MECHANISMS OF KINESIN MOTOR PROTEIN INHIBITION SUMMARY The spatiotemporal regulation of organelle positioning is critical for proper cellular function, especially as the cell responds to a changing environment. The kinesin superfamily of motor proteins is responsible for various cellular processes that range from long-range axonal transport to orchestrating the mitotic spindle during cell division. The regulation of kinesin motor proteins occurs via inhibitory and activation-based mechanisms, where kinesin motor proteins are subject to autoinhibition when not bound to a cargo. Recently, the discovery of kinesin-binding protein (KIFBP) revealed a novel form of kinesin inhibition whereby KIFBP binds to kinesin motor domains to block microtubule-binding. In this grant, we will determine the molecular basis for KIFBP-mediated kinesin inhibition in trans and how kinesin light chains in cis-lead to kinesin inhibition. Based on structural studies of KIFBP bound to two different kinesin motor domains, we developed a model of how KIFBP remodels kinesin motors and how KIFBP selectively engages motors. We will introduce site-specific mutagenesis based on crosslinking mass spectrometry and patient-derived mutants to dissect kinesin binding and remodeling by KIFBP (Aim 1). In parallel, we will determine how light chains regulate full-length kinesin motor proteins via autoinhibition. Despite decades of work into the regulation of full-length kinesin motor proteins, there remain conflicting results regarding inhibition of kinesin motor domains. We are poised to answer this question by exploiting a combination of crosslinking mass spectrometry, protein engineering, and cryo-EM to determine how kinesin light chains stabilize a compact, inhibited kinesin motor (Aim 2). We will study both kinesin-1 heterotetramers (KIF5B:KLC1) in addition to kinesin-2 heterotrimers (KIF3A:3B:KAP) to compare and contrast how these different kinesin motor complexes are autoinhibited. Taken together, this work will expand our understanding of kinesin regulation, establishing modes of inhibition to provide a complete view of kinesin activity.