Biology subjects

Stubbs, M. T.

Publications and source records attributed to Stubbs, M. T..

7 recordsLinked to original sources

bioRxiv2026

Structural basis of Arf1-driven membrane tubulation

Membrane tubules form at Golgi compartments to facilitate membrane and cargo flow in intracellular trafficking. Here we show that the small GTPase Arf1, an inducer of membrane curvature and key regulator of trafficking, is able to form strongly curved tubules in the presence of lipids and GTP{gamma}S in vitro without the need for further coat components. Using cryo-electron microscopy, we determined the structures of tubular Arf1-scaffolds with diameters of 195 and 215 [A] at 3.1 and 3.8 [A] resolutions, respectively. The nucleotide-bound globular domains of Arf1 form polar helical lattices (i.e. directional assemblies with distinct start/finish orientations), with conserved interfaces and a consistent back-to-face orientation along the filaments. The rigid coat is tethered to the membrane by a flexible linker and anchored by an amphipathic helix (AH) that is free to diffuse and make space within the leaflet, allowing for accommodation of transmembrane cargo. The diversity of tubular diameters observed would allow various cargo sizes to be accommodated in the lumen, while maintaining the local coat architecture. Apart from serving as tubular transport intermediates, Arf1-scaffolds may also play a role at the neck of COPI vesicle on the route to scission.

biophysics↗

bioRxiv2025

A flexible Janus head: molecular determinants of a viral protein's RNAi suppressor and capsid forming activities

Viral suppressors of RNA silencing (RNAi) expressed by plant viruses, VSRs, are exceptional proteins. Although not conserved, even within virus families, most VSRs bind small interfering RNAs, siRNAs, thereby blocking antiviral RNAi. Turnip crinkle virus, a member of the Tombusviridae family, encodes a VSR, TCV P38, which also forms the viral capsid. Biochemical studies of the purified protein revealed that the TCV P38 VSR functions as a metastable dimer that binds double-stranded (ds) RNA with high affinity via an induced-fit mechanism of both binding partners. Consistent with its role as a VSR that interferes with antiviral RNAi at various stages, P38 distinguishes between siRNAs of different lengths. Consistent with its capsid-forming function, the protein binds longer dsRNAs cooperatively. Structural data obtained from an RNA-free capsid-like icosahedral crystal and modeling of Tombusviridae capsid proteins suggest that flexible interactions between the P (protruding)-domains of P38 are important determinants for forming both the VSR dimer and the capsid structure. Studies with protein mutants confirmed this and also revealed the central role of the S (shell)- and R (RNA-binding)-domains of TCV P38 in adaptive substrate binding. Our study provides comprehensive insights into the molecular and structural properties of a versatile viral "Janus head" protein. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/690650v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@185f6b1org.highwire.dtl.DTLVardef@6e773org.highwire.dtl.DTLVardef@1456981org.highwire.dtl.DTLVardef@11a63e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Strategies for Mitigating Radiation Damage and Improving Data Completeness in 3D Electron Diffraction of Protein Crystals

While 3D electron diffraction (3D-ED or microcrystal electron diffraction, MicroED) has emerged as a promising method for protein structure determination, its applicability is hindered by high susceptibility to radiation damage, leading to decreasing signal-to-noise ratio in consecutive diffraction patterns that limits the quality (resolution and redundancy) of the data. In addition, data completeness may be restricted due to the geometrical limitations of current sample holders and stages. In this work, we introduce an approach that addresses these issues using a commonly available 200keV cryo-electron microscope. The multi-position acquisition technique we present here combines (a) multiple data acquisitions from a single crystal over several tilt ranges and (b) merging data from a small number of crystals, each tilted about a different axis. The robustness of this approach is demonstrated by the de novo elucidation of a protein-peptide complex structure from only two orthorhombic microcrystals.

biochemistry↗

bioRxiv2024

Mechanism of SHP2 activation by bis-Tyr-phosphorylated Gab1

The non-receptor tyrosine phosphatase SHP2 (PTPN11) is a regulator of diverse cellular functions including mitogenic activation and cell migration. SHP2 consists of two tandem SH2 domains followed by the catalytic domain, and is autoinhibited by the N-terminal SH2 domain that blocks access to the active site. Mutations that influence auto-inhibition have been implicated in cancer and other diseases, and allosteric inhibitors have been developed that stabilise the inactive state. The mechanism of SHP2 activation remains unclear, however. Here, we show that the intrinsically disordered bis-phosphorylated SHP2-activating peptide pY627pY659-Gab1 binds to both SH2 domains, undergoing a partial disorder-to-order transition in the process. In addition to eliciting changes in SH2 domain dynamics, the peptide reorganises their relative orientations to provide a new SH2-SH2 interface. Our data suggest an active conformation for SHP2 that is also applicable to the hematopoietic cell-specific SHP1 (PTPN6), shedding light on the activation mechanism of both enzymes and paving the way for the development of novel compounds that modulate SHP2 activity.

biochemistry↗

bioRxiv2023

Self-assembly of Grb2 meshworks revealed by Grb2-Gab1497-528 complex structure

The ubiquitously expressed adaptor protein Growth factor receptor bound protein 2 (Grb2) plays an essential role in signal transduction by binding to activated receptor tyrosine kinases through its SH2 domain and to downstream effectors via its N- and C-terminal SH3 domains (nSH3, cSH3). Here we present the first structure of ligand-bound full length Grb2. The crystal structure of Grb2 in complex with a bidentate nSH3-cSH3-binding peptide, derived from the multi-site docking protein Grb2- associated binder-1 (Gab1), provides molecular insight into effector recognition by Grb2 and reveals the assembly of a two-dimensional meshwork, consisting of multimeric filament-like Grb2 chains linked to each other by the bivalent bound Gab1497-528 peptide. Dominant contacts between Grb2 molecules in the multimer are provided by an intermolecular SH2/cSH3 domain interface that is also present in the closed dimer of ligand-free Grb2. We further show that Grb2 is able to self-assemble to form phase-separated condensates in solution. The Grb2 SH2 domain phosphotyrosine binding site is freely accessible in the multimeric assembly, and phase separation is fostered by addition of Gab1497- 528, as expected from the crystal structure. Multimeric assembly is also observed using a Grb2 SH2- cSH3 didomain construct, and suppressed using a Grb2 Tyr60Glu mutant, a mimic of the in vivo phosphorylated Tyr160 central to the SH2/cSH3 interface, demonstrating that an intact SH2/cSH3 interface is needed for Grb2 assembly in solution.

biochemistry↗

bioRxiv2022

Delineating organizational principles of the endogenous L-A virus by cryo-EM and computational analysis of native cell extracts

The high abundance of most viruses in infected host cells benefits their structural characterization; endogenous viruses are present in low copy numbers, however, and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus. The determined cryo-EM structure discloses capsid-stabilizing cation-{pi} stacking and an interplay of non-covalent interactions from ten distinct capsomere interfaces. The capsid-embedded mRNA decapping active site trench is supported by a constricting movement of two opposite-facing loops. tRNA-loaded polysomes and other biomacromolecules, presumably mRNA, are found in virus proximity while stacked dsRNA bundles and the sub-stoichiometric polymerase localize underneath the capsid surface. Mature viruses participate in larger viral communities resembling their rare in-cell equivalents in terms of size, composition, and inter-virus distances. Our results collectively describe a 3D-architecture of a viral milieu, opening the door to cellextract-based high-resolution structural virology.

biochemistry↗

bioRxiv2019

2.7 A cryo-EM structure of vitrified M. musculus H-chain apoferritin from 200 keV \"screening microscope\"

Here we present the structure of mouse H-chain apoferritin at 2.7 [A] (FSC=0.143) solved by single particle cryogenic electron microscopy (cryo-EM) using a 200 kV device. Data were collected using a compact, two-lens illumination system with a constant power objective lens, without the use of energy filters or aberration correctors. Coulomb potential maps reveal clear densities for main chain carbonyl oxygens, residue side chains (including alternative conformations) and bound solvent molecules. We argue that the advantages offered by (a) the high electronic and mechanical stability of the microscope, (b) the high emission stability and low beam energy spread of the high brightness Field Emission Gun (x-FEG), (c) direct electron detection technology and (d) particle-based Contrast Transfer Function (CTF) refinement have contributed to achieving resolution close to the Rayleigh limit. Overall, we show that basic electron optical settings for automated cryo-electron microscopy imaging, widely thought of as a "screening cryo-microscope", can be used to determine structures approaching atomic resolution. HighlightsO_LIThe 2.7 [A] structure of mouse apoferritin was solved using a 200 keV screening cryo-microscope C_LIO_LIThe apoferritin reconstruction was resolved without an energy filter, aberration correctors, or constant-power condenser lenses C_LIO_LIComparison to available crystallographic and cryo-EM structures from high-end cryo-microscopes demonstrates consistency in resolved water molecules, metals and side chain orientations C_LIO_LIAlthough radiation damage is more prominent at 200 keV compared to 300 keV, this type of instrumentation is more accessible to research laboratories due to its compactness and simplicity C_LI

biophysics↗