Molecular mechanisms of calcium/calmodulin-dependent kinase localisation activation and inhibition

In this research project the three dimensional structures, and molecular interactions and flexibility of a human signaling enzyme will be characterized at a resolution that is most useful for drug discovery. This 40 kDa kinase is known as Ca2+/calmodulin-dependent protein kinase I delta (CaMK1D), and adds phosphates to substrate proteins on serine or threonine residues. Our protein target has recently been found to be critically involved in the transformation and invasiveness of breast cells. Its gene is frequently amplified and hyperactive in basal layer cells of breast carcinomas, causing cells to grow and divide without control, and suggesting that CaMK1D inhibitors could have direct relevance to improving human health and well being. However, these targets still remain very challenging for analysis in terms of their solution structures and conformational dynamics under physiological conditions, warranting further fundamental research and technological developments to render them more amenable to experimental investigation and uncover their mechanisms at a predictive level. We will use a method known as nuclear magnetic resonance spectroscopy using our national facility's superconducting magnets, which can be used to detect a unique signal for the individual thousands of atomic nuclei in the macromolecule. The method provides an unprecedented level of information about the shape, conformation, motions and chemical interactivity of a protein in three dimensional space and over a range of timescales from picoseconds to seconds. We have assigned most of the protein's backbone signals, and now plan to extend these to its resolved complexes and sidechains to understand its molecular functions better than any other protein kinase. We will study the interactions of CaMK1D with other proteins that activate the enzyme as well as metabolites and inhibitors which we have recently discovered as ligands and begun to map the interactions of. We have also predicted a novel site that could bind lipids and membranes, and will use spin label molecules and computer methods to validate and define the nature of this proposed mechanism to localize the protein to its sites of activity in cells. Together with our collaborators we will provide the first comprehensive structural, functional and chemical insights into how this kinase acts and signals at a molecular level, allowing us to much more accurately manipulate its behaviour in vitro and in vivo. The endpoints of the project include structures of the enzyme bound to the lipids, metabolites, substrates and protein ligands that regulate its activity in cells, a deeper understanding of the dynamics and kinetics of these binding events, and a rational basis for designing inhibitors and mutations for in vitro and in vivo analysis of this emerging target for drug discovery.

Our specific aims for CaMK1D are to: 1. Assign the backbone and sidechain 1H, 15N and 13C resonances of CaMK1D kinase and regulatory domains in their free and bound states, providing a basis to map interaction sites of biological ligands that emerge from our ThermoFluor and NMR-based screening. The tertiary structure of the physiological monomer will be elucidated using 900 MHz NMR and SAXS data, leveraging the SGC's recent dimer structure. 2. Characterize the binding properties and conformational changes induced by novel ligands, substrate peptides, biological signalling molecules, inhibitors and fragments, allowing the identification and exploitation of new sites that modulate kinase activity. HADDOCK will be used to rapidly calculate the complexed structures, defining the binding modes and specific contacts responsible for selective recognition and ligand competition within physiological mixtures, 3. Map and define structural and functional effects of kinase phosphorylation, which we propose initiate its release from cis-inhibition by the autoinhibitory domain, defining the local and long range effects on kinase structure, dynamics and ligand binding activity, 4. Elucidate how calmodulin and calmodulin binding domain of the kinase interact, and calculate the structure of their complexed states, showing how the kinase is activated by our proposed dramatic unfolding of the C-terminal regulatory elements, 5. Prove our proposed membrane interaction site, define lipid binding specificities and micelle insertion depth and angle, validate with novel membrane systems including bicelles and bilayer discs, model the protein-micelle complex using PRE and RDC based NMR restraints, and 6. Design and test mutations and interactions of specific chemical probes, small molecule inhibitors and novel ligands for their ability to manipulate CaMK1D enzymatic and signalling activity and localization in vitro and in cell based systems with our collaborators.

Broader Scientific Community: Scientific groups will be engaged by primary journal articles, reviews, web sites, exchange visits and access to research products and services. Overduin's group has produced 30 manuscripts since 2004, including papers in high impact journals like JACS, EMBO J and PNAS and scholarly reviews including Nature Reviews and Methods In Molecular Biology in 2009. Open access journals will continue to be the primary medium for communicating our research results. Overduin also operates websites at www.proteinexpress.org, www.lipidprism.org and www.nmr.bham.ac.uk to disseminate scientific knowledge and practice. Conferences and meetings will be used to transfer knowledge and skills, with Overduin being the organizer of four BBSRC-funded JPA workshops on protein expression (2007-2010) as well as annual meetings for the LipidPrism project on membrane-protein interactions, and with HWB-NMR on NMR applications and methods including software, sample preparation and challenging systems. Commercial Sector: Pharmaceutical and biotech companies will learn about the tools and methods being developed here through their visits to Birmingham as HWB-NMR users, as well as through collaborations, with GSK supporting a PhD studentship and academic research activities in Overduin's group. The kinase superfamily includes ~79 drug targets including the first mechanism-based cancer drug target (Gleevec), and new targets including CaMK1D are now emerging, with the latter being implicated in invasive breast carcinomas and inducing tumorigenesis when overexpressed. Our research on the fundamental mechanisms of kinase activity will thus have broad applicability to the pharmaceutical industry. Wider Public: Overduin will continue to play an active role in promoting the public understanding of science, and has contributed to articles in the Guardian (Nov 2007), Birmingham Post (Mar 2009, Nov 2006), Telegraph (Nov 2005) and Research TV (Nov 2004). Overduin is a member of the steering group of the British Science Association, which will be holding a Science Festival in Birmingham in Sept 2010, and every four years thereafter. Lab and facility tours will continue to given by Overduin and members of his group to high school classes and the public, and several high school students have performed summer research projects in his lab in recent years. Overduin also volunteers as Chair of the Science and Medicine Forum of the Lunar Society, a scientific body which was originally founded in the West Midlands in 1775. He also serves as its Honorary Secretary, helping to to organize monthly lectures and public events with attendances of up to 600 people. Recent speakers he has hosted include the Nobel Laureate Paul Nurse, President, Rockefeller University and Sir Liam Donaldson, Chief Medical Officer. This provides an avenue to present results on pharmaceutical research and genetics, proteomics and systems biology of disease, helping to overcome public anxiety about human mutations and breast cancer, and demonstrating the benefits of new technologies and drug discovery through academic - industrial collaborations. As requested by the guidance, the costs of these public engagement activities are estimated at 2 hours per month of Overduin's time and effort, as well as the associated local travel costs (~£20/month), all of which is given freely for this publicly funded research. Business engagement: Overduin is a member of the University of Birmingham's Regional Advisory Group which is tasked with exploring strategic collaborations with regional partners. He is helping to establish Science Capital as a new organization to connect internationally recognized scientists with the public and businesses to present and discuss innovations and priorities for growth and investment.