SAXS STUDY OF REGULATION OF THE KINESIN-1 MOTOR BY THE KINESIN LIGHT CHAINS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Kinesin-1 is a ubiquitous motor protein that hydrolyzes ATP to drive the transport of intracellular cargo. It is composed of two heavy chains (KHCs) and two light chains (KLCs). Proper regulation of kinesin-1 prevents its mislocalization and allows for coordination with other molecular motors. Regulated kinesin-1 is folded over on itself so that the KLCs and regulatory KHC tail domains come in contact with the enzymatically active KHC heads. This KHC/KLC complex is central to the regulatory mechanism of kinesin-1, yet the structure remains undetermined because the complex is not amenable to traditional structure determination techniques. To overcome this, we used small-angle X-ray scattering (SAXS) to visualize the never-before-seen structure of a regulated kinesin-1 holoenzyme in solution at ~3nm resolution. The regulated conformation of kinesin-1 appears to be a compact and rigid structure, therefore we expect to obtain valuable information about its molecular shape from SAXS. We will use the SAXS structure to determine where the various kinesin-1 elements interact with each other in this regulated complex. Since our previous annual report, we have successfully collected the sample data and concluded our experiments. The raw data is currently being processed and analyzed, and we expect the final model to provide significant insight into the nature of kinesin-1 regulation. We were also able to extend our original studies by performing SAXS on complexes of head and tail fragments. The smaller size of these fragments allowed us to achieve higher SAXS resolution compared to the full-length molecule.