Aims: The project focuses on the structural and functional characterization of a multi-protein transcription factor complex which is key regulator of haematopoiesis and whose aberrant regulation can lead to leukemia.
Background: In the hematopoietic system, lineage commitment and differentiation is controlled by the combinatorial action of transcription factors that contain basic helix-loop-helix (bHLH) domains, such as the hematopoietic master regulator SCL (also known as TAL-1). Like other tissue-specific bHLH factors, SCL forms E-box (CANNTG) binding heterodimers with ubiquitous bHLH partners known as E-proteins, which include products of the E2A gene (E12 and E47). In erythroid cells, SCL is found in a multifactorial complex (SCL complex) with E47, LMO2, Ldb1, and GATA-1. Although the network of interactions has been established the molecular basis of how this complex controls gene expression has not been elucidated. Furthermore, not much is known about the dynamics of the SCL complex as during differentiation the complex will undergo dynamic changes in role and composition. In order to characterise at a molecular level the protein complex nucleated by SCL and to get insights into the working of this transcriptional network of proteins in the early stages of haematopoiesis as well as in erythroid and megakaryocytic development, we aim to solve the crystal structure of SCL multi-protein complexes bound to an DNA.
Biological and Medical Relevance: SCL is expressed in hematopoietic stem cells, as well as multipotent, erythroid, and megakaryocytic progenitors. In normal development SCL is essential for the establishment of the hematopoietic system while the scl gene is the most frequent target of chromosomal rearrangements in patients with T-cell acute lymphoblastic leukaemia (T-ALL). As well as shedding light on haemopoiesis, this work may therefore also provide clues to the development of leukaemia and suggest novel targeted therapies.
The student will be a member of the vibrant Division of Structural Biology which now includes over 40 graduate students. This project provides an opportunity for training in every aspect of modern X-ray Crystallography: protein cloning, expression and purification; protein analysis - including chromatography and light scattering; protein crystallization, mounting of crystals and collection of diffraction data both on in-house sources and at synchrotrons (DIAMOND and ESRF); determination of structures from diffraction data. Training in various biochemical and biophysical techniques such as Analytical Ultracentrifugation, Biacore and Isothermal Titration Calorimetry and EM microscopy will also be provided.
Protein Science & Structural Biology and Physiology, Cellular & Molecular Biology
Project reference number: 109
| Name | Department | Institution | Country | |
|---|---|---|---|---|
| Dr Erika J Mancini | Structural Biology | Oxford University | UK | erika@strubi.ox.ac.uk |
| Catherine Porcher | WIMM | UK |
2010. Purification, crystallization and preliminary X-ray analysis of a fusion of the LIM domains of LMO2 and the LID domain of Ldb1. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun., 66 (Pt 11), pp. 1466-9. Read abstract | Read more
LMO2 (LIM domain only 2), also known as rhombotin-2, is a transcriptional regulator that is essential for normal haematopoietic development. In malignant haematopoiesis, its ectopic expression in T cells is involved in the pathogenesis of leukaemia. LMO2 contains four zinc-finger domains and binds to the ubiquitous nuclear adaptor protein Ldb1 via the LIM-interaction domain (LID). Together, they act as scaffolding proteins and bridge important haematopoietic transcription factors such as SCL/Tal1, E2A and GATA-1. Solving the structure of the LMO2:Ldb1-LID complex would therefore be a first step towards understanding how haematopoietic specific protein complexes form and would also provide an attractive target for drug development in anticancer therapy, especially for T-cell leukaemia. Here, the expression, purification, crystallization and data collection of a fusion protein consisting of the two LIM domains of LMO2 linked to the LID domain of Ldb1 via a flexible linker is reported. The crystals belonged to space group C2, with unit-cell parameters a = 179.9, b = 51.5, c = 114.7 Å, β = 90.1°, and contained five molecules in the asymmetric unit. Multiple-wavelength anomalous dispersion (MAD) data have been collected at the zinc X-ray absorption edge to a resolution of 2.8 Å and the data were used to solve the structure of the LMO2:Ldb1-LID complex. Refinement and analysis of the electron-density map is in progress. Hide abstract
2011. Structure of the leukemia oncogene LMO2: implications for the assembly of a hematopoietic transcription factor complex. Blood, 117 (7), pp. 2146-56. Read abstract | Read more
The LIM only protein 2 (LMO2) is a key regulator of hematopoietic stem cell development whose ectopic expression in T cells leads to the onset of acute lymphoblastic leukemia. Through its LIM domains, LMO2 is thought to function as the scaffold for a DNA-binding transcription regulator complex, including the basic helix-loop-helix proteins SCL/TAL1 and E47, the zinc finger protein GATA-1, and LIM-domain interacting protein LDB1. To understand the role of LMO2 in the formation of this complex and ultimately to dissect its function in normal and aberrant hematopoiesis, we solved the crystal structure of LMO2 in complex with the LID domain of LDB1 at 2.4 Å resolution. We observe a largely unstructured LMO2 kept in register by the LID binding both LIM domains. Comparison of independently determined crystal structures of LMO2 reveals large movements around a conserved hinge between the LIM domains. We demonstrate that such conformational flexibility is necessary for binding of LMO2 to its partner protein SCL/TAL1 in vitro and for the function of this complex in vivo. These results, together with molecular docking and analysis of evolutionarily conserved residues, yield the first structural model of the DNA-binding complex containing LMO2, LDB1, SCL/TAL1, and GATA-1. Hide abstract