| Abstract |
The classical view of protein function was that after protein synthesis in the cell, the protein chain needed to 'fold' up into a highly specific three-dimensional structure in order to perform its function. For example, enzymes fold into a structure that allows them to selectively bind to their substrate molecules and to catalyze the conversion into products by means of precisely positioned amino acid residues in the 'active site'. Interest has recently centred on a large class of proteins which do not form a single well-defined three-dimensional structure. These so-called 'intrinsically unstructured proteins' (IUPs) account for about 30 % of the coding regions of the human genome. They frequently play a role in transcription or signalling and often occur as modules in larger proteins that facilitate the assembly of protein-protein complexes. Many IUPs do form a structure when they bind to their target molecule in a coupled folding-binding process, which allows high specificity via a large interaction surface, but low affinity because of the entropic cost of binding. This project aims to study the mechanism of coupled folding and binding for several intrinsically unstructured proteins using computer simulations of atomistic models of the protein - that is, where all of the atoms of the protein are represented. We will begin by developing the energy functions to be used in the simulations, building on work already done by the PI, since an accurate energy function is critical for obtaining the correct mechanism. Next, we will develop simulation methods suitable for studying coupled folding and binding. The major hurdle to this application is the gap between the time scales which can be accessed in the simulations (0.1 - 1 microseconds) and those which can be accessed experimentally (milliseconds to seconds or even longer). We will do this by means of a combination of enhanced sampling methods to be developed as part of the project. We will apply the novel methods developed to study the coupled binding and folding of two systems. Firstly, we study a system which has become a paradigm for binding-induced folding, and which has been very well characterized experimentally. This involves the binding of a transcription factor (a protein which triggers initiation of the transcription of genes by the cellular machinery) to its coactivator (a protein which enhances the effect of the transcription factor). Binding of the intrinsically unstructured pKID domain of the transcription factor CREB to the KIX domain of its coactivator CBP causes pKID to assume a folded two-helix structure. We will characterize the thermodynamics and kinetics of binding by simulation and compare these with experiment. The main purpose of this system is as a test of the simulation methods, however it will also provide an atomistic picture of the binding mechanism and may suggest further experiments to probe this mechanism. Our second application is to a transcription factor-coactivator pair that is implicated in tumour growth. The response of cells to low oxygen conditions inside a tumour is triggered by the binding of the CAD domain of the transcription factor Hif-1alpha to the TAZ-1 domain of the coactivator CBP. In the bound structure, the intrinsically unstructured CAD domain wraps almost completely around TAZ-1, forming three short helices. We will determine the mechanism and association rate of this pair of proteins and compare them with experimental data. In this case, we also intend to use the simulation results to suggest ways in which the binding mechanism could be interfered with for the development of novel anti-tumour drugs. We hope to deduce some general principles for the mechanism of coupled folding and binding based on the examples studied. Furthermore, the methods developed as part of this proposal should be generally applicable to other instances of coupled folding and binding. |
