Structural basis for partial redundancy in a class of transcription factors, the LIM homeodomain proteins, in neural cell type specification

Abstract

Combinations of LIM homeodomain proteins form a transcriptional "LIM code" to direct the specification of neural cell types. Two paralogous pairs of LIM homeodomain proteins, LIM homeobox protein 3/4 (Lhx3/Lhx4) and Islet-1/2 (Isl1/Isl2), are expressed in developing ventral motor neurons. Lhx3 and Isl1 interact within a well characterized transcriptional complex that triggers motor neuron development, but it was not known whether Lhx4 and Isl2 could participate in equivalent complexes. We have identified an Lhx3-binding domain (LBD) in Isl2 based on sequence homology with the Isl1(LBD) and show that both Isl2(LBD) and Isl1(LBD) can bind each of Lhx3 and Lhx4. X-ray crystal- and small-angle x-ray scattering-derived solution structures of an Lhx4·Isl2 complex exhibit many similarities with that of Lhx3·Isl1; however, structural differences supported by mutagenic studies reveal differences in the mechanisms of binding. Differences in binding have implications for the mode of exchange of protein partners in transcriptional complexes and indicate a divergence in functions of Lhx3/4 and Isl1/2. The formation of weaker Lhx·Isl complexes would likely be masked by the availability of the other Lhx·Isl complexes in postmitotic motor neurons.

FIGURE 1.

LIM homeodomain complexes in development. A, the proteins Ldb1 (gray) and Lhx3 (black) assemble into a binary complex. B, with the addition of Isl1 (white), the ternary complex is preferentially formed (12). Note that the self-association domain of Ldb1 forms trimers in vitro (21) and that the homeodomains from Lhx3 and Isl1 bind DNA. The spacing between the homeodomain-binding sites (dashed lines) is not defined and may represent close or distant binding sites. C, details of domain contacts within the binary and ternary complexes. The LIM interaction domain (LID) from Ldb1 makes contacts with the LIM domains (LIM) from Lhx3 in the binary complex, but in the ternary complex, Ldb1_LID contacts the LIM domains of Isl1, and the LBD from Isl1 binds the LIM domains from Lhx3. SD, self-association domain; HD, homeodomain.

FIGURE 2.

Identification of the Isl2 Lhx3-binding domain, Isl2_LBD. A, sequence alignment of Isl1_LBD and Isl2_272–301. Dots are identical residues. B and C, pairwise interactions between Isl1/Isl2 and Lhx3/Lhx4 identified by yeast two-hybrid analysis. AH109 yeast cells co-transformed with pGBT9/pGAD10 vectors shown on the right were tested for growth under different selection conditions (−H + X-α-gal + 3-AT) or (−H-A); 0 indicates no dilution of yeast cells (A_{600 nm} = 0.2), 1 indicates a 1:10 dilution (A_{600 nm} = 0.02), and 2 indicates a 1:100 dilution (A_{600 nm} = 0.002). pGBT9 encodes the GAL4_DBD and pGAD10 encodes the GAL4_AD.

FIGURE 3.

Construction and characterization of the tethered Lhx3/4-Isl1/2 complexes. A and B, schematics illustrating the generation of the tethered constructs (A) and arrangement of domains (B) in the constructs, using Lhx4-Isl2 as an example. C, experimental (MM_exp) and predicted (MM_pr) molecular masses of the complexes as determined by multi-angle laser light scattering. D, far-UV CD spectra of the tethered complexes.

FIGURE 4.

The crystal structure of Lhx4-Isl2. A, ribbon diagram of chain B of the Lhx4-Isl2 complex. Lhx4 is shown in orange, and Isl2 is in purple. The position of the linker is shown as a black dashed line. B, overall shape comparison of tethered LIM homeodomain and LIM-only complexes. Molecules are aligned over the backbone residues of the LIM2 domains of Lhx4-Isl2 (3MMK), Lhx3-Isl1 (2RGT), and LMO4(LIM-only protein 4)_LIM1+2-Ldb1_LID (1RUT) complexes. For clarity, only the LIM domains are shown; Lhx4 is in orange, Lhx3 is in red, and LMO4 is in blue. Images were prepared and alignment of molecules was performed using MolMol (46).

FIGURE 5.

The solution structures of Lhx3/4-Isl1/2. A, P(r) profiles from experimental scattering data for Lhx4-Isl2 (green squares) and calculated scattering profiles from the Lhx4-Isl2 crystal structure (purple line) and the generated BUNCH model (green line). B, alignment using the atoms of Lhx4_LIM2 of the Lhx4-Isl2 crystal structure (purple) with the best-fit BUNCH model (green) and ab initio DAMMIF reconstruction (transparent green surface). C, P(r) profiles from experimental scattering data for Lhx3-Isl1 (blue triangles) and Lhx3-Isl2 (orange squares) and calculated scattering profiles from the Lhx3-Isl1 crystal structure (red dashed line), the Lhx3-Isl1 BUNCH model (dark blue line), and the Lhx3-Isl2 BUNCH model (dark orange line). D, alignment using the atoms of Lhx3_LIM2 of the Lhx3-Isl1 crystal structure (red) with the best-fit BUNCH model (blue) and ab initio DAMMIF reconstruction (transparent blue surface). E, the best-fit BUNCH model (orange) and ab initio DAMMIF reconstruction (transparent orange surface) for Lhx3-Isl2. LIM domains are shown as ribbons, and LBDs as are shown as spheres.

FIGURE 6.

Binding and apparent stability in Lhx3/4·Isl1/2 complexes. A, summary of alanine mutagenic screening assayed by yeast two-hybrid analysis from supplemental Table S4. The sequence of Isl1_LBD (262–291) is shown. The sequence of Isl2_LBD (272–301) shows where residues are conserved (*) or different. Colored boxes indicate where mutation had a strong (red), moderate (orange), or minor effect (yellow). White boxes indicate residues mutated in triple-alanine constructs that had a minor effect on growth of yeast. Gray boxes indicate no effect on yeast growth. B, comparison of wild-type and mutant LBDs from Isl1/Isl2 binding to the LIM domains of Lhx3/Lhx4 by yeast two-hybrid analysis. AH109 yeast cells co-transformed with pGBT9/pGAD10 vectors shown on the right were tested for growth under different selection conditions (−H + X-α-gal + 3-AT) or (−H−A); 0 indicates no dilution of yeast cells (A_{600 nm} = 0.2), 1 indicates a 1:10 dilution (A_{600 nm} = 0.02), and 2 indicates a 1:100 dilution (A_{600 nm} = 0.002). C, resistance of Lhx3-Isl1/2 complexes to denaturation by guanidine hydrochloride (Gdn.HCl). □, Lhx3-Isl1; ■, Lhx3-Isl2. D, resistance of Lhx4-Isl1/2 complexes to denaturation by guanidine hydrochloride. ○, Lhx4-Isl1; ●, Lhx4-Isl2. For C and D, λ_max reports maximum emission wavelength in the range 320–380 nm with excitation at 295 nm.

FIGURE 7.

Comparison of Lhx3/4·Isl1/2 interaction interfaces. A and B, backbone alignment of Lhx3 (red) and Lhx4 (orange) using the LIM1 (A) and LIM2 (B) domains. The backbones of Isl1 (blue) and Isl2 (purple) are also shown. An asterisk indicates loops in Lhx3/Lhx4 that show the most variation in the backbone alignment. C and D, interaction maps indicating the residues from Lhx3-Isl1 (C) and Lhx4-Isl2 (D) that make contacts as identified by LIGPLOT (47). Gray boxes are residues of Isl1_LBD or Isl2_LBD. Colored boxes for the Lhx residues define the type of interaction as indicated; solid lines represent conserved contacts between the two complexes, and dashed lines indicate non-conserved contacts between the two complexes. Note that the numbering between Lhx3 and Lhx4 differs by four (e.g. Lhx4_F130 corresponds to Lhx3_F134) and by 10 between Isl1 and Isl2 (e.g. Isl1_M265 corresponds to Isl2_L275). E and F, the key residues in the two complexes from Lhx3 and Lhx4 LIM1 domains (E) and Lhx3 and Lhx4 LIM2 domains (F). The side chains of critical residues from Isl1 (blue) and Isl2 (purple) identified using yeast two-hybrid analysis and the residues they contact are shown (Lhx3 in red and Lhx4 in orange), as well as the backbone atoms of non-critical residues from Isl1_LBD and Isl2_LBD. Non-Isl-binding residues from Lhx4_LIM1 and Lhx4_LIM2 are shown in surface representation (white). The residues Isl1_M265 and Isl2_L275 adopt equivalent rotamers, mtp and mt, respectively.