Biology subjects

Dodding, M. P.

Publications and source records attributed to Dodding, M. P..

7 recordsLinked to original sources

bioRxiv2026

Kinase-gated coincidence detection controls kinesin-driven lysosome transport

Kinesin motors drive long-range intracellular transport through coordinated cargo recognition and conformational autoregulation, yet the mechanisms that selectively control cargo engagement remain unclear. Here, we identify a phosphorylation-dependent gate on kinesin-1 activity mediated by the carboxy-terminal domain (CTD) of kinesin light chain 2 (KLC2). The KLC2 CTD is constitutively phosphorylated on multiple serine residues, suppressing membrane association via its amphipathic helix. We identify the NIMA-related kinase NEK10 as a KLC2-selective regulator of kinesin-1, restraining motor activation, cargo engagement, and lysosome transport; conversely, loss of NEK10 increases membrane association and, together with low-affinity adaptor interactions, promotes lysosome motility. These findings reveal a phosphorylation-regulated protein-lipid coincidence-detection mechanism - a kinesin-kinase code - that integrates adaptor binding with membrane cues to control kinesin-1-mediated transport and provide a mechanistic basis for understanding paralogue and isoform diversity in the kinesin-1 family.

cell biology↗

bioRxiv2025

Molecular mechanism for cargo-mediated initiation of kinesin-1 activation

Kinesin-1 is a dynamic heterotetrameric assembly of two heavy and two light chains (KHC and KLC) that mediates microtubule-based intracellular transport of many different cargoes. The complex adopts a compact, autoinhibited state that is activated by cargo-adaptor proteins containing specific short linear peptide motifs (SLiMs). These motifs interact with the tetratricopeptide repeat (TPR) domains of the KLCs. The mechanism coupling SLiM recognition to activation-associated conformational changes in the complex is unknown. Here we combine protein design, computational modelling, biophysical analysis, and electron microscopy to examine the structural and mechanistic consequences of SLiM binding to the KLC-TPR domain within the complete heterotetrameric holoenzyme. We show that coiled coil 1 (CC1) of the KHC docks KLC TPR domains in the autoinhibited complex, forming the shoulder feature observed in electron microscopy. Disrupting this interaction or binding an activating SLiM dislocates the TPR shoulder, freeing the motor domains and promoting transition between its closed, inactive, and open states. Opening the kinesin-1 complex facilitates binding to the microtubule-associated kinesin-1 cofactor, MAP7. Therefore, cargo-mediated dislocation of the TPR shoulder serves as a key initial step in kinesin-1 activation, allosterically linking cargo binding to motor dynamics.

biochemistry↗

bioRxiv2023

Allosteric regulation of a molecular motor through de novo protein design

Many enzymes are allosterically regulated. Our ability to manipulate these structural changes is limited. Here we install an allosteric switch into the kinesin-1 microtubule motor in vitro and in cells. Kinesin-1 is a heterotetramer that accesses open active and closed auto-inhibited states. The equilibrium between these centres on a flexible elbow within a complex coiled-coil architecture. We target the elbow to engineer a closed state that can be opened with a de novo designed peptide. The alternative states are modelled computationally and confirmed by biophysical measurements and electron microscopy. In cells, peptide-driven activation increases kinesin transport, demonstrating a primary role for conformational switching in regulating motor activity. The designs are enabled by our understanding of ubiquitous coiled-coil structures, opening possibilities for controlling other protein activities. One Sentence SummaryDe novo peptide and protein design are used to engineer an allosteric switch into kinesin-1 motors in vitro and directly in cells.

synthetic biology↗

bioRxiv2022

Molecular architecture of the autoinhibited kinesin-1 lambda particle

Despite continuing progress in kinesin enzyme mechanochemistry and emerging understanding of the cargo recognition machinery, it is not known how these functions are coupled and controlled by the alpha-helical coiled coils encoded by a large component of kinesin protein sequences. Here, we combine computational structure prediction with single-particle negative stain electron microscopy to reveal the coiled-coil architecture of heterotetrameric kinesin-1, in its compact state. An unusual flexion in the scaffold enables folding of the complex, bringing the kinesin heavy chain-light chain interface into close apposition with a tetrameric assembly formed from the region of the molecule previously assumed to be the folding hinge. This framework for autoinhibition is required to uncover how engagement of cargo and other regulatory factors drive kinesin-1 activation. Summary statementIntegration of computational structure prediction with electron microscopy reveals the coiled-coil architecture of the autoinhibited compact conformer of the microtubule motor, kinesin-1.

cell biology↗

bioRxiv2021

Molecular mechanism for kinesin-1 direct membrane recognition

The cargo-binding capabilities of cytoskeletal motor proteins have expanded during evolution through both gene duplication and alternative splicing. For the light chains of the kinesin-1 family of microtubule motors, this has resulted in an array of carboxy-terminal domain sequences of unknown molecular function. Here, combining phylogenetic analyses with biophysical, biochemical and cell biology approaches we identify a highly conserved membrane-induced curvature-sensitive amphipathic helix within this region of a newly defined subset of long kinesin light chain paralogues and splice isoforms. This helix mediates the direct binding of kinesin-1 to lipid membranes. Membrane binding requires specific anionic phospholipids and is important for kinesin-1 dependent lysosome positioning, a canonical activity that until now has been attributed exclusively the recognition of organelle-associated cargo adaptor proteins. This leads us to propose a new protein-lipid coincidence detection framework for kinesin-1 mediated organelle transport.

cell biology↗

bioRxiv2019

In situ cryo-electron tomography reveals filamentous actin within the microtubule lumen

Microtubules and filamentous (F-) actin engage in complex interactions to drive many cellular processes from subcellular organisation to cell division and migration. This is thought to be largely controlled by proteins that interface between the two structurally distinct cytoskeletal components. Here, we use cryo-electron tomography to demonstrate that the microtubule lumen can be occupied by extended segments of F-actin in small-molecule induced, microtubule-based cellular projections. We uncover an unexpected versatility in cytoskeletal form that may prompt a significant development of our current models of cellular architecture and offer a new experimental approach for the in-situ study of microtubule structure and contents.

cell biology↗

bioRxiv2018

Structural basis for isoform-specific kinesin-1 recognition of Y-acidic cargo adaptors

The light chains (KLCs) of the heterotetrameric microtubule motor kinesin-1, that bind to cargo adaptor proteins and regulate its activity, have a capacity to recognize short peptides via their tetratricopeptide repeat domains (KLCTPR). Here, using X-ray crystallography, we show how kinesin-1 recognizes a novel class of adaptor motifs that we call Y-acidic (tyrosine flanked by acidic residues), in a KLC-isoform specific manner. Binding specificities of Y-acidic motifs (present in JIP1 and in TorsinA) to KLC1TPR are distinct from those utilized for the recognition of W-acidic motifs found in adaptors that are KLC-isoform non-selective. However, a partial overlap on their receptor binding sites implies that adaptors relying on Y-acidic and W-acidic motifs must act independently. We propose a model to explain why these two classes of motifs that bind to the concave surface of KLCTPR with similar low micromolar affinity can exhibit different capacities to promote kinesin-1 activity.

biochemistry↗