Structural basis for TOR activation by the HSP90-R2TP-TTT molecular chaperone complex

The normal and healthy functioning of cells is controlled by a plethora of nano-molecuiar machines which are made up of protein molecules, each of whose shape and mechanism of action is defined by the genetic information encoded in DNA. Assembly of many of these nano-machines, requires help from specialised nano-machines called chaperones, which look after component proteins until they are required, and ensure that the correct components are combined into the final working nano-machine.

One of the most important of these chaperones is called HSP90. This is responsible for assisting the assembly of many of the nano-machines that monitor the health of the cell and transmit messages between and within cells. One of these - TOR - is particularly important for monitoring the nutritional health of the cell, and over-activity or defects in TOR are associated with diseases such as cancer, diabetes and unhealthy ageing.

We wish to understand how the HSP90 chaperone, with the collaboration of two other nano-machines R2TP and TTT, facilitates the assembly of nano-machines based around TOR. To do this we will use very high magnification electron microscopes and intense X-ray beams, to look at the atomic structures of the different nano-machines involved, to work out which parts are critical for activity, and which parts of different nano-machines make contact with each other during the assembly process. This information will help us understand how TOR is regulated by these chaperones, and will point at new ways of controlling TOR activity with novel drugs.

HSP90 is a molecular chaperone implicated in folding, assembly and in vivo stabilisation of 'client' proteins and complexes, such as protein kinases, steroid hormone receptors, TLR innate immunity receptors, viral and cellular RNA polymerases, and PI3-kinase-related kinases (PIKKs). Specificity for diverse client protein classes is provided by co-chaperones. These act as adaptors coupling client proteins to HSP90, but also regulate the inherent ATPase coupled conformational cycle of the chaperone, which is critical to its biological function.

R2TP (Rvb1p-Rvb2p-Tah1p-Pih1p in yeast and RUVBL1-RUVBL2-RPAP3-PIH1D1 in humans) is the most complicated HSP90 co-chaperone yet described. It is implicated in stabilisation and activation of PIKKs such Tor/mTOR and SMG1, RNA polymerase II and small nucleolar ribonucleoproteins. Recruitment of PIKKs to R2TP, is itself mediated by the TTT complex (Tel2p/TEL2, Tti1p/TTI1 and Tti2p/TTI2) which binds to Pih1p/PIH1D1 via a conserved CK2-phosphorylated motif in Tel2p/TEL2. It is currently unknown which components of TTT mediate recruitment of PIKKs, how and where PIKKs interact with HSP90 and components of the R2TP sub-complex, and what role these interactions play in assembly of active PIKK complexes.

To resolve these questions we will focus on the role of HSP90-TTT-R2TP in the activation of TOR - a PIKK responsible for integrating multiple signalling pathways that promote cell survival. We will biochemically reconstruct and characterise functional complexes of the yeast and human proteins; define how chaperone interactions regulate TOR dimerisation and interaction with co-factor proteins Lst8/MLST8 and Kog1/RAPTOR; and determine structures of HSP90-R2TP-TTT-TOR complexes, and tractable sub-complexes thereof at the highest resolution we can achieve using single-particle cryo-EM and X-ray crystallography, and use these to define the key interactions that mediate the specificity and mechanism of the system.

We aim to elucidate the mechanism of chaperone mediated assembly and activation of complexes of TOR, a key signalling molecule that integrates multiple environmental, metabolic and stress processes within the eukaryotic cell. The central position of TOR complexes as cellular regulators makes them the focus of interest for drug discovery against a range of non-infectious diseases, including cancer, diabetes and obesity, neurodegeneration and dementia, and the general process of ageing. Additionally, TOR is implicated in the immune response to infectious diseases and is manipulated by infectious agents within host-pathogen interactions. Consequently, improved understanding of the molecular function of cellular molecular chaperones such as HSP90, and how they contribute to the regulation and assembly of TOR-centred signalling complexes, has the potential to achieve substantial impact across a range of discovery and translational research that falls comfortably under the BBSRC priority of 'Healthy Ageing'. Previous discovery science focussed on HSP90 structure and function, in which we played a substantial role, has led to considerable downstream translational activity in academia and in industry, in the UK and abroad. Translation of discoveries made in this current project are also expected to translate into opportunities for drug discovery, but in the nature of that process this will be some way downstream.

The early impacts of this research will primarily come through Communications and Engagement, and Training and Collaboration. Communication of our work to the broader academic and industrial research community will come through publication in peer-reviewed journals, and pre-print servers such as bioRxiv. All publications will be 'open access' and will be deposited in Europe PubMed Central, and in the University of Sussex's own online repository. Our results will be communicated by oral presentations and/posters at selected national and international meetings, especially those focussed on molecular chaperones and/or signalling pathways, as well as at relevant sessions within broader conferences such as NCRI, AACR, The EMBO Meeting, FEBS Congress etc. Detailed presentations will be made at invited research seminars at academic institutions and industrial research organisations, in the UK and abroad, which the PI is regularly invited to present.

This project requires the full spectrum of the enabling techniques that underpin modern structural biology, and therefore provides a superb training opportunity. The PDRA and RA involved in the project will develop state-of-the-art expertise in the application of these techniques in a laboratory with an international reputation for its research and track-record in training researchers. The majority of these techniques are on the critical path of modern structure-guided drug discovery, and expertise in these is highly valued within the biotechnology, pharmaceutical and agrochemical industries worldwide, as well as in academic laboratories.

Our laboratory has been very actively engaged in translating the results of their discovery research towards the development of new pharmaceutical approaches to human and animal disease, for many years. In particular our work on the HSP90 molecular chaperone and its role in activation of oncogenic protein kinases has resulted in several international granted patents. As our understanding of the molecular basis for interactions within the HSP90-R2TP-TTT-TOR nexus develops, we will identify 'druggable' opportunities that could form the basis of assays/screens for identification of initial 'hit' material. These will be developed into high-throughput format and used in preliminary in vitro and in silico screens. Where these are promising we will utilise our extensive network of industrial contacts to identify opportunities for collaborative development of these targets, and prepare initial patent filings, where appropriate.