Biology subjects

Hayes, B. K.

Publications and source records attributed to Hayes, B. K..

5 recordsLinked to original sources

bioRxiv2025

Structures of tRNA-bound CRISPR-Cas13 reveal universal HEPN RNase mechanisms

Ribonucleases (RNases) are ubiquitous drivers of RNA metabolism across life. Higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain-containing RNases form a nuclease superfamily whose mechanisms of substrate selection and catalysis remain poorly understood. Here, we report substrate preferences for diverse Cas13 HEPN RNases, revealing precise cleavage of tRNA anticodons and acceptor stems. Using cryo-EM, we present a series of activated Leptotrichia buccalis Cas13a (LbuCas13a) structures engaged with substrate tRNA. We show LbuCas13a captures tRNA through shape- and sequence-specific recognition, cleaving U-rich anticodons independently of tRNA modifications. Leveraging these insights, we reprogram Cas13 specificity and engineer variants with accelerated RNase activity. These findings establish the molecular basis for Cas13 tRNase activity and indicate that the HEPN RNase superfamily obeys universally conserved mechanisms of substrate recognition and catalysis.

biochemistry↗

bioRxiv2024

De novo design of potent CRISPR-Cas13 inhibitors

CRISPR-Cas systems are transformative tools for gene editing which can be tuned or controlled by anti-CRISPRs (Acrs) - phage derived inhibitors that regulate CRISPR-Cas activity. However, Acrs that are capable of inhibiting biotechnologically relevant CRISPR systems are relatively rare and challenging to discover. To overcome this limitation, we describe a highly successful, rapid, and generalisable approach that leverages de novo protein design to develop new-to-nature proteins for controlling CRISPR-Cas activity. Using CRISPR-Cas13 as a representative example, we demonstrate that AI-designed anti-CRISPRs (AIcrs) are capable of highly potent and specific inhibition of CRISPR-Cas13 proteins. We present a comprehensive workflow for design validation and demonstrate AIcrs functionality in controlling CRISPR-Cas13 activity in bacteria. The ability to design bespoke inhibitors of Cas effectors will contribute to the ongoing development of CRISPR-Cas tools in diverse applications across research, medicine, agriculture, and microbiology.

biochemistry↗

bioRxiv2024

Delivery determinants of an Acinetobacter baumannii type VI secretion system bifunctional peptidoglycan hydrolase.

Acinetobacter baumannii is a Gram-negative opportunistic pathogen that is a common cause of nosocomial infections. The increasing development of antibiotic resistance in this organism is a global health concern. The A. baumannii clinical isolate AB307-0294 produces a type VI secretion system (T6SS) that delivers three antibacterial cargo effector proteins (Tse15, Tde16 and Tae17) that give this strain a competitive advantage against other bacteria in polymicrobial environments. These effectors are delivered via specific non-covalent interactions with the T6SS needle tip proteins VgrG15, VgrG16 and VgrG17, respectively. Here we determine the molecular function of the Tae17 effector protein and define the regions of Tae17 and VgrG17 essential for its delivery. Specifically, we show that Tae17 is a multidomain, bifunctional peptidoglycan-degrading enzyme. Tae17 has both lytic transglycosylase activity, which targets the peptidoglycan sugar backbone, and amidase activity, which targets the sugar-peptide bonds. Moreover, we show that the transglycosylase activity was more important for killing Escherichia coli. Using deletion constructs and bacterial two-hybrid analyses, we identify that amino acids 1051-1085 of the VgrG17 needle tip protein and amino acids 1-162 of the Tae17 effector protein are necessary for the Tae17:VgrG17 interaction. Furthermore, we identify the VgrG17 amino acids G1069 and W1075 as crucial for the delivery of Tae17; the first time such specific delivery determinants of T6SS cargo effectors have been defined. This study provides molecular insight into how the T6SS allows A. baumannii strains to gain dominance in polymicrobial communities and thus improve their chances of survival and transmission. IMPORTANCEWe have shown that the Acinetobacter baumannii T6SS effector Tae17 is a modular, bifunctional, peptidoglycan-degrading enzyme that has both lytic transglycosylase and amidase activity. Both activities contribute to the ability to degrade peptidoglycan, but the glycosyltransferase activity was more important for the interbacterial killing of Escherichia coli. We have defined the specific regions of Tae17 and its cognate delivery protein VgrG17 that are necessary for the non-covalent interactions and, for the first time, identified specific amino acids essential for delivery. This work contributes to our molecular understanding of bacterial competition strategies in polymicrobial environments and may provide a window to the design of new therapeutic approaches for combating infection by A. baumannii.

microbiology↗

bioRxiv2023

Structure of a Rhs effector clade domain identifies new mechanistic insights into type VI secretion system toxin delivery.

The type VI secretion system (T6SS) is a molecular machine utilised by many Gram-negative bacteria to deliver antibacterial toxins directly into adjacent bacteria, often providing a competitive advantage. Rearrangement hotspot (Rhs) effectors are known T6SS cargo effectors, but the precise delivery and activation of these toxins is poorly defined. We present the structure of a novel T6SS Rhs effector (Tse15) from the multidrug resistant nosocomial pathogen Acinetobacter baumannii. Tse15 forms a triple layered y-cocoon Rhs domain with an N-terminal -helical clade domain and an unfolded C-terminal toxin domain located entirely inside the Rhs cage. We identify that Tse15 is cleaved into three domains, through two independent auto-cleavage events involving aspartyl protease activity for toxin self-cleavage and a nucleophilic glutamic acid for the cleavage of the N-terminal clade domain. Proteomic analyses showed that the N-terminal clade and toxin domains, but not the y-strand rich Rhs cage, are delivered outside of the cell, suggesting a novel mechanism for Rhs toxin delivery and activation. Our findings suggest that this delivery mechanism requires an interaction between the N-terminal clade and toxin domains, with the clade domain acting as the internal chaperone to mediate tethering of the toxin to the T6SS machinery. Conservation of the clade domain in other Gram-negative bacteria suggest this may be a common mechanism for T6SS toxin delivery.

microbiology↗

bioRxiv2020

Mapping the substrate sequence and length of the Plasmodium M1 and M17 aminopeptidases

During malarial infection, Plasmodium parasites digest human hemoglobin to obtain free amino acids for protein production and maintenance of osmotic pressure. The Plasmodium M1 and M17 aminopeptidases are both postulated to have an essential role in the terminal stages of the hemoglobin digestion process and are validated drug targets for the design of new dualtarget anti-malarial compounds. In this study, we profiled the substrate specificity fingerprints and kinetic behaviors of M1 and M17 aminopeptidases from Plasmodium falciparum and Plasmodium vivax, and the mouse model species, Plasmodium berghei. We found that although the Plasmodium M1 aminopeptidases share a largely similar, broad specificity at the P1 position, the P. falciparum M1 displays the greatest diversity in specificity and P. berghei M1 showing a preference for charged P1 residues. In contrast, the Plasmodium M17 aminopeptidases share a highly conserved preference for hydrophobic residues at the P1 position. The aminopeptidases also demonstrated intra-peptide sequence specificity, particularly the M1 aminopeptidases, which showed a definitive preference for peptides with fewer negatively charged intrapeptide residues. When tested with a panel of peptides of increasing length, each aminopeptidase exhibited unique catalytic behavioral responses to the increase in peptide length, although all six aminopeptidases exhibited an increase in cooperativity as peptide length increased. Overall the P. vivax and P. berghei enzymes were generally faster than the P. falciparum enzymes, which we postulate is due to subtle differences in structural dynamicity. Together, these results build a kinetic profile that allows us to better understand the catalytic nuances of the M1 and M17 aminopeptidases from different Plasmodium species.

biochemistry↗