Biology subjects

Woolfson, D.

Publications and source records attributed to Woolfson, D..

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

Coiled-coil scaffolds for Targeted Protein Degradation

In targeted protein degradation (TPD) the cells quality control machinery is co-opted to degrade proteins of interest. Currently TPD is limited by the availability of small-molecule binders for targets and the many potential human E3 ubiquitin ligases. Consequently, other approaches are needed to tackle new targets and to exploit degradation pathways fully. The natural binding epitopes for E3s and other degradation machineries are often short linear peptide motifs, which offer routes to new biologics-based degraders. Here, targeting the anti-apoptotic BCL-xL and initiation of apoptosis, we show that several protein-degradation pathways--ubiquitination, direct proteasome, autophagy-lysosome recruitment--can be leveraged by integrating target-binding and degradation motifs into single-polypeptide constructs. This exploits versatile and varied de novo coiled-coil scaffolds as adaptable molecular glues by presenting motifs multivalently and in diverse arrangements. The constructs are small ({approx}80 residues) and provide a plug-and-play platform to map and optimise target-degradation space using rational design. We call this Coiled Coils for induced Proximity (CoCoProx) and propose its use for targeted degradation and other induced-proximity reactions.

synthetic biology↗

bioRxiv2025

Tumbleweed: an artificial motor protein that walks along a DNA track

Summary ParagraphMolecular motors are fundamental to life1-6 because of their ability to convert chemical energy into mechanical work, an ability that is conferred by the chemical and structural complexity of their constituent proteins. Scientists have long sought to create artificial protein motors that may reveal insights into how biological motors function. While artificial molecular motors based on small molecules7 and DNA8,9 have been developed, creating an artificial motor protein has remained an elusive goal in synthetic biology10. Here we demonstrate the realization of an artificial protein motor called Tumbleweed (TW) that walks directionally along a DNA track under external control. TW consists of three legs, each with a ligand-gated DNA-binding domain that enables selective interaction with specific sites along a DNA track11. Using single-molecule fluorescence assays and a programmable microfluidic device, we show that TW steps directionally along a designed DNA track in response to a defined sequence of ligand inputs. We built our TW molecular walker using a modular approach, combining existing proteins with known properties to achieve emergent motor function, similar to how Nature evolves new proteins. Our design strategy thus offers a a platform for engineering advanced and dynamic protein functionality. Our demonstration of TW walking represents a step toward developing fully autonomous protein motors and opens new avenues for uncovering and leveraging the principles by which biological motors transduce chemical energy into motion.

biophysics↗

bioRxiv2023

Structural flexibility dominates over binding strength for supramolecular crystallinity

Supramolecular crystallinity is abundantly present in nature and results from directional, weak non-covalent interactions between components. Bottom-up nanotechnology aims to exploit such phenomena to control the self-assembly of ordered networks and complex objects from rationally designed monomers. Like all crystalline materials, 2D supramolecular crystals develop from an initial nucleation site, followed by growth, based on directional interactions. Traditionally, the binding strength and directionality of interactions is thought to dictate the nucleation and crystal growth, whereas structural flexibility favours defects. Usually, macromonomers present multiple binding units with relative intramolecular flexibility that affects their intermolecular interactions. Thus far, the effects of such flexibility on supramolecular assembly have not been explored. Here we introduce the concept of "interface flexibility" and demonstrate its critical importance in the nucleation and growth of supramolecular crystalline networks. We show that tuning the interface flexibility greatly expands the available design space for synthetic supramolecular crystalline materials.

bioengineering↗

bioRxiv2023

Rationally seeded computational protein design

Computational protein design is advancing rapidly. Here we describe efficient routes to two families of -helical-barrel proteins with central channels that bind small molecules. The designs are seeded by the sequences and structures of defined de novo oligomeric barrel-forming peptides. Adjacent helices are connected using computational loop building. For targets with antiparallel helices, short loops are sufficient. However, targets with parallel helices require longer connectors; namely, an outer layer of helix-turn-helix-turn-helix motifs that are packed onto the barrels computationally. Throughout these pipelines, residues that define open states of the barrels are maintained. This minimises sequence sampling and accelerates routes to successful designs. For each of 6 targets, just 2 - 6 synthetic genes are made for expression in E. coli. On average, 80% express to give soluble monomeric proteins that are characterized fully, including high-resolution structures for most targets that match the seed structures and design models with high accuracy.

synthetic biology↗

bioRxiv2023

Labile assembly of a tardigrade protein induces biostasis

Tardigrades are microscopic animals that survive desiccation by inducing biostasis. To survive drying tardigrades rely on intrinsically disordered CAHS proteins that form gels. However, the sequence features and mechanisms underlying gel formation and the necessity of gelation for protection have not been demonstrated. Here we report a mechanism of gelation for CAHS D similar to that of intermediate filaments. We show that gelation restricts molecular motion, immobilizing and protecting labile material from the harmful effects of drying. In vivo, we observe that CAHS D forms fiber-like condensates during osmotic stress. Condensation of CAHS D improves survival of osmotically shocked cells through at least two mechanisms: reduction of cell volume change and reduction of metabolic activity. Importantly, condensation of CAHS D is reversible and metabolic rates return to control levels after CAHS condensates are resolved. This work provides insights into how tardigrades induce biostasis through the self-assembly of CAHS gels.

physiology↗

bioRxiv2023

CC + : A Searchable Database of Validated Coiled coils in PDB Structures and AlphaFold2 Models

-Helical coiled coils are common tertiary and quaternary elements of protein structure. In coiled coils, two or more helices wrapped around each other to form bundles. This apparently simple structural motif can generate many architectures and topologies. Understanding the variety of and limits on coiled-coil assemblies and their sequence-to-structure relationships impacts on protein structure, design, and engineering. Coiled coil-forming sequences can be predicted from heptad repeats of hydrophobic and polar residues, hpphppp, although this is not always reliable. Alternatively, coiled-coil structures can be identified using the program SOCKET, which finds knobs-into-holes (KIH) packing between side chains of neighboring helices. SOCKET also classifies coiled-coil architecture and topology, thus allowing sequence-to-structure relationships to be garnered. In 2009, we used SOCKET to create a relational database of coiled-coil structures, CC+, from the RCSB Protein Data Bank (PDB). Here we report an update of CC+ following the recent explosion of structural data and the success of AlphaFold2 in predicting protein structures from genome sequences. With the most-stringent SOCKET parameters, CC+ contains {approx}12,000 coiled-coil assemblies from experimentally determined structures, and {approx}120,000 potential coiled-coil structures within single-chain models predicted by AlphaFold2 across 48 proteomes. CC+ allows these and other less-stringently defined coiled coils to be searched at various levels of structure, sequence, and side-chain interactions. The identified coiled coils can be viewed directly from CC+ using the Socket2 application, and their associated data can be downloaded for further analyses. CC+ is available freely at http://coiledcoils.chm.bris.ac.uk/CCPlus/Home.html. It will be regularly updated automatically. FOR THE BROADER AUDIENCEProtein assemblies and protein-protein interactions are key to all biological processes. -Helical coiled coils are one of the most common modes of directing and stabilising these interfaces. Here, we report an updated CC+ database of structurally validated coiled coils from experimental protein structures and AlphaFold2 models. CC+ contains many thousands of coiled-coil structures and models, associated parameters, and sequences. It enables the compilation of rich datasets for advancing protein structure, design, and engineering research.

bioinformatics↗