Biology subjects

Egelman, E.

Publications and source records attributed to Egelman, E..

7 recordsLinked to original sources

bioRxiv2025

Cryo-EM analysis of the Bacillus thuringiensis extrasporal matrix identifies F-ENA as a widespread family of endospore appendages across the Firmicutes phylum

For over 100 years, Bacillus thuringiensis (Bt) has been used as an agricultural biopesticide to control pests caused by insect species in the orders of Lepidoptera, Diptera and Coleoptera. Under nutrient starvation, Bt cells differentiate into spores and associated toxin crystals that can adopt biofilm-like aggregates. We reveal that such Bt spore/toxin biofilms are embedded in a fibrous extrasporal matrix (ESM), and using cryoID, we resolved the structure and molecular identity of an uncharacterized type of pili, referred to here as Fibrillar ENdospore Appendages or F-ENA. F-ENA are monomolecular protein polymers tethered to the exosporium of Bt and are decorated with a flexible tip fibrillum. Phylogenetic analysis reveals that F-ENA is widespread not only in the class Bacilli, but also in the class Clostridia, and the cryoEM structures of F-ENA filaments from Bacillus, Anaerovorax and Paenibaccilus reveal subunits with a generic head-neck domain structure, where the {beta}-barrel neck of variable length latch onto a preceding head domain through short N-terminal hook peptides. In Bacillus, two collagen-like proteins (CLP) respectively tether F-ENA to the exosporium (F-Anchor), or constitute the tip fibrillum at the distal terminus of F-ENA (F-BclA). Sedimentation assays point towards F-ENA involvement in spore-spore clustering, likely mediated via F-BclA contacts and F-ENA bundling through the antiparallel interlocking of the head-neck units.

molecular biology↗

bioRxiv2025

The atypical filament assembly underpins the inflammasome-independent functions of IFI16.

Inflammasomes trigger cell death upon sensing various intracellular maladies. These supra-structures relay upstream signals by sequentially assembling architecturally congruent filaments via their pyrin domains (PYDs). Interferon Inducible Protein 16 (IFI16) is an innate immune sensor that detects dysregulated nucleic acids. Once presumed as an inflammasome receptor due to its PYD, the role of IFI16 has been much more appreciated in other innate immune pathways such as regulating interferon production and viral replication restriction. Here, a cryo-EM structure of the filament assembled by the PYD of IFI16(PYD) shows a helical architecture distinct from inflammasome PYD filaments. In silico Rosetta interaction energy calculations suggest that the helical architecture of the IFI16PYD filament is incompatible with those assembled by the central inflammasome adaptor ASC and its interacting partners. Cellular experiments further support that IFI16 fails to interact with ASC. Together, we provide the structural basis for the inflammasome-independent functions of IFI16.

biophysics↗

bioRxiv2024

Donor Strand Complementation and Calcium Ion Coordination Drive the Chaperone-free Polymerization of Archaeal Cannulae

Cannulae are tubular protein filaments that accumulate on the extracellular surface of the hyperthermophilic archaeon Pyrodictium abyssi during cell division. Cannulae have been postulated to act as a primitive extracellular matrix through which cells could communicate or exchange material, although their native biological function remains obscure. Here, we report cryoEM structural analyses of ex vivo cannulae and of in vitro protein assemblies derived from recombinant cannula-like proteins. Three-dimensional reconstructions of P. abyssi cannulae revealed that the structural interactions between protomers in the native and recombinant filaments were based on donor strand complementation, a form of non-covalent polymerization in which a donor {beta}-strand from one subunit is inserted into an acceptor groove in a {beta}-sheet of a neighboring subunit. Donor strand complementation in cannulae is reinforced through calcium ion coordination at the interfaces between structural subunits in the respective assemblies. While donor strand complementation occurs during the assembly of chaperone-usher pili, this process requires the participation of accessory proteins that are localized in the outer membrane. In contrast, we demonstrate that calcium ions can induce assembly of cannulae in the absence of other co-factors. Crystallographic analysis of a recombinant cannula-like protein monomer provided evidence that calcium ion binding primes the precursor for donor strand invasion through unblocking of the acceptor groove. Bioinformatic analysis suggested that structurally homologous cannula-like proteins occurred within the genomes of other hyperthermophilic archaea and were encompassed within the TasA superfamily of biomatrix proteins. CryoEM structural analyses of tubular filaments derived from in vitro assembly of a recombinant cannula-like protein from an uncultured Hyperthermus species revealed a common mode of assembly to the Pyrodictium cannulae, in which donor strand complementation and calcium ion binding stabilized longitudinal and lateral assembly in tubular 2D sheets.

microbiology↗

bioRxiv2024

Cryo-EM structure of the conjugation H-pilus reveals the cyclic nature of the TrhA pilin

Conjugation, the major driver of the spread of antimicrobial resistance genes, relies on a conjugation pilus for DNA transfer. Conjugative pili, such as the F-pilus, are dynamic tubular structures, composed of a polymerized pilin, that mediate the initial donor-recipient interactions, a process known as mating pair formation (MPF). IncH are low-copy-number plasmids, traditionally considered broad host range, which are found in bacteria infecting both humans and animals. The reference IncHI1 plasmid R27, isolated from Salmonella enterica serovar Typhi, encodes the conjugative H-pilus subunit TrhA containing 74 residues after cleavage of the signal sequence. Here, we show that the H-pilus forms long filamentous structures that mediate MPF, and describe its cryo electron-microscopic (cryo-EM) structure at 2.2 [A] resolution. Like the F pilus, the H-pilin subunits form helical assemblies with phospholipid molecules at a stochiometric ratio of 1:1. While there were previous reports that the T-pilus from Agrobacterium tumefaciens was composed of cyclic subunits, three recent cryo-EM structures of the T-pilus found no such cyclization. Here, we report that the H-pilin is cyclic, with a covalent bond connecting the peptide backbone between the N- and C-termini. Both the cryo-EM map and mass spectrometry revealed cleavage of the last five residues of the pilin, followed by cyclization via condensation of the amine and carboxylate residues. The cyclic nature of the pilin could stabilize the pilus and may explain the high incidence of IncH plasmid dissemination. SignificanceA major medical challenge is the spread of bacteria which are resistant to antibiotics. The resistance genes are spread via mobilized DNA, mainly via a process named conjugation. During conjugation, a resistant bacterium (donor), transfers the resistance DNA to another bacterium (recipient) in a contact-dependent manner. The initial donor-recipient interaction is mediated by a hollow filament expressed by the donor, named the conjugation pilus, that binds the recipient. This pilus is built via polymerization of a small protein subunit, pilin. Here, we report the atomic structure of the H-pilus, whose pilin subunit has an unusual cyclic structure where the N- and C-termini of the protein are covalently linked by a peptide bond.

microbiology↗

bioRxiv2023

Stabilization of F-actin by Salmonella effector SipA resembles the structural effects of inorganic phosphate and phalloidin

Entry of Salmonella into host enterocytes strictly relies on its pathogenicity island 1 effector SipA. We found that SipA binds to F-actin in a unique mode in a 1:2 stoichiometry with picomolar affinity. A cryo-EM reconstruction revealed that SipAs globular core binds at the grove between actin strands, whereas the extended C-terminal arm penetrates deeply into the inter-strand space, stabilizing F-actin from within. The unusually strong binding of SipA is achieved via a combination of fast association via the core and very slow dissociation dictated by the arm. Similarly to Pi, BeF3, and phalloidin, SipA potently inhibited actin depolymerization by ADF/cofilin, which correlated with the increased filament stiffness, supporting the hypothesis that F-actins mechanical properties contribute to the recognition of its nucleotide state by protein partners. The remarkably strong binding to F-actin maximizes the toxins effects at the injection site while minimizing global influence on the cytoskeleton and preventing pathogen detection by the host cell.

biochemistry↗

bioRxiv2023

Large pilin subunits provide distinct structural and mechanical properties for the Myxococcus xanthus type IV pilus

Type IV pili (T4P) are ubiquitous bacterial cell surface filaments important for surface motility, adhesion to biotic and abiotic surfaces, DNA uptake, biofilm formation, and virulence. T4P are built from thousands of copies of the major pilin subunit and tipped by a complex composed of minor pilins and in some systems also the PilY1 adhesin. While the major pilins of structurally characterized T4P have lengths of up to 161 residues, the major pilin PilA of Myxococcus xanthus is unusually large with 208 residues. All major pilins have a highly conserved N-terminal domain and a highly variable C-terminal domain, and the additional residues in the M. xanthus PilA are due to a larger C-terminal domain. We solved the structure of the M. xanthus T4P (T4PMx) at a resolution of 3.0 [A] using cryo-electron microscopy (cryo-EM). The T4PMx follows the structural blueprint observed in other T4P with the pilus core comprised of the extensively interacting N-terminal 1-helices while the globular domains decorate the T4P surface. The atomic model of PilA built into this map shows that the large C-terminal domain has much more extensive intersubunit contacts than major pilins in other T4P. As expected from these greater contacts, the bending and axial stiffness of the T4PMx is significantly higher than that of other T4P and supports T4P-dependent motility on surfaces of different stiffnesses. Notably, T4PMx variants with interrupted intersubunit interfaces had decreased bending stiffness and strongly reduced motility on all surfaces. These observations support an evolutionary scenario whereby the large major pilin enables the formation of a rigid T4P that expands the environmental conditions in which the T4P system functions.

microbiology↗

bioRxiv2022

Archaeal bundling pili of Pyrobaculum calidifontis reveal similarities between archaeal and bacterial biofilms

While biofilms formed by bacteria have received great attention due to their importance in pathogenesis, much less research has been focused on the biofilms formed by archaea. It has been known that extracellular filaments in archaea, such as Type IV pili, hami and cannulae, play a part in the formation of archaeal biofilms. We have used cryo-electron microscopy to determine the atomic structure of a previously uncharacterized class of archaeal surface filaments from hyperthermophilic Pyrobaculum calidifontis. These filaments, which we call archaeal bundling pili (ABP), assemble into highly ordered bipolar bundles. The bipolar nature of these bundles most likely arises from the association of filaments from at least two different cells. The component protein shows homology, both at the sequence and structural level, to the bacterial protein TasA, a major component of the extracellular matrix in bacterial biofilms, contributing to biofilm stability. We show that ABP forms very stable filaments in a manner similar to the donor-strand exchange of bacterial TasA fibers and chaperone-usher pathway pili where a {beta}-strand from one subunit is incorporated into a {beta}-sheet of the next subunit. Our results reveal mechanistic similarities and evolutionary connection between bacterial and archaeal biofilms, and suggest that there could be many other archaeal surface filaments that are as yet uncharacterized. SignificanceBiofilms are communities of microbes where cells attach to each other as well as to surfaces, and bacterial biofilms have been intensively studied due to their importance in many infections. Much less has been known about archaeal biofilms, where archaea are a third domain of life. Using cryo-electron microscopy, we have determined the atomic structure of a surface filament in archaea that forms bi-polar bundles connecting cells. We show that this protein has common ancestry with a protein known to be an important component of bacterial biofilms. This adds to our understanding of the evolutionary relationship between bacteria and archaea and may provide new insights into bacterial biofilms.

biophysics↗