Browsing by Author "Matthews, JM"

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    LIM domain binding proteins 1 and 2 have different oligomeric states
    (Elsevier, 2010-05-28) Cross, AJ; Jeffries, CM; Trewhella, J; Matthews, JM
    LIM domain binding (Ldb) proteins are important regulators of LIM homeodomain and LIM-only proteins that specify cell fate in many different tissues. An essential feature of these proteins is the ability to self-associate, but there have been no studies that characterise the nature of this self-association. We have used deletion mutagenesis with yeast two-hybrid analysis to define the minimal self-association domains of Ldb1 and Ldb2 as residues 14–200 and 21–197, respectively. We then used a range of different biophysical methods, including sedimentation equilibrium and small-angle X-ray scattering to show that Ldb114–200 forms a trimer and Ldb221–197 undergoes a monomer–tetramer–octamer equilibrium, where the association in each case is of moderate affinity (105 M− 1). These modes of association represent a clear physical difference between these two proteins that otherwise appear to have very similar properties. The levels of association are more complex than previously assumed and emphasise roles of avidity and DNA looping in transcriptional regulation by Ldb1/LIM protein complexes. The abilities of Ldb1 and Ldb2 to form trimers and higher oligomers, respectively, should be considered in models of transcriptional regulation by Ldb1-containing complexes in a wide range of biological processes. © 2010, Elsevier Ltd.
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    Solution structure of the LIM-homeodomain transcription factor complex Lhx3/Ldb1 and the effects of a pituitary mutation on key Lhx3 interactions
    (Public Libary of Science, 2012-07-25) Bhati, M; Lee, C; Gadd, MS; Jeffries, CM; Kwan, AH; Whitten, AE; Trewhella, J; Mackay, JP; Matthews, JM
    Lhx3 is a LIM-homeodomain (LIM-HD) transcription factor that regulates neural cell subtype specification and pituitary development in vertebrates, and mutations in this protein cause combined pituitary hormone deficiency syndrome (CPHDS). The recently published structures of Lhx3 in complex with each of two key protein partners, Isl1 and Ldb1, provide an opportunity to understand the effect of mutations and posttranslational modifications on key protein-protein interactions. Here, we use small-angle X-ray scattering of an Ldb1-Lhx3 complex to confirm that in solution the protein is well represented by our previously determined NMR structure as an ensemble of conformers each comprising two well-defined halves (each made up of LIM domain from Lhx3 and the corresponding binding motif in Ldb1) with some flexibility between the two halves. NMR analysis of an Lhx3 mutant that causes CPHDS, Lhx3(Y114C), shows that the mutation does not alter the zinc-ligation properties of Lhx3, but appears to cause a structural rearrangement of the hydrophobic core of the LIM2 domain of Lhx3 that destabilises the domain and/or reduces the affinity of Lhx3 for both Ldb1 and Isl1. Thus the mutation would affect the formation of Lhx3-containing transcription factor complexes, particularly in the pituitary gland where these complexes are required for the production of multiple pituitary cell types and hormones. © 2012 Bhati et al.