Atomic-resolution structure of kinesin-1 motor domain in complex with microtubules by magic angle spinning NMR spectroscopy
Date
2021
Authors
Journal Title
Journal ISSN
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Publisher
University of Delaware
Abstract
Microtubules (MTs) and their associated proteins play essential roles in maintaining cell structure, organelle transport, cell motility and cell division. Critical in linking cargo and the MT network are two motor families, kinesin and cytoplasmic dynein. Members of both families utilize ATP hydrolysis to produce the force needed for cellular polarization. While numerous studies have produced important information and led to viable models to explain their preference for traveling a specific direction, there remains a number of questions how hydrolysis of one motor subunit affects the activity of the second subunit. Kinesin superfamily of motor proteins walk along MTs primarily towards the plus end to transport cargos using the energy of ATP hydrolysis. Based on the currently proposed mechanisms of processive movement for kinesin on MTs, three major kinesin states have been identified, namely apo-state (nucleotide free), ADP-state and ATP-state. ☐ This thesis focuses on the structural analysis of apo-state kinesin-1 motor domain (apo-KIF5B) in complex with paclitaxel-stabilized polymeric microtubules (MTs) by magic angle spinning (MAS) NMR spectroscopy. The main challenge of this work is the intrinsically low sensitivity caused by the low weight fraction of the isotopically labeled protein in the MAS NMR rotor. To determine the atomic-resolution structure of KIF5B in complex with polymerized MTs, we had to develop unique MAS NMR experiments, structure calculation, and sample preparation protocols, as well as employ the novel CPMAS CryoProbe technology developed by our collaborators from Bruker BioSpin. ☐ The thesis is organized as follows. ☐ In Chapter 1, we first introduce the structure and dynamics of MTs. Then we introduce the current proposed mechanisms of microtubule-associated proteins (MAPs)’ walk on MTs and the current structure models of MAPs, with a special focus on kinesin. ☐ In Chapter 2, we discuss protein structure determination by MAS NMR. Strategies and methods of different steps during the structure determination process, as well as methods for sensitivity and resolution enhancement by MAS NMR are introduced. ☐ In Chapter 3, we present the experimental results on KIF5B/MT assemblies acquired with the novel CPMAS CryoProbe. Compared to Efree probe, CPMAS CryoProbe has a sensitivity enhancement of 2- to 4- fold for 2D experiments and even 6- to 7- fold for 3D heteronuclear experiments. The newly collected dataset of KIF5B/MT assemblies allows the increasing completeness of backbone assignments as well as distance restraints to a large extent. ☐ In Chapter 4, we present the first atomic-resolution structure of apo-state kinesin-1 motor domain (apo-KIF5B) in complex with paclitaxel-stabilized polymeric microtubules using magic-angle-spinning (MAS) NMR spectroscopy. The structure derived from these measurements defines position and conformation of the functionally important neck linker. Moreover, pronounced conformational and dynamic changes are observed in the nucleotide binding motif and neck linker regions upon ADP binding. Critically, these atomic-level data provide unique structural insight into how information is transmitted between each motor domain, enabling the complex to generate force and transport cargo. Remarkably, the structure reveals the conformation of the neck linker in the undocked position that is missing in previous studies, presumably due to conformational flexibility. Pronounced structural and dynamic changes were observed in the apo- and ADP- states of KIF5B bound to microtubules. Broadly, this study underscores the power of integrating MAS NMR with medium-resolution cryo-EM maps in deriving atomic-resolution structures of microtubule-associated proteins bound with polymerized MTs.
Description
Keywords
Integrated structural biology, Kinesin-1 motor domain, Magic-angle spinning NMR, Microtubules