Phosphodependent helix switches in cellular signalling

Cells must be able to respond to changes in the human body. They grow, divide, change their shape and sometimes even die as the body requires. The millions of individual molecules that make up a cell contribute to the cell's response, and their individual actions are coordinated through an intricate cellular messaging system. One of the most common types of message is chemical modification of proteins, the presence of which may affect the location, stability or activity of that protein. The modification can also be detected by other proteins, which can then pass on the message. By analogy with written messages, the protein that carries out the modification is called a 'writer' and the protein that detects modification is called a 'reader'. Perhaps the most common modification is phosphorylation, which is written by a family of proteins called kinases, of which there are more than 500 in humans. Phosphorylation is commonly used to pass on the message that a cell is prepared to reproduce, and reaches a peak as a cell is just about to divide.

Several different reader proteins are known to read the phosphorylation message. They recognize the shape and electrical charge of the phosphate group and the features of a region of the protein surrounding it. Alternatively, phosphorylation can alter the shape of the protein, and this new shape is then recognized by a reader. We recently discovered an example that works through a simple mechanism: a protein that has a fluctuating, undefined shape becomes a helix when it is phosphorylated. The helix is a rigid shape that fits into the groove of a specific reader protein.

Strikingly, the molecular and structural features of the protein that are involved in helix formation are also required for the kinase to bind and phosphorylate it. Analysis of other proteins that are subject to phosphorylation suggests that this might cause many of them to form a helix. We now aim to clarify the details of what features of a protein are needed for phosphorylation to switch it from a dynamic shape to a defined helical shape. Based on this analysis, we will predict which other proteins are most likely to be switched, carry out experiments to test the predictions, and identify how they are read. Finally, we will use these insights to develop chemicals that block the interaction of the phosphorylated protein and its reader (chemical inhibitors). This will help us to understand what happens to cancer cells when the binding is blocked - in other words when the message is not passed on. If the interaction is essential for cancer cells to divide, the chemical inhibitors could be studied further for development into new cancer therapies.

There are more than 100,000 sites of phosphorylation in a human cell. Many phosphorylated proteins interact with canonical 'reader' domains that recognise a specific motif incorporating a phosphorylated sidechain. We have recently identified a novel form of recognition, in which the phosphorylation induced a short region of the TACC3 protein to adopt a helical conformation, which is then recognised by another protein (CHC). Here we aim to investigate the structure, dynamics and specificity of this phosphodependent helix switch using structural, computational, chemical and cell biological approaches.

1. The key structural and dynamic features of the TACC3 phosphoswitch. Crystallographic and NMR studies show that phosphorylated TACC3 forms a helix in solution that is recognised by CHC. We will investigate the structural and dynamic properties of this peptide using NMR spectroscopy and molecular simulation, and determine the relationship between amino acid sequence and phospho-dependent helix formation using sequence variants of the peptide.
2. Developing peptide-based inhibitors of the helix phosphoswitch. The interaction between CHC and TACC3 is not driven by the presence of the phosphate group per se, but by the helical conformation of TACC3. Therefore, stable helix mimetics of TACC3 should effectively inhibit the TACC3-CHC interaction because they are independent of phosphorylation. We will develop stably helical peptides using hydrocarbon staples and rational design enabled by crystallography.
3. Identification of new helix phosphoswitches. Based on understanding the key features of the helix phosphoswitch, we will identify further phospho-protein sequences that have these properties, test whether they act as switches in biochemical assays, and then identify the physiological binding partners that recognise their phosphorylated forms.

IMPACT SUMMARY (4000 chars)

Who might benefit from this research, and how?

The research scientists we train benefit from our contribution to their research career development. Training the next generation of biomedical research scientists is an important component of our work that has lasting impact. Our aim is that the researchers who work on this project will be in a strong position to apply for Fellowships or lectureships either directly afterwards, or perhaps after gaining further experience. This approach is highlighted by former Bayliss lab postdocs who have gone on to establish their own research groups: Charlotte Dodson is a lecturer at the University of Bath after holding research fellowships in Oxford and Imperial; Anja Winter moved directly from her post in the Bayliss lab to a lectureship at Keele. Indeed, the postdoctoral researchers funded by our previous joint BBSRC grant have also progressed in their careers: Elena Roskova (moved to another postdoctoral research position at KCL) and Manjeet Mukherjee (who is a permanent research scientist at Astex, a structure-based biotechnology company in Cambridge). We plan to develop the careers of the researchers who work with us through careful evaluation of their individual needs, by providing feedback and opportunities for training, by helping them to network and build their CVs. Researchers who work with us are always part of the 'family' and will receive career advice and more direct assistance (e.g. from grant writing) as long as they need it to realize their ambitions.

Pharmaceutical and biotechnology industry. Academic researchers have an important role to play in the identification of novel therapuetic targets and in the development of new methods to accelerate the process of drug discovery. Our proposal aims to develop inhibitors of a protein-protein interaction (TACC3-CHC) that is under investigation as a cancer drug target in the laboratory of our collaborator, Dr Stephen Royle at the University of Warwick. If we can help him to validate this target with a chemical tool, this will open up a new target to be exploited by industry. Of much broader scope, our central concept is that there exists a class of phospho-dependent protein-protein interaction that is based on a helix-groove interaction and therefore potentially druggable. This contrasts with the difficult-to-drug, canonical mode of phospho-dependent protein-protein interaction that depends on the recognition of the phosphate group in the context of an extended peptide. Protein kinases (and therefore protein phosphorylation) are dysregulated in many human diseases, notably cancer, and kinases have proven to be a successful drug target. Industry is looking for new ways to target phosphorylation-driven pathways, and we hope to open up a whole new class of protein-protein interactions for them to aim for. We of course realize there is a long way to go to realize this ambition, and we see this grant proposal as a critical but early step towards it.

Informing the public and exciting potential future scientists. We believe that outreach events play a crucial role in demonstrating to the public that the funds they raise, through taxation or charity, are being used to carry out world-class research for public good. Another goal of public engagement is to excite the next generation to become scientists. We work with local hospitals, charities and patient groups, giving tours of our laboratories and presenting our work. We will develop bespoke, hands-on activities using molecular models that provide real insights into the mechanisms under investigation in the project, and related topics. Our activities will form part of local/regional events supported by the University such as Be Curious, Astbury Conversation, Leeds Science Festival. The PIs and researchers working on this project directly in such events, giving them the opportunity to showcase their work to the public.