Structural and functional analysis of a novel proline-rich dimerisation/oligomerisation domain

Proteins are long chains of units called amino acids and are essential components of all living things. Genes are segments of DNA that encode information on how to make the proteins that are required by the cell. This information is decoded by a set of proteins known as the transcription complex and the first step in the decoding process is known as transcription. To ensure that the information encoded by a gene is only transcribed in the correct circumstances there are a group of proteins called transcription factors that regulate the transcription process. Transcription factors are made up of a large and variable number of functional modules that allow them to interact with each other, with the transcription complex and with DNA. One group of modules that occur frequently both in transcription factors and also in a variety of other proteins is proline-rich. These modules are characterised by the presence of a large number of proline residues. Proline residues have a unique structure that cannot form many of the associations that occur between the other amino acids. One consequence of the unusual structure of proline is that proteins that contain a high density of proline residues are folded into their three dimensional shape (structure) in a way that is only poorly understood. The reason for having so many proline-rich modules in transcription factors and the structure of these modules are only poorly understood. The Proline-Rich Homeodomain (PRH) protein is a transcription factor that contains a high density of proline residues clustered in one region of the protein. PRH is an important protein in determining how cells divide and what sort of cells they will become in the future. The proline-rich region of PRH is important for regulating the transcription of genes, and for regulating cell division. Our present results show that the proline-rich region of PRH can bind to DNA and also to itself either to form small complexes called dimers that contain 2 PRH proteins or large complexes called oligomers that contain many PRH proteins. We want to understand how the proline-rich sequence in PRH influences the dimerisation and oligomerisation of the entire PRH protein. We also want to understand whether the ability of the PRH to bind to itself and to DNA using the proline-rich region is important for regulating transcription. Since other transcription factors that function in a similar manner to PRH also have similar proline-rich sequences, the information that we learn about PRH is very likely to be applicable to many other proteins.

Proline-rich sequences in proteins have been identified as; protein-protein interaction sequences, sequences that confer structural rigidity, and sequences that allow transport across cell membranes. Short proline-rich motifs in proteins can function as ligands for a variety of binding proteins, that include; signal transduction proteins, transcription factors and actin regulating proteins. Proteins that contain a very high density of proline residues within a large domain often appear to have a structural role, for example collagen and the oral salivary proteins, whilst peptides that are proline-rich can cross the cell membrane independently of conventional membrane transport proteins. Surprisingly the role of large proline-rich domains as opposed to short proline-rich motifs in transcription factors has not been extensively studied and the structure of proline-rich domains that also contain non-proline residues such as the RNA Polymerase C-Terminal Domain (CTD) is still ambiguous. Although the proline-rich domains in the tumor suppressor protein p53 and in RNA Polymersase II CTD are known to be important for multiple protein-protein interactions and multiple cellular functions, little is known about how a proline-rich region can participate in so many processes. The Proline-Rich Homeodomain (PRH/Hex) protein is a multifunctional protein that is a critical regulator of embryonic development in all vertebrates. PRH also regulates cell proliferation and differentiation during haematopoiesis in the adult and aberrant expression or sub-cellular localisation of PRH leads to leukaemia. The N-terminal domain of PRH is 20 percent rich in prolines and we have shown that the PRH N-terminal domain represses transcription, interacts with a variety of proteins that regulate transcription, and can prevent cell transformation by the E26 Myb-Ets viral oncoproteins. Here we show that like a number of other transcription repressor proteins PRH is an oligomeric protein in solution and that the proline-rich N-terminus contains a novel proline-rich dimerisation region. Moreover we show that the proline-rich N terminal domain interacts with DNA and also with the PRH homeodomain. This suggests that the proline-rich N-terminal domain is involved both in dimerisation and oligomerisation and in stabilising DNA binding by the full length PRH protein. The proline-rich N-terminal domain contains five copies of a P(X2-3)PS/TP motif. Multiple copies of this motif are found in a number of proteins that can repress transcription including p53, Mi-2 (from the NurD complex), N-CoR2 (Nuclear receptor corepressor 2), Mint (Msx2 repressor interacting protein), Mll (Mixed Lineage Leukaemia) protein, Pcl (Polycomb like) as well as some kinases including CAM kinase II and MAPKKK. In this proposal we will carry out a biophysical analysis of this novel proline-rich dimerisation and oligomerisation domain using CD spectroscopy in collaboration with Professor A. R. Clarke and using NMR in collaboration with Dr M. Crump. We will also examine the role of the N-terminal proline-rich domain in the DNA binding activity of PRH using EMSA and in vitro footprinting techniques. Finally we will use site-directed and random mutagenesis of the PRH N-terminal domain and homeodomain to examine the role of dimerisation and oligomerisation in transcriptional repression and the sub-cellular localisation of PRH. This body of work will reveal the role of proline-rich sequences in the structure and function of this novel protein domain and will be of great value to understanding the role of these sequences in many other regulatory proteins.