-DNA ternary complexes (Figure 6), it was found that three 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). In their protein-bound states, the shapes of both sites are distinct (dark gray, concave; green, convex surfaces). The distinct shapes of the two DNA binding sites, shown as minor groove width in the crystal structures of the complexes (blue plots), are already present when the protein is not bound to the DNA, with two minima in ''fkh250'' (C) vs. one minimum in ''fkh250con'' (D), as inferred by Monte Carlo simulations (green plots). Minor groove width (blue plots) and electrostatic potential (red plots) correlate and form two binding pockets in ''fkh250'' (E) and only a binding site for Arg5 in ''fkh250con'' (F).<br/>

==High-throughput Analysis of Hox-DNA Binding Specificity==