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.

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LMO (LIM-only) and LIM-HD (LIM-homeodomain) proteins form a family of proteins that is required for myriad developmental processes and which can contribute to diseases such as T-cell leukaemia and breast cancer. The four LMO and 12 LIM-HD proteins in mammals are expressed in a combinatorial manner in many cell types, forming a transcriptional 'LIM code'. The proteins all contain a pair of closely spaced LIM domains near their N-termini that mediate protein-protein interactions, including binding to the approximately 30-residue LID (LIM interaction domain) of the essential co-factor protein Ldb1 (LIM domain-binding protein 1). In an attempt to understand the molecular mechanisms behind the LIM code, we have determined the molecular basis of binding of LMO and LIM-HD proteins for Ldb1(LID) through a series of structural, mutagenic and biophysical studies. These studies provide an explanation for why Ldb1 binds the LIM domains of the LMO/LIM-HD family, but not LIM domains from other proteins. The LMO/LIM-HD family exhibit a range of affinities for Ldb1, which influences the formation of specific functional complexes within cells. We have also identified an additional LIM interaction domain in one of the LIM-HD proteins, Isl1. Despite low sequence similarity to Ldb1(LID), this domain binds another LIM-HD protein, Lhx3, in an identical manner to Ldb1(LID). Through our and other studies, it is emerging that the multiple layers of competitive binding involving LMO and LIM-HD proteins and their partner proteins contribute significantly to cell fate specification and development.

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A stable intramolecular complex comprising the LIM domains of the LIMhomeodomain protein Lhx3 tethered to a peptide region of Isl1 has been engineered, purified and crystallized. The monoclinic crystals belong to space group C2, with unit-cell parameters a = 119, b = 62.2, c = 51.9 Å , = 91.6 , and diffract to 2.05 Å resolution.