Implementing the LIM code: the structural basis for cell type-specific assembly of LIM-homeodomain complexes

Abstract

LIM-homeodomain (LIM-HD) transcription factors form a combinatorial 'LIM code' that contributes to the specification of cell types. In the ventral spinal cord, the binary LIM homeobox protein 3 (Lhx3)/LIM domain-binding protein 1 (Ldb1) complex specifies the formation of V2 interneurons. The additional expression of islet-1 (Isl1) in adjacent cells instead specifies the formation of motor neurons through assembly of a ternary complex in which Isl1 contacts both Lhx3 and Ldb1, displacing Lhx3 as the binding partner of Ldb1. However, little is known about how this molecular switch occurs. Here, we have identified the 30-residue Lhx3-binding domain on Isl1 (Isl1 LBD ). Although the LIM interaction domain of Ldb1 (Ldb1 LID ) and Isl1 LBD share low levels of sequence homology, X-ray and NMR structures reveal that they bind Lhx3 in an identical manner, that is, Isl1 LBD mimics Ldb1 LID . These data provide a structural basis for the formation of cell type-specific protein-protein interactions in which unstructured linear motifs with diverse sequences compete to bind protein partners. The resulting alternate protein complexes can target different genes to regulate key biological events.

Solution structure of a tethered Lmo2LIM2/Ldb1LID complex

“…Together these parameters indicate that the solution structure of Lmo2 LIM2 -Ldb1 LID is of medium to high resolution. 24 The structure of this tethered complex closely resembles that of other LIM-Ldb1 LID complexes, 22,23,[25][26][27] [e.g. Fig.…”

“…2(B)]; order angle parameters >0.9, TALOSþ, and random coil index (RCI) analysis all support structure in this region and chemical shift index analysis and the presence of non-sequential NOEs indicate helical structure (Supporting Information data). In related complexes that contain the LIM1 domains from Lmo2, Lmo4, or Lhx3 in complex with Ldb1, these same residues form extended or b structure 22,23,[25][26][27] [e.g., Fig. 2(A,C), orange boxes], indicating that for these residues in Ldb1 a conformational change is brought about through binding to the LIM1 domains of Lmo2 and related proteins.…”

“…This phenylalanine residue is highly conserved in all other LIM-only and LIM-homeodomain proteins, and adopts the same conformation in all of the known structures from this group of LIM domain proteins (Lmo4, Lhx3, and Lhx4). 22,[25][26][27]30 Thus, the lack of Lmo2 F88 likely destabilized Lmo2 LIM1 -Zn2 in the NMR structure. The solution structure of an Lmo4 LIM1 -Ldb1 LID construct (PDB accession code 1M3V) that does contain Lmo4 F81 (which is equivalent to Lmo2 F88 ) is considerably more ordered than the Lmo2 LIM1 -Ldb1 LID structure, 22 supporting an important structural role for this aromatic sidechain.…”

“…These ordered regions from the single domain NMR structures correspond well with the results of mutagenic studies that indicated a single binding hotspot in Ldb1 LID , centered on Ldb1 I322 , that drives binding to the LIM domains of Lmo2, Lmo4, Lhx3, and Isl1. 21,25,27 The only significant difference in the structures of the LIM2 domain constructs lies at start of this domain. Residues Lmo2 G92/L93 form a well defined short b-strand in the double LIM domain X-ray structures, but are disordered at the start of the single LIM domain NMR structure [Fig 2(A), blue box]; Lmo2 A20-M23 is also poorly ordered in the NMR structure, Lmo2 (Fig 1, cyan).…”

“…Mutagenic studies indicate two binding hotspots that form an extended interface in Ldb1 LID for the LIM2 domains from Lmo2, Lmo4, and Lhx3. 21,25,27 For Lmo2 LIM1þ2 / Ldb1 interactions, the number of residues from Ldb1 for which electron density is defined varies between each of the six molecules from the X-ray structures, but apart from a minor hinging ($11 ) at Ldb1 E313 , 23 Ldb1 adopts the same structure in each conformer [ Fig. 2(C)].…”