Kinesin Regulation via Kinesin Binding Protein and Autoinhibition

Abstract

Cells are complex systems that continuously adapt and respond to their ever-changing environments. Integral to this adaptability is the precise organization and reorganization of cellular structures, among which the cytoskeleton plays a foundational role. The cytoskeleton provides structural integrity to the cell, but beyond that, it also serves as a network for motor proteins to transport cargo.

A key player in cargo transport is the kinesin superfamily of motor proteins. Kinesins participate in a diverse range of cellular processes, from facilitating 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 the kinesin-binding protein (KIFBP) revealed a novel form of kinesin inhibition, in which KIFBP binds to kinesin motor domains to block microtubule-binding.

Both autoinhibition and KIFBP-mediated inhibition are critical for proper cellular function. However, there remains little consensus and conflicting reports on the molecular mechanism of kinesin autoinhibition due to a lack of structural information on full-length kinesin motors. Moreover, the mechanism governing the KIFBP inhibition is poorly defined. In my Ph.D. research, I applied integrative structural analysis to determine how autoinhibition and KIFBP-mediated inhibition occurs at the molecular level.

My research project on kinesin-1 autoinhibition revised the established model in the field by offering the first comprehensive description of kinesin-1 autoinhibition (Tan et al. eLife 2023). Kinesin-1, discovered nearly 40 years ago, was the first identified kinesin and serves as a model for understanding kinesin function. A widely accepted model of kinesin-1 autoinhibition is that the C-terminal tail of kinesin-1 drives autoinhibition to block motility. However, published data suggested that the tail may not be the sole determinant of autoinhibition. To uncover the molecular mechanism of kinesin-1 autoinhibition, I combined AlphaFold protein structure prediction, electron microscopy and cross-linking mass spectrometry to reveal the molecular architecture of autoinhibited kinesin-1. My findings showed that multiple intramolecular contacts within the kinesin-1 lead to an inhibited state. My work provides a framework to understand why kinesin-1 activation requires cargo adaptors and microtubule-associated proteins to bind to and compete with intramolecular association to produce an open, active motor.

My second project aimed to investigate kinesin binding protein (KIFBP), a newly identified regulatory binding partner of kinesins. KIFBP exhibits the ability to associate and inhibit specific members of the kinesin superfamily. Mutations to KIFBP in humans have been linked to the neurological disorder Goldberg-Shprintzen (GOSHS) syndrome. Despite the clinical relevance of KIFBP, it remains unclear how KIFBP binds kinesins and how it achieves specificity for a subset of kinesins. To characterize the mechanism of kinesin regulation through KIFBP, I determined cryo-EM structures of KIFBP alone at 3.8Å and KIFBP bound to two different kinesin motors at 4.5-4.9Å. My structures provided the first atomistic view of kinesin engaged by KIFBP, showing that, KIFBP remodels and displaces the microtubule binding helix from kinesins to block microtubule binding. My work (Solon*, Tan*, et al. Science Advances 2021) paves the way for a deeper understanding of how KIFBP regulates its subset of kinesins, how it achieves specificity in binding, and how it contributes to GOSHS.