Biology subjects

Brilot, A. F.

Publications and source records attributed to Brilot, A. F..

6 recordsLinked to original sources

bioRxiv2025

The CAGE complex: a hollow, megadalton, protein assembly in prokaryotic and eukaryotic microbes

We report the discovery and structure of a previously unknown [~]1 MDa hollow protein assembly, identified during a survey of ciliary complexes from the ciliate Tetrahymena thermophila. By combining mass spectrometry, structure prediction, and cryo-electron microscopy, we define a homotetrameric cage-like complex with a distinctive elliptical architecture and a large internal cavity. A sequence survey revealed several thousand homologs spanning diverse unicellular eukaryotes--including green algae, fungi, amoebozoans, choanoflagellates, and SAR lineages--as well as predominantly gram-negative bacteria, indicating an ancient evolutionary origin and arguing against a eukaryote-specific function. We determined a near-atomic resolution structure of the complex from the slime mold Dictyostelium discoideum, demonstrating conservation of overall architecture and cavity despite low sequence identity. Together, these results establish the CAGE complex (Conserved Assembly in Gram-negative bacteria and Eukaryotes) as a new class of large protein cage broadly distributed across the tree of life. While its biological function remains unknown, its size, architecture, and conservation suggest possible roles in transport or protein/RNA homeostasis.

molecular biology↗

bioRxiv2024

Structure and organization of full-length Epidermal Growth Factor Receptor in extracellular vesicles by cryo-electron tomography

We report here transport of the Epidermal Growth Factor Receptor (EGFR), Insulin Receptor, 7-pass transmembrane receptor Smoothened, and 13-pass Sodium-iodide symporter to extracellular vesicles (EVs) for structural and functional studies. Mass spectrometry confirmed the transported proteins as the most abundant in EV membranes, and the presence of many receptor-interacting proteins demonstrates the utility of EVs for characterizing membrane protein interactomes. Cryo-electron tomography of EGFR-containing EVs reveals that EGFR forms clusters in the presence of EGF with a [~]3 nm gap between the inner membrane and cytoplasmic density. EGFR extracellular regions do not form regular arrays, suggesting that clustering is mediated by the intracellular region. Subtomogram averaging of the EGFR extracellular region (ECR) yielded a 15 [A] map into which the crystal structure of the ligand-bound EGFR ECR dimer fits well. These findings refine our understanding of EGFR activation, clustering, and signaling, and they establish EVs as a versatile platform for structural and functional characterization of human membrane proteins in a native-like environment. Significance StatementAtomic or near-atomic resolution structural studies of proteins embedded in cell membranes have proven challenging. We show that transporting integral membrane proteins to cell-derived extracellular vesicles enables structural and functional studies of human membrane proteins in a native membrane environment. We have used this approach to visualize an active form of full-length Epidermal Growth Factor Receptor (EGFR) and show that it forms clusters in the membrane and projects its cytoplasmic signaling domains [~]3 nm away from the membrane surface. EGFR is essential for normal development, but abnormal EGFR activity is associated with several human cancers and is the target of many anticancer therapies. Our studies refine current models of how ligand binding to EGFR transmits signals across cell membranes.

biophysics↗

bioRxiv2022

The {varphi}PA3 Phage Nucleus is Enclosed by a Self-Assembling, 2D Crystalline Lattice

A growing number of jumbo bacteriophages, with genomes exceeding 200 kb, have been found to establish a Phage Nucleus--a micron-scale, proteinaceous structure encompassing the replicating phage DNA. Bacteriophage and host proteins associated with replication and transcription are concentrated inside the Phage Nucleus while nucleotide synthesis, translation, and numerous other host and exogenous proteins are effectively excluded, including CRISPR-Cas and restriction endonuclease host defense systems. Here, we show that fragments of the Phage Nucleus isolated from {phi}PA3 infected Pseudomonas aeruginosa cells form a square lattice and demonstrate that the recombinantly purified primary Phage Nuclear Enclosure (PhuN) protein spontaneously assembles into sheets also constructed from a square lattice which we resolve to 3.8 [A] by cryo-EM. Our structure reveals that the flexible termini and large loops mediate adaptable inter-tetramer contacts that drive shell assembly into a C2-symmetric lattice. While the interfaces between subunits are mostly well packed, two of the interfaces are open, forming clear channels that likely have important functional implications for the transport of proteins, mRNA, and small molecules.

biochemistry↗

bioRxiv2021

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

biophysics↗

bioRxiv2020

CM1-driven assembly and activation of Yeast γ-Tubulin Small Complex underlies microtubule nucleation

Microtubule (MT) nucleation is regulated by the {gamma}-tubulin ring complex ({gamma}TuRC), conserved from yeast to humans. In Saccharomyces cerevisiae, {gamma}TuRC is composed of seven identical {gamma}-tubulin small complex ({gamma}TuSC) sub-assemblies which associate helically to template microtubule growth. {gamma}TuRC assembly provides a key point of regulation for the MT cytoskeleton. Here we combine cross-linking mass spectrometry (XL-MS), X-ray crystallography and cryo-EM structures of both monomeric and dimeric {gamma}TuSCs, and open and closed helical {gamma}TuRC assemblies in complex with Spc110p to elucidate the mechanisms of {gamma}TuRC assembly. {gamma}TuRC assembly is substantially aided by the evolutionarily conserved CM1 motif in Spc110p spanning a pair of adjacent {gamma}TuSCs. By providing the highest resolution and most complete views of any {gamma}TuSC assembly, our structures allow phosphorylation sites to be mapped, surprisingly suggesting that they are mostly inhibitory. A comparison of our structures with the CM1 binding site in the human {gamma}TuRC structure at the interface between GCP2 and GCP6 allows for the interpretation of significant structural changes arising from CM1 helix binding to metazoan {gamma}TuRC.

biochemistry↗

bioRxiv2018

The Atomic Structure of the Microtubule-Nucleating γ-Tubulin Small Complex and its Implications for Regulation

The microtubule cytoskeleton is essential in mediating a number of critical cellular processes, affecting cell shape, transport, organelle organization, and chromosomal segregation during mitosis. Microtubule network dynamics are controlled by many factors including the efficiency and localization of the nucleation machinery. Microtubule nucleation is dependent on the universally conserved {gamma}-tubulin small complex ({gamma}TuSC), a 300 kDa heterotetramer composed of two copies of {gamma}-tubulin and one each of accessory proteins GCP2 and GCP3. In yeast, nucleation is mediated by a heptameric ring of {gamma}TuSC, which presents 13 {gamma}-tubulins to form a template for microtubule nucleation.\n\nWe have obtained single-particle structures of the {gamma}TuSC as a monomer and dimer at resolutions of 3.6-4.6[A], allowing us to build an atomic model for this important complex. By comparison with a crystal structure of isolated {gamma}-tubulin, it is clear that {gamma}-tubulin is activated upon assembly into the {gamma}TuSC, in a manner analogous to the bent to straight transition in {beta}-tubulin upon assembly into the microtubule lattice. Our structures allow us to map phosphorylation sites, revealing several at key interfaces, highly suggestive of their role in regulating spindle pole body attachment and assembly into rings. When combined with previous lower resolution structures of helical assemblies, we observe that additional conformational changes occur during ring assembly and activation.

biophysics↗