Flexibility and Disorder in Gene Regulation: LacI/GalR and Hox Proteins

Abstract

To modulate transcription, a variety of input signals must be sensed by genetic regulatory proteins. In these proteins, flexibility and disorder are emerging as common themes. Prokaryotic regulators generally have short, flexible segments, whereas eukaryotic regulators have extended regions that lack predicted secondary structure (intrinsic disorder). Two examples illustrate the impact of flexibility and disorder on gene regulation: the prokaryotic LacI/GalR family, with detailed information from studies on LacI, and the eukaryotic family of Hox proteins, with specific insights from investigations of Ultrabithorax (Ubx). The widespread importance of structural disorder in gene regulatory proteins may derive from the need for flexibility in signal response and, particularly in eukaryotes, in protein partner selection.

Keywords: DNA; DNA operator; DNA-binding protein; Hox proteins; LacI/GalR proteins; Ultrabithorax; gene regulation; intrinsic disorder; protein flexibility; repressor protein; transcription factor.

FIGURE 1.

LacI/GalR protein structure and DNA targets. A, graphic of a LacI/GalR homodimer. D indicates DNA-binding domains (gold/cyan ovals); L indicates linkers that contain the hinge helix (dark gold/dark cyan bars); and R indicates regulatory domains (large, stippled shapes). B, structure of LacI tetramer. Each monomer is a different color. The arrows labeled H point to the linker hinge helices. Inducer-binding sites are labeled with black stars; DNA is shown at the top of the figure as a twisted ladder. The tetramerization domain (T) is a four-helix bundle at the bottom of the figure; note the flexible linkers connecting this domain to each regulatory domain. C, overlay of monomers from DNA-bound (Protein Data Bank (PDB) 1LBG, gold) and inducer (IPTG)-bound (PDB 1LBH, black) LacI structures (14). Note that the hinge helix and N-terminal DNA-binding domain are not resolved in the presence of inducer, presumably due to flexibility that arises from hinge helix unfolding; the DNA-binding domain may also become less structured. D and E, linker sequence variation and DNA sites for subsets of LacI/GalR homologs that contain (top) and lack (bottom) the YPAL motif (13). In YPAL homologs, structures show that amino acids in positions P+1 through L+2 fold into an α helix (14, 15, 17). YPAL homologs recognize operators with contiguous DNA half-sites (panel E, top). Homologs that lack the YPAL motif (e.g. E. coli CytR) bind DNA with half-sites that are more widely spaced (panel E, bottom) (13, 27). When examined individually, the sequences of non-YPAL linkers resemble those of intrinsically disordered proteins (13). Logos were created with WebLogo (95).

FIGURE 2.

Hox protein structure and DNA targets. A, bar schematics depicting the functional domains of representative Hox proteins; note that domain organization differs for each protein. Black regions represent the homeodomain (HD); light gray regions indicate the activation domain(s); dark gray regions show the conserved hexapeptide motif. Ubx (389 aa) and Abd-A (330 aa) are derived from D. melanogaster; HoxB7 (431 aa), and HoxB3 (217 aa) are from Homo sapiens (96) (adapted from Ref. 37). B, bar schematics depicting structural and functional domains in Ubx. Yellow bars (top) indicate disordered regions of Ubx (32). Orange bars (bottom) represent a region of Ubx that is 35% glycine, including 13 consecutive glycines (dark orange) (32). The HD is shown as a black bar, and the hexapeptide motif is shown as a dark gray bar. Various domains are depicted as structures below. From left to right: (i) model of N terminus (91); (ii) molecular models, based on molecular dynamics simulations of 13 glycines in a free peptide, for the conserved Ubx polyglycine sequence that demonstrates the range of possible conformations (Justin Drake and B. Montgomery Pettitt, UTMB-Galveston, personal communication); (iii) conserved FYPWMA hexapeptide motif (from PDB file 1B8I) (34); and (iv) Ubx homeodomain (from PDB file 1B8I) (34). C, examples of Ubx DNA-binding sites. Dll is a composite site bound by a single Ubx protein with partner proteins Homothorax (Hth) and Exd to regulate the distalless gene (44). Sequences recognized by Ubx are in bold, and sequences recognized by Hth (left underline) and Exd (right underline) are indicated. The UA-binding region contains four Ubx-binding sites (bold) and is part of the ubx gene; a linear schematic of a portion of the ubx gene is shown beneath, showing a second cluster of binding sites designated as UB (66). When multiple Ubx proteins bind to UA and UB sequences in the ubx gene, they interact to create a DNA loop. A loop schematic is shown on the lower left, and an electron micrograph of a loop is shown at the lower right (reprinted with permission from Ref. 66).

FIGURE 3.

Schematics for different search modes of DNA binding. The LacI structure (dimer or tetramer) is used for most of these examples, with requisite flexible regions highlighted with gold ovals. A, proteins can slide along the DNA backbone in search of specific binding sites. B, proteins can dissociate from DNA and reassociate with the same or a different DNA in a “hopping” process. C, intersegment transfer and brachiation allow proteins with multiple binding sites to move from one DNA segment to another via transient contacts to both DNA strands. This type of movement can be accomplished by an oligomeric protein with two DNA-binding sites (e.g. LacI tetramer, left) or by a monomer with two separate DNA-binding regions within a single domain (e.g. Ubx homeodomain, right, green with flexible N-terminal arm in gold oval). D, stable loops can be formed when two DNA-binding domains simultaneously form specific complexes at DNA target sites. The two sites can be separated by stretches of DNA that vary widely in length. Prokaryotic looping (left) generally involves single proteins (e.g. LacI tetramer) or a protein assisted by a nearby DNA bending protein (e.g. two GalR dimers and the bending protein HU) (60, 65). In contrast, multi-protein complexes at eukaryotic promoters (right) can be highly complex and comprise multiple loops of varying stability that can encompass up to ∼10⁶ bp (as in for example Ref. 97).

FIGURE 4.

Functional regions in LacI/GalR and Ubx. A, adaptable regions of the LacI/GalR proteins are shown on a graphic of the homodimer superimposed on a crystal structure (PDB file 1LBG) (14). The flexibility of these regions is critical to strong transcription repression and allosteric regulation. B, the flexible interface between the LacI/GalR linkers and regulatory domains can facilitate allosteric response to multiple ligands (79). The LacI DNA-binding domain/linker can be fused to the regulatory domains of other homologs (shown as schematic dimers) to create functional chimeric repressors (red bars in graph) with intact allosteric response to small effector ligands (blue bars). Note that the “LLHP” chimera has the opposite allosteric response of LacI (79). DEL control indicates the activity of reporter enzyme in the absence of repressor. C, regions within Ubx important for modulating its DNA binding. The upper red bracket indicates the Ubx region that contains phosphorylation sites (38). Blue brackets below indicate sequences that interact with Ubx partner proteins (69). Yellow regions are intrinsically disordered (32). The gold and brown striped region is both spliced and disordered (32, 38). The conserved hexapeptide motif important for Exd interaction is dark gray, and the HD is black. D, example structures for three families of proteins that interact with Ubx. For the six partners of the DNA/RNA-binding three-helical bundle family, the engrailed homeodomain is shown (PDB file 1ENH) (98); for the five partners of the α-α superhelix family, β-catenin is shown (PDB file 1QZ7) (99); and for the six partners of the zinc finger C2H2/C2H2 family, Zif268 is shown (PDB file 1A1I) (100). Intrinsic disorder of Ubx regions may be key for recognizing this wide range of partners (69).