Biology subjects

Manka, S. W.

Publications and source records attributed to Manka, S. W..

8 recordsLinked to original sources

bioRxiv2026

STRUCTURAL PLASTICITY ALLOWS DOUBLECORTIN AND DRUG-STABILIZED MICROTUBULES TO ACCOMMODATE EACH OTHER

Microtubule architecture and dynamics are modulated by small molecules, but how these compounds alter lattice properties and interactions with cellular regulators remains incompletely understood. We used the neuronal microtubule-associated protein doublecortin (DCX), previously reported to preferentially recognize 13-protofilament microtubules, to probe lattices stabilized by different taxanes, including the fluorescent taxane Fchitax-3. We show that DCX binds 15-protofilament Fchitax-3-stabilized microtubules, demonstrating that DCX is not a specific marker of 13-protofilament architecture. Cryo-electron microscopy revealed that the fluorescent moiety of Fchitax-3 inserts into inter-protofilament lateral contacts, explaining its effects on microtubule architecture and dynamics. DCX binds Fchitax-3 microtubules through its N-terminal DC domain, which accommodates the narrower inter-protofilament binding site in the 15-protofilament lattice. Remarkably, although Fchitax-3 induces the longitudinal lattice expansion characteristic of taxanes, DCX remodels its binding site towards its preferred compacted configuration. Thus, taxanes can modify microtubule structure in distinct ways while preserving lattice plasticity and the capacity to undergo conformational remodelling by microtubule-associated proteins. This contrasts with the comparatively rigid lattice of GMPCPP-stabilized microtubules. Consequently, interpretations of microtubule lattice states based on comparisons between taxane- and GMPCPP-stabilized microtubules may conflate structural differences with different degrees of lattice plasticity. Our findings establish the microtubule lattice as a malleable structural substrate whose drug-induced conformations can be further remodelled by cellular binding partners.

biophysics↗

bioRxiv2022

Prion propagation is dependent on key amino acids in Charge cluster 2 within the prion protein

Prions consist of assemblies of aberrantly folded cellular prion protein (PrPC) upon template-assisted conversion and propagation of disease-associated PrP. To dissect the N-terminal residues critical for efficient prion propagation, we generated a library of point, double, or triple alanine replacements within residues 23-111 of PrP, stably expressed them in cells silenced for endogenous mouse PrPC and challenged the reconstituted cells with four mouse prion strains. Amino acids (aa) 105-111 of Charge Cluster 2 (CC2), which is disordered in PrPC, were required for propagation of all four prion strains; other residues had no effect or exhibited strain-specific effects. Replacements in CC2, including aa105-111, dominantly inhibited prion propagation in the presence of endogenous wild type PrPC whilst other changes were not inhibitory. Single alanine replacements within aa105-111 identified leucine 108 and valine 111 or the cluster of lysine 105, threonine 106 and asparagine 107 as critical for prion propagation. These residues mediate specific ordering of CC2 in the prion fibrils from Rocky Mountain Laboratory (RML) and ME7 mouse prion strains.

molecular biology↗

bioRxiv2022

A structural basis for prion strain diversity

Recent cryo-EM studies of infectious, ex vivo, prion fibrils from hamster 263K and mouse RML prion strains revealed a comparable, parallel in-register intermolecular {beta}-sheet (PIRIBS) amyloid architecture. Rungs of the fibrils are composed of individual prion protein (PrP) monomers that fold to create distinct N- and C-terminal lobes. However, disparity in the hamster/mouse PrP sequence precludes understanding how divergent prion strains emerge from an identical PrP substrate. Here, we determined the near-atomic resolution cryo-EM structure of infectious, ex vivo mouse prion fibrils from the ME7 prion strain and have compared this with the RML fibril structure. This structural comparison of two biologically distinct mouse-adapted prion strains suggests defined folding sub-domains of PrP rungs and the way in which they are interrelated, providing the first structural definition of intra-species prion strain-specific conformations.

biochemistry↗

bioRxiv2021

2.7 A cryo-EM structure of ex vivo RML prion fibrils

Mammalian prions are lethal infectious agents that propagate as distinct strains and are composed of multichain assemblies of misfolded host-encoded prion protein (PrP), often referred to as prion rods. The structural features that define infectious prion rods and the molecular determinants of prion strain diversity are poorly understood. Here, we present a near-atomic resolution cryo-EM structure of PrP fibrils present in highly infectious prion rod preparations isolated from the brains of RML prion-infected mice. We found that prion rods comprise single-protofilament helical amyloid fibrils that coexist with twisted pairs of the same protofilaments. Each rung of the protofilament is formed by a single PrP monomer with the ordered core comprising PrP residues 94-225, which folds to create two asymmetric lobes with the N-linked glycans and the glycosylphosphatidylinositol anchor projecting from the C-terminal lobe. The overall architecture is comparable to that of recently reported PrP fibrils isolated from the brain of hamsters infected with the 263K prion strain. However, there are marked conformational variations that could result from differences in PrP primary sequence and/or represent distinguishing features of the distinct prion strains. These conformational changes impact the overall geometry of the fibrils and may also impact fibril pairing, one or both of which may critically influence PrP glycoform selection that occurs during strain-specific prion propagation.

biochemistry↗

bioRxiv2020

Thermodynamic and mechanistic insights into coupled binding and unwinding of collagen by matrix metalloproteinase 1

Local unwinding of the collagen triple helix is a necessary step for initiating the collagen degradation cascade in extracellular matrices. A few matrix metalloproteinases (MMPs) are known to support this key process, but its energetic aspects remain unknown. Here, we captured the thermodynamics of the triple helix unwinding by monitoring interactions between a collagen peptide and MMP-1(E200A) - an active-site mutant of an archetypal vertebrate collagenase - at increasing temperatures, using isothermal titration calorimetry (ITC). Coupled binding and unwinding manifests as a curved relationship between the total enthalpy change and temperature of the reaction, producing increasingly negative heat capacity change ({Delta}{Delta}Cp {approx} -36.3 kcal/molK2). A specially designed solid-phase binding and cleavage assay (SPBCA) reported strain in the catalytically relevant unwound state, suggesting that this state is distinct from the horizon of sampled conformations of the collagenase-susceptible site. MMP-1 appears to blend selected fit with induced fit mechanisms to catalyse collagen unwinding prior to cleavage of individual collagen chains. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/272104v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@15e6c94org.highwire.dtl.DTLVardef@2a9c8aorg.highwire.dtl.DTLVardef@18b3571org.highwire.dtl.DTLVardef@61c0c5_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIThe unwinding of the collagen triple helix by matrix metalloproteinases (MMPs) is critical for collagen catabolism, but how MMPs harness bioavailable energy for this energetically expensive process is enigmatic C_LIO_LIRising temperature causes linear increases of the negative heat capacity change associated with the interaction of MMP-1 with a collagenase-susceptible triple-helical peptide, indicating enrichment of hydrophobic contacts during the triple helix unwinding C_LIO_LIThe complex of MMP-1 with locally unwound collagen holds considerable structural strain, which becomes relieved upon cleavage of the unwound collagen strand(s) C_LIO_LIMMP-1 consumes heat to drive specific (structurally distinct) collagen unwinding by increasing entropy associated with evolving hydrophobic anchor points between the enzyme and the substrate C_LIO_LIThe prototypic collagenase MMP-1 appears to blend conformational selection with induced fit, shedding light on the thermodynamic principles by which MMPs trigger collagen breakdown and supporting general mechanistic conclusions concerning conformational changes coupled to protein-protein binding C_LI

biophysics↗

bioRxiv2019

Pseudo-repeats in doublecortin make distinct mechanistic contributions to microtubule regulation

Doublecortin (DCX) is a neuronal microtubule-associated protein (MAP) indispensable for brain development. Its flexibly linked DC domains - NDC and CDC - mediate microtubule (MT) nucleation and stabilisation, but it is unclear how. Using high-resolution time-resolved cryo-EM, we mapped NDC and CDC interactions with tubulin at different MT polymerisation stages and studied their functional effects on MT dynamics using TIRF microscopy. Although coupled, each DC repeat appears to have a distinct role in MT nucleation and stabilisation by DCX: CDC is a conformationally plastic tubulin binding module that appears to facilitate MT nucleation by binding tubulin oligomers and stabilising tubulin-tubulin contacts in the nascent MT lattice, while NDC appears to be favoured along the mature lattice, providing enhanced and durable MT stabilisation. Our near-atomic resolution structures of MT-bound DC domains also explain in unprecedented detail the DCX mutation-related brain defects observed in the clinic. This modular composition of DCX reflects a common design principle among MAPs where pseudo-repeats of tubulin/MT binding elements chaperone or stabilise distinct conformational transitions to regulate distinct stages of MT dynamic instability.

biophysics↗

bioRxiv2019

Structural studies of MMP-3 interaction with triple-helical collagen introduce the enzyme’s new roles in tissue remodelling

Matrix metalloproteinase-3 (MMP-3 or stromelysin 1) participates in normal extracellular matrix (ECM) turnover during embryonic development, organ morphogenesis and wound healing, and in tissue-destructive diseases, such as aneurysm, cancer, arthritis and heart failure. Despite its ability to hydrolyse numerous proteins in the ECM, MMP-3 fails to cleave the triple helix of interstitial fibrillar collagens. Nonetheless, it can still bind to these collagens although the mechanism, location and role of binding are not known. We used the Collagen Toolkits, libraries of triple-helical peptides that embrace the entire helical domains of collagens II and III, to map MMP-3 interaction sites. The enzyme recognises five sites on collagen II and three sites on collagen III. They share a glycine-phenylalanine-hydroxyproline/alanine (GFO/A) motif that is recognised by the enzyme in a context-dependent manner. Neither MMP-3 zymogen (proMMP-3) nor the individual catalytic (Cat) and hemopexin (Hpx) domains of MMP-3 interact with the peptides, revealing cooperative binding of both domains to the triple helix. The Toolkit binding data combined with molecular modelling enabled us to deduce the putative collagen-binding mode of MMP-3, where all three collagen chains make contacts with the enzyme in the valley running across both Cat and Hpx domains. The observed binding pattern casts light on how MMP-3 could regulate collagen turnover and compete with various collagen-binding proteins regulating cell adhesion and proliferation.

biochemistry↗

bioRxiv2019

A microtubule RELION-based pipeline for cryo-EM image processing

Microtubules are polar filaments built from {beta}-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of - and {beta}-tubulin. The determination of the {beta}-tubulin register and seam location during image processing is essential for alignment accuracy that enables determination of biologically relevant structures. Here we present a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Microtubule RELION-based Pipeline (MiRP). The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing near-atomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins.\n\nAbbreviationsMiRP, Microtubule RELION-based Pipeline; cryo-EM, cryo-electron microscopy; MT, microtubule; CTF, contrast transfer function; PF, protofilament.

molecular biology↗