Peptide disruptors and facilitators of protein-protein interactions for mediating intracellular transport

• Main Author: Cross, Jessica A. A
• Format: Dissertation
• Language: English
• Published: University of Bristol 2022

Kinesin-1 is an ATP-dependent motor protein that plays a key role in the spatial and temporal organisation of the cell by the anterograde transport of a diverse range of cargo including proteins and membrane bound organelles on microtubules. The motor complex is a heterotetramer of two heavy chains (KHCs) and two light chains (KLCs), built on a dimeric KHC coiled-coil scaffold. Understanding the structure and mechanisms of activation of this protein is important to gain insights into the normal and pathological roles it plays in intracellular transport. In addition, the development of new strategies to manipulate kinesin-1 and other motor proteins could allow us to target transport systems in human disease. To date, models of cargo transport have predominantly focussed on motor-microtubule interactions. The work described in this thesis sought to use peptide-design approaches to explore the mechanisms of cargo attachment and how this is linked to a transition from a regulated, autoinhibited state to an active form capable of motility on the cytoskeleton. In Chapter 3, a new structure-guided, fragment-linking, peptide-design approach is developed to deliver a high-affinity peptide ligand that targets the kinesin-1:cargo interface through binding to tetratricopeptide repeat (TPR) domains of KLCs. In Chapter 4, the de novo peptide, KinTag, is shown to be effective in hijacking the transport function of endogenous kinesin motors in cells and can deliver cargo into axons of primary neurons with a high efficiency that correlates with its enhanced binding affinity. This demonstrates for the first time that the more tightly the motor holds onto its cargo adaptor, the more efficiently the cargo is transported. To enable inducible manipulation of transport in the cell, a de novo cell-penetrating peptide tag is described that delivers KinTag to the cytosol and dimerizes with a genetically encoded bait sequence, localized to an organelle of choice. In Chapter 5, to understand better how conformational changes linked to cargo-binding in the KLCs are coupled to downstream activation of the motor domains, the interaction between the heavy and light chains in the tetrameric motor is investigated. The KHC-KLC interface is formed by a short region within the coiled-coil stalk of the KHCs and a purported coiled-coil region near the N terminus of the KLCs. This interaction is required for the autoinhibition and activity of kinesin-1, however, the structure and organisation remains unresolved. Solution-phase biophysical characterisation alongside model building and X-ray crystallography is employed and reveals the KHC coils as a dynamic platform for protein-protein interactions. In addition to its role in organelle transport, kinesin-1 performs a function in sliding pairs of microtubules relative to one another for regulation of the microtubule network. In Chapter 6, the organelle transport and microtubule remodelling activities are linked in a shared, acidification-induced activation mechanism. Mutations in full-length proteins in cells reveal that a Glu residue in the core of the KHC-KLC interface may play an important role in the pH sensing capacity of kinesin-1. This begins to develop a model for the long-unexplained pH dependence of kinesin-1 activity, opening the door for future studies using peptide design to target this interface.