Biology subjects

Sonani, R. R.

Publications and source records attributed to Sonani, R. R..

4 recordsLinked to original sources

bioRxiv2026

Structural determinants of endopilus assembly, stability and functional specificity in bacterial type II secretion

Gram-negative bacteria employ the type II secretion system (T2SS) to transport folded protein effectors across the outer membrane. This multiprotein nanomachine assembles the endopilus, a periplasmic helical polymer composed of one major and four minor pilin subunits. Endopili are structurally related to type IV pili but exhibit distinct features including a conserved calcium-binding site stabilizing their major pilin subunits. Endopilus polymerisation is coupled to substrate translocation through a dedicated outer membrane channel. To investigate the structural basis of endopilus assembly, stability and functional specificity, we performed a comparative analysis of the Out T2SS from the plant pathogen Dickeya dadantii and the Pul T2SS from the human pathogen Klebsiella oxytoca. Although their major pilins OutG and PulG share over 77% sequence identity, these bacteria differ markedly in ecological niche and range of secreted effectors. We report here the NMR structure of calcium-bound OutG monomer and cryo-EM structures of OutG and PulG endopili at 3.6 [A] resolution. The integration of structural, mutational, and biophysical analyses with in vivo assays, allowed us to identify the molecular determinants of secretion specificity and endopilus stability. These findings demonstrate how subtle sequence variations in conserved nanomachines have evolved to optimize their function and adapt to their local environments.

microbiology↗

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

Noncanonical interactions and conformational dynamics in cargo-Pex5-Pex14 ternary complex for peroxisomal import

Peroxisomes are membrane enclosed organelles hosting diverse metabolic processes in eukaryotic cells. Having no protein synthetic abilities, peroxisomes import all required enzymes from the cytosol through a peroxin (pex) import system. Peroxisome targeting sequence 1 (PTS1)-tagged cargo is recognized by soluble receptor, Pex5. The cargo-Pex5 complex docks at the peroxisomal membrane translocon, composed of Pex14 and Pex13, facilitating translocation into the peroxisomal lumen. Despite its significance, the structural basis of this process is poorly understood. Here, we present a cryo-electron microscopy (cryo-EM) structure of cargo-Pex5-Pex14 ternary complex from Trypanosoma cruzi, the causative agent of human Chagas disease. The atomic model of the Pex5 (residues 327-653) bound to malate dehydrogenase (MDH, cargo) tetramer and Pex14NTD (residues 21-85) reveals dynamic motions and secondary interfaces. Specifically, we observe a swinging motion of Pex5 at the MDH-Pex5 interface. Additionally, the noncanonical contact surfaces are observed at the MDH-Pex5 and Pex5-Pex14 interfaces. Mutational analysis of the former interface indicates that it does not significantly enhance the affinity, underscoring the dynamic nature of cargo-Pex5 interactions. The latter interface constitutes an extended binding site of Pex14NTD over Pex5. We discuss the implications of these findings for understanding peroxisomal transport mechanisms. HIGHLIGHTSO_LIThe structure of the peroxisomal import ternary complex (MDH-Pex5TPR-Pex14NTD) is determined using cryo-EM. C_LIO_LIPex5 interface with the cargo (MDH) is characterized by significant conformational dynamics. C_LIO_LIThe extended binding site of Pex14NTD on Pex5 is revealed. C_LI

biochemistry↗