Biology subjects

Martin, F. J. O.

Publications and source records attributed to Martin, F. J. O..

4 recordsLinked to original sources

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↗

bioRxiv2024

Exchange, promiscuity, and orthogonality in de novo designed coiled-coil peptide assemblies

De novo protein design is delivering new peptide and protein structures at a rapid pace. Many of these synthetic polypeptides form well-defined and hyperthermal-stable structures. Generally, however, less is known about the dynamic properties of the de novo designed structures. Here, we explore one aspect of dynamics in a series of de novo coiled-coil peptide assemblies: namely, peptide exchange within and between different oligomers from dimers through to heptamers. First, we develop a fluorescence-based reporter assay for peptide exchange that is straightforward to implement, and, thus, would be useful to others examining similar systems. We apply this assay to explore both homotypic exchange within single species, and heterotypic exchange between coiled coils of different coiled-coil oligomer states. For the former, we provide detailed study for the dimeric coiled coil CC-Di finding a half-life for exchange of 4.2 {+/-} 0.3 minutes when the concentration of CC-Di is 200 {micro}M. Interestingly, more broadly when assessing exchange across all of the oligomeric states, we find that some of the designs are faithful and only undergo homotypic strand exchange, whereas others are promiscuous and exchange to form unexpected hetero-oligomers. Finally, we develop two design strategies to improve the orthogonality of the different oligomers: (i) using alternate positioning of salt bridge interactions; and (ii) incorporating of non-canonical repeats into the designed sequences. In so doing, we reconcile the promiscuity and deliver a set of faithful homo-oligomeric de novo coiled-coil peptides. Our findings have implications for the application of these and other coiled coils as modules in chemical and synthetic biology.

systems biology↗

bioRxiv2023

Structure and mechanism of Zorya anti-phage defense system

Zorya is a recently identified and widely distributed bacterial immune system, which protects against phage invasion. It consists of a predicted membrane-embedded complex (ZorAB) and soluble components that differ among Zorya subtypes, notably ZorC and ZorD, in type I Zorya systems. Here, we reveal the molecular basis of the Zorya defense system using cryo-electron microscopy, mutagenesis, fluorescence microscopy, proteomics, and functional studies. We demonstrate that ZorAB shares the stoichiometry of other 5:2 inner membrane ion-driven rotary motors. Additionally, ZorA5B2 features a dimeric ZorB peptidoglycan binding domain and a pentameric -helical coiled-coil tail made of ZorA that projects approximately 700 [A] into the cytoplasm. We further characterize the structure and function of the soluble Zorya components, ZorC and ZorD, and find that they harbour DNA binding and nuclease activity, respectively. Comprehensive functional and mutational analyses demonstrates that all Zorya components work in concert to protect bacterial cells against invading phages. We present evidence that ZorAB operates as an ion-driven motor that becomes activated and anchors to the cell wall upon sensing of cell envelope perturbations during phage invasion. Subsequently, ZorAB transfers the phage invasion signal through the ZorA cytoplasmic tail to the soluble effectors, which function to prevent phage propagation. In summary, our study elucidates the foundational mechanisms of Zorya function and reveals a novel triggering signal for the rapid activation of an anti-phage defense system.

molecular biology↗

bioRxiv2021

How coiled-coil assemblies accommodate multiple aromatic residues

Rational protein design requires understanding the contribution of each amino acid to a targeted protein fold. For a subset of protein structures, namely the ;-helical coiled coils (CCs), knowledge is sufficiently advanced to allow the rational de novo design of many structures, including entirely new protein folds. However, current CC design rules center on using aliphatic hydrophobic residues predominantly to drive the folding and assembly of amphipathic helices. The consequences of using aromatic residues--which would be useful for introducing structural probes, and binding and catalytic functionalities--into these interfaces is not understood. There are specific examples of designed CCs containing such aromatic residues, e.g., phenylalanine-rich sequences, and the use of polar aromatic residues to make buried hydrogen-bond networks. However, it is not known generally if sequences rich in tyrosine can form CCs, or what CC assemblies these would lead to. Here we explore tyrosine-rich sequences in a general CC-forming background and resolve new CC structures. In one of these, an antiparallel tetramer, the tyrosine residues are solvent accessible and pack at the interface between the core and the surface. In the other more-complex structure, the residues are buried and form an extended hydrogen-bond network.

biochemistry↗