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King’s College London Health Schools Studentships 2015 Division: Randall Division of Cell and Molecular Biophysics PROJECT DETAILS
Title of project Unravelling the structural complexity in cargo recognition by the kinesin‐1 molecular motor Supervisor 1 Roberto A Steiner ([email protected]) Supervisor 2 Mark P Dodding ([email protected]) Project description (max 500 words) The sorting and transport of intracellular components is of central importance to all eukaryotes. Proteins, nucleic acids, vesicles as well as whole organelles must be directed from their site of production to locations appropriate for their function. After use, these components are often transported for recycling or degradation. Long‐distance movement of intracellular components is principally mediated by the dynein and kinesin protein families. These molecular motors have two fundamental properties. Firstly, they bind to the microtubule (MT) network, produce force and translocate upon it. Secondly, they attach to specific intracellular components known as cargoes. In a pathological context, disruption of these transport functions can contribute to neurodegenerative disease. Motor transport functions are also usurped by bacteria and viruses to aid in their replication. We still know relatively little about the molecular basis of how these motors associate with their cargo or how this association is regulated. Similarly, we do not fully understand the range of cellular proteins and process that require the activity of a specific molecular motor in order to fulfil function. Kinesin‐1 (also known as conventional kinesin) is the best‐studied member of the kinesin superfamily with diverse roles in protein, ribonuclear protein, vesicular and organelle transport by virtue of its ability to interact with many cargoes. Kinesin‐1 is a tetramer composed of two heavy‐
chains (KHCs) and two light‐chains (KLCs). The KHCs contain an ATP‐driven motor domain used to move cargoes to the plus‐end of MTs and several coiled‐coil regions. In the inactive state KHCs are folded in an auto‐inhibited. In the active state inhibitory interactions are relieved resulting in a more elongated structure able to move along MTs. How kinesin‐1 is activated is not fully understood although it clearly occurs in response to cargo binding and, at least some cases, with the contribution of other regulatory factors. Until recently relatively little was also known about the molecular basis of cargo recognition. Cargoes can interact with KHCs and KLCs, both of which are encoded by multiple genes. We have recently solved the first crystal structure of a kinesin‐1:cargo complex providing the structural basis for the recognition process for cargoes containing tryptophan‐acid motif(s). We propose to build on that success to further our understanding of recognition and activation mechanisms. In particular, we intend to pursue a structural and functional investigation of kinesin‐1 1 KHCs as these chains are also actively involved in cargo recognition/transport. The work will involve the overexpression (in bacteria and insect cells) and purification of KHC protein domains for structural studies (X‐ray crystallography and SAXS). Our approach will also integrate biophysical techniques and the analysis of human cells in culture to obtain a full range of biological insights on kinesin‐mediated transport. The student will join the productive collaborative environment between the Steiner and Dodding groups and will be trained in various sought‐after techniques. Various constructs yielding soluble protein material are already available in the lab. Please indicate the type of programme 4 years 1+ 3 years (lab rotations) MRes + 3 years 2