Biology subjects

Siborova, M.

Publications and source records attributed to Siborova, M..

7 recordsLinked to original sources

bioRxiv2026

Piercing mechanism in the minimal contractile tail of bacteriophage P2.

Bacteriophage P2 is a long-standing model for contractile injection systems, yet high-resolution structures of the intact virion and the mechanism of baseplate activation have remained unresolved. Here, we combine high-resolution cryo-electron microscopy and in situ cryo-electron tomography to characterize P2, using its lytic mutant P2vir, in its pre- and post-contraction states and during host infection. We identify a previously undescribed fold in the head-to-tail adaptor, which mediates the transition from 12-fold portal symmetry to sixfold tail symmetry through interlocking {beta}-strand handshake interactions. Our structures further reveal a compact, threefold-symmetric baseplate established by a distinctive loop-helix module formed by the hub and tube initiator, which bioinformatic and phylogenetic analyses identify as a characteristic architectural feature of the Peduovirus lineage. We also propose a rope-like arrangement that anchors the tail fibre to the baseplate triplex component gpI and obtain evidence that the tape-measure protein adopts a C3 arrangement within the tail lumen, while computational predictions suggest a possible hexameric, pore-like organization after release. Comparison of pre- and post-contraction particles, supported by in situ imaging of infection, suggests that sheath contraction destabilizes the baseplate, promoting its disassembly. The inner tail tube appears to penetrate the host outer membrane but does not appear to reach the inner membrane, leading us to propose that the reorganized tape-measure protein forms a secondary conduit for genome translocation. These findings support a distinct model of contractile tail activation and expand the known architectural diversity of viral injection systems.

microbiology↗

bioRxiv2025

Structure of a novel contractile injection system in subsp.

Extracellular contractile injection systems (eCISs) are phage-derived nanomachines used by bacteria to deliver effectors into target cells. Well-studied examples include the Photorhabdus asymbiotica virulence cassettes and the antifeeding prophage from Serratia entomophila, which have been engineered for heterologous cargo delivery. Recent genomic analyses identified previously uncharacterized eCIS gene clusters in the opportunistic human pathogen Salmonella enterica subspecies salamae, but their structure, function, and biotechnological potential remain unexplored. Here, we report a high-resolution cryo-electron microscopy structure of the S. enterica eCIS. Our atomic models reveal a unique sheath architecture, an expansive cage-like shell around a central spike, and an associated transmembrane hydrolase. We identify a putative effector encoded within the operon exhibiting periplasmic toxicity and provide evidence that the S. enterica eCIS deviates from canonical eCISs by interacting with the inner membrane. Guided by these structural features, we uncover a previously unannotated cluster of contractile injection systems (CISs). Together, our findings expand the known diversity of CISs structures and functions and lay the groundwork for engineering customizable protein delivery platforms.

microbiology↗

bioRxiv2025

Structural basis for postfusion-specific binding to Respiratory Syncytial Virus F protein by the canonical antigenic site I antibody 131-2a

The Respiratory Syncytial Virus (RSV) Fusion (F) protein is a major target of antiviral antibodies following natural infection or vaccination and responsible for mediating fusion between the viral envelope and the host membrane. The fusion process is driven by a large-scale conformational change in F, switching irreversibly from the metastable prefusion state to the stable postfusion conformation. Previous research has identified six distinct antigenic sites in RSV-F, termed sites O, I, II, III, IV, and V. Of these, only antigenic site I is fully specific to the postfusion conformation of F. A monoclonal antibody 131-2a that specifically targets postfusion F has been widely used as a research tool to probe for postfusion F and to define antigenic site I in serological studies, yet its sequence and precise epitope have remained unknown. Here we use mass spectrometry-based de novo sequencing of 131-2a to reverse engineer a recombinant product and study the epitope to define antigenic site I with molecular detail, revealing the structural basis for the antibodys specificity towards postfusion RSV-F.

immunology↗

bioRxiv2024

Cell attachment and tail contraction of S. aureus phage phi812

Phages with contractile tails employ elaborate mechanisms to penetrate bacterial cell walls and deliver their genomes into the host cytoplasm. Here, we used cryo-EM to show that the baseplate of phage 812, a member of the Kayvirus genus, which infects Gram-positive Staphylococcus strains, is formed of a core, wedge modules, and baseplate arms carrying receptor-binding proteins 1 and 2 and tripod complexes. Upon binding to a host cell, the receptor-binding proteins of phage 812 baseplate reorient and undergo conformational changes. The changes to the tripod complexes trigger the release of the central spike and weld proteins, which expose peptidoglycan-degrading domains of the hub proteins. Changes in the positions of baseplate arms are transmitted through wedge modules to tail sheath initiator proteins. The ring of the tail sheath initiator proteins expands and triggers the contraction of the tail sheath, which shortens to 50% and pushes the tail tube 10-30 nm into the bacterial cytoplasm. Homologous molecular mechanisms are probably shared by phages of the Herelleviridae family with contractile tails to infect Gram-positive bacteria.

microbiology↗

bioRxiv2024

Simultaneous polyclonal antibody sequencing and epitope mapping by cryo electron microscopy and mass spectrometry - a perspective.

Antibodies are a major component of adaptive immunity against invading pathogens. Here we explore possibilities for an analytical approach to characterize the antigen-specific antibody repertoire directly from the secreted proteins in convalescent serum. This approach aims to perform simultaneous antibody sequencing and epitope mapping using a combination of single particle cryo-electron microscopy (cryoEM) and bottom-up proteomics techniques based on mass spectrometry (LC-MS/MS). We evaluate the performance of the deep-learning tool ModelAngelo in determining de novo antibody sequences directly from reconstructed 3D volumes of antibody-antigen complexes. We demonstrate that while map quality is a critical bottleneck, it is possible to sequence antibody variable domains from cryoEM reconstructions with accuracies of up to 80-90%. While the rate of errors exceeds the typical levels of somatic hypermutation, we show that the ModelAngelo-derived sequences can be used to assign the used V-genes. This provides a functional guide to assemble de novo peptides from LC-MS/MS data more accurately and improves the tolerance to a background of polyclonal antibody sequences. Following this proof-of-principle, we discuss the feasibility and future directions of this approach to characterize antigen-specific antibody repertoires.

biochemistry↗

bioRxiv2023

Reverse engineering the anti-MUC1 hybridoma antibody 139H2 by mass spectrometry-based de novo sequencing

Mucin 1 (MUC1) is a transmembrane mucin expressed at the apical surface of epithelial cells at different mucosal surfaces including breast and intestine. In the gastrointestinal tract, MUC1 has a barrier function against bacterial invasion, but can also serve as an entry receptor for pathogenic Salmonella bacteria. Moreover, MUC1 is well known for its aberrant expression and glycosylation in adenocarcinomas The MUC1 extracellular domain contains a variable number of tandem repeats (VNTR) of 20 amino acids, which are heavily O-linked glycosylated.. Monoclonal antibodies against the MUC1 VNTR can be powerful tools because of their multiplicity of binding and possible applications in the diagnosis and treatment of MUC1-expressing cancers. One such antibody is the hybridoma mouse monoclonal 139H2, which is also widely used as a research tool to study non-cancer MUC1. Here we report direct mass spectrometry-based sequencing of hybridoma-derived 139H2 IgG, which enabled reverse engineering of a recombinant 139H2. The performance of the reverse engineered 139H2 IgG and its Fab fragment were validated by comparison to the hybridoma-derived product in Western blot and immunofluorescence microscopy. The reverse engineering of 139H2 allowed us to characterize binding to the VNTR peptide epitope by surface plasmon resonance (SPR) and solve the crystal structure of the 139H2 Fab fragment in complex with the MUC1 VNTR peptide. These analyses reveal the molecular basis for 139H2 binding specificity to MUC1 and its tolerance to O-glycosylation of the VNTR. The available sequence of 139H2 will allow further development of MUC1-related diagnostics, targeting and treatment strategies.

biochemistry↗

bioRxiv2023

CD5L is a canonical component of circulatory IgM

Immunoglobulin M (IgM) is an evolutionary conserved key component of humoral immunity, and the first antibody isotype to emerge during an immune response. IgM is a large (1 MDa), multimeric protein, for which both hexameric and pentameric structures have been described, the latter additionally containing a joining (J) chain. Using a combination of single-particle mass spectrometry and mass photometry, proteomics and immunochemical assays, we here demonstrate that circulatory (serum) IgM exclusively exists as a complex of J-chain-containing pentamers covalently bound to the small CD5 antigen-like (CD5L, 36 kDa) protein. In sharp contrast, secretory IgM in saliva and milk is principally devoid of CD5L. Unlike IgM itself, CD5L is not produced by B cells, implying that it associates with IgM in the extracellular space. We demonstrate that CD5L integration has functional implications, i.e., it diminishes IgM binding to two of its receptors, the Fc{micro}R and the polymeric Immunoglobulin receptor (pIgR). On the other hand, binding to Fc{micro}R as well as complement activation via C1q seem unaffected by CD5L integration. Taken together, we redefine the composition of circulatory IgM as a J-chain containing pentamer, always in complex with CD5L.

molecular biology↗