Biology subjects

Chappie, J. S.

Publications and source records attributed to Chappie, J. S..

10 recordsLinked to original sources

bioRxiv2026

Structural characterization the LlaI anti-phage defense system reveals insights into the evolution of nucleotide specificity and the organization of DNA binding in McrBC restriction complexes

Canonical McrBC enzymes are nucleotide-powered, motor-driven endonucleases that bind and cleave modified bacteriophage DNA. Non-canonical McrBC homologs like LlaI and BsuMI are distinguished by a unique three-gene organization and the ability to target DNA site-specifically. Here, we report the atomic-resolution crystal structures of the DNA-binding module LlaI.R1 and AAA+ motor LlaI.R2 from the Lactococcus lactis LlaI anti-phage defense system. The crystallized LlaI.R2 hexamer traps two distinct active site conformations that correlate to different states of the nucleotide hydrolysis cycle and reveal that the organization of the critical catalytic machinery present in canonical McrB homologs is also conserved in non-canonical R2 proteins. Although canonical McrB homologs are strictly GTP-specific, we find that the R2 proteins from LlaI and BsuMI do not discriminate between different nucleotides, even when in complex with their respective R1 partners. Using mutagenesis, we define surfaces on the LlaI.R1 structure that are critical for DNA-binding and interaction with LlaI.R2. These observations support computational modelling of the assembled LlaI restriction system bound to DNA. Together, our data provide new insights into the evolution of nucleotide specificity in McrBC restriction complexes and the molecular mechanisms governing McrBC-catalyzed DNA translocation and cleavage.

biochemistry↗

bioRxiv2025

Viral silencing suppressor activity in plants modifies aphid antiviral immunity and fecundity

Plants defend themselves from viral infection using RNA interference (RNAi), an evolutionarily conserved mechanism that degrades viral RNA through the production of small interfering RNAs. As a counter defense, plant viruses evolved suppressors of RNA silencing (VSRs) to inhibit plant RNAi machinery, aiding viral replication and transmission. P0, a VSR encoded by the potato leafroll virus (PLRV), family Polerovirus, suppresses RNAi by targeting the plant protein ARGONATE 1 for degradation through its F-box motif interaction with a Skp1 subunit of the family of E3 ubiquitin ligases. Our previous work shows PLRV P0 suppresses antiviral immunity in its aphid vector Myzus persicae, leading to an increase in aphid infection by the insect virus Myzus persicae densovirus (MpDNV). Here, we expand on these findings and show that the P0 protein also regulates aphid fecundity. Using a series of F-box mutants, we demonstrate that a functional P0 F-box motif is required for inhibition MpDNV antiviral immunity but not modulation of aphid fecundity. We further show that silencing suppressors from non-aphid-borne plant viruses that target other components of the plants RNAi machinery also modulate aphid fecundity but not MpDNV titer. Collectively, the results show that aphids have been favored by selection to modulate their anti-viral immunity and fecundity in response to changes in the plant RNAi pathways induced by plant viral infection. These data highlight the intricate co-evolution of plant viruses, their vectors, and host defenses. This knowledge may open new avenues for managing vector-borne plant diseases by targeting viral proteins to manipulate insect vectors.

microbiology↗

bioRxiv2023

The crystal structure of bacteriophage lambda RexA provides novel insights into the DNA binding properties of Rex-like phage exclusion proteins

RexA and RexB function as an exclusion system that prevents bacteriophage T4rII mutants from growing on E. coli {lambda} phage lysogens. Recent data established that RexA is a non-specific DNA binding protein that can act independently of RexB to bias the {lambda} bistable switch toward the lytic state, preventing conversion back to lysogeny. The molecular interactions underlying these activities are unknown, owing in part to a dearth of structural information. Here we present the 2.05-[A] crystal structure of the {lambda} RexA dimer, which reveals a two-domain architecture with unexpected structural homology to the recombination-associated protein RdgC. Modelling suggests that our structure adopts a closed conformation and would require significant domain rearrangements to facilitate DNA binding. Mutagenesis coupled with electromobility shift assays, limited proteolysis, and double electron-electron spin resonance spectroscopy support a DNA-dependent conformational change. In vivo phenotypes of RexA mutants suggest that DNA binding is not a strict requirement for phage exclusion but may directly contribute to modulation of the bistable switch. We further demonstrate that RexA homologs from other temperate phages also dimerize and bind DNA in vitro. Collectively, these findings advance our mechanistic understanding of Rex functions and provide new evolutionary insights into different aspects of phage biology.

microbiology↗

bioRxiv2022

Polerovirus N-terminal readthrough domain structures reveal novel molecular strategies for mitigating virus transmission by aphids

Poleroviruses, enamoviruses, and lutoeviruses are icosahedral, positive sense RNA viruses that cause economically important diseases in food and fiber crops. They are transmitted by phloem-feeding aphids in a circulative manner that involves the movement across and within insect tissues. The N-terminal portion of the viral readthrough domain (NRTD) has been implicated as a key determinant of aphid transmission in each of these genera. Here, we report crystal structures of the NRTDs from the poleroviruses turnip yellow virus (TuYV) and potato leafroll virus (PLRV) at 1.53-[A] and 2.22-[A] resolution, respectively. These adopt a two-domain arrangement with a unique interdigitated topology and form highly conserved dimers that are stabilized by a C-terminal peptide that is critical for proper folding. We demonstrate that the PLRV NRTD can act as an inhibitor of virus transmission and identify NRTD mutant variants that are lethal to aphids. Sequence conservation argues that enamovirus and luteovirus NRTDs will follow the same structural blueprint, which affords a novel approach to block the spread of these agricultural pathogens in a generalizable manner.

plant biology↗

bioRxiv2021

Crystal structure of the potato leafroll virus coat protein and implications for viral assembly

Luteoviruses, poleroviruses, and enamoviruses are insect-transmitted, agricultural pathogens that infect a wide array of staple food crops. Previous cryo-electron microscopy studies of virus-like particles indicate that luteovirid viral capsids are built from a structural coat protein that organizes with T=3 icosahedral symmetry. Here we present the crystal structure of a truncated version of the coat protein monomer from potato leafroll virus at 1.57-[A] resolution. In the crystal lattice, monomers pack into flat sheets that preserve the two-fold and three-fold axes of icosahedral symmetry and show minimal structural deviations when compared to the full-length subunits of the assembled virus-like particle. These observations have important implications in viral assembly and maturation, suggesting that the CP N-terminus and its interactions with RNA serve as a key driver for generating capsid curvature.

biochemistry↗

bioRxiv2021

Bacteriophage Lambda RexA and RexB Functions Assist the Transition from Lysogeny to Lytic Growth

The CI and Cro repressors of bacteriophage {lambda} create a bistable switch between lysogenic and lytic growth. In {lambda} lysogens, CI repressor expressed from the PRM promoter blocks expression of the lytic promoters PL and PR to allow stable maintenance of the lysogenic state. When lysogens are induced, CI repressor is inactivated and Cro repressor is expressed from the lytic PR promoter. Cro repressor blocks PRM transcription and CI repressor synthesis to ensure that the lytic state proceeds. RexA and RexB proteins, like CI, are expressed from the PRM promoter in {lambda} lysogens; RexB is also expressed from a second promoter, PLIT, embedded in rexA. Here we show that RexA binds CI repressor and assists the transition from lysogenic to lytic growth, using both intact lysogens and defective prophages with reporter genes under control of the lytic PL and PR promoters. Once lytic growth begins, if the bistable switch does return to the immune state, RexA expression lessens the probability that it will remain there, thus stabilizing the lytic state and activation of the lytic PL and PR promoters. RexB modulates the effect of RexA and may also help establish phage DNA replication as lytic growth ensues.

microbiology↗

bioRxiv2020

Structural asymmetry governs the assembly and GTPase activity of McrBC restriction complexes

McrBC complexes are motor-driven nucleases functioning in bacterial self-defense by cleaving foreign DNA. The GTP-specific AAA+ protein McrB powers translocation along DNA and its hydrolysis activity is stimulated by its partner nuclease McrC. Here, we report cryo-EM structures of Thermococcus gammatolerans McrB and McrBC, and E. coli McrBC. The McrB hexamers, containing the necessary catalytic machinery for basal GTP hydrolysis, are intrinsically asymmetric. This asymmetry directs McrC binding so that it engages a single active site, where it then uses an arginine/lysine-mediated hydrogen-bonding network to reposition the asparagine in the McrB signature motif for optimal catalytic function. While the two McrBC complexes use different DNA-binding domains, these contribute to the same general GTP-recognition mechanism employed by all G proteins. Asymmetry also induces distinct inter-subunit interactions around the ring, suggesting a coordinated and directional GTP-hydrolysis cycle. Our data provide novel insights into the conserved molecular mechanisms governing McrB family AAA+ motors.

biochemistry↗

bioRxiv2019

Cyclin N-Terminal Domain-Containing 1 (CNTD1) coordinates meiotic crossover formation with cell cycle progression in a cyclin-independent manner

During meiotic prophase I, programmed DNA double-strand breaks repair as non-crossover or crossover events, the latter predominantly occurring via the Class I crossover pathway and requiring the cyclin family member CNTD1. Using an epitope-tagged Cntd1 allele, we show that mouse CNTD1 exists in vivo as a short isoform that lacks the predicted N-terminal cyclin domain and does not bind cyclin-dependent kinases. Instead, we find that CNTD1 associates with Replication Factor C to drive crossover formation and the Skp1-Cullin1-F-Box complex to regulate ubiquitination and subsequent degradation of the WEE1 kinase, thereby indirectly modulating cell cycle progression. We propose that these interactions enable CNTD1 to orchestrate the steps of prophase I and coordinate crossover formation with cellular division.

cell biology↗

bioRxiv2019

A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex

Endocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma membrane. Membrane-activated complexes are also phosphorylated, but the significance of this mark is debated. We recently proposed that NECAP negatively regulates AP2 by binding open and phosphorylated complexes (Beacham et al., 2018). Here, we report high-resolution cryo-EM structures of NECAP bound to phosphorylated AP2. The site of AP2 phosphorylation is directly coordinated by residues of the NECAP PHear domain that are predicted from genetic screens in C. elegans. Using membrane mimetics to generate conformationally open AP2, we find that a second domain of NECAP binds these complexes and cryo-EM reveals both domains of NECAP engaging closed, inactive AP2. Assays in vitro and in vivo confirm these domains cooperate to inactivate AP2. We propose that phosphorylation marks adaptors for inactivation.

cell biology↗

bioRxiv2018

Endopeptidase regulation as a novel function of the Zur-dependent zinc starvation response

The cell wall is a strong, yet flexible, meshwork of peptidoglycan (PG) that gives a bacterium structural integrity. To accommodate a growing cell, the wall is remodeled by both PG synthesis and degradation. Vibrio cholerae encodes a group of three nearly identical zinc-dependent endopeptidases (EPs) that hydrolyze PG to facilitate cell growth. Two of these (shyA and shyC) are housekeeping genes and form a synthetic lethal pair, while the third (shyB) is not expressed under standard laboratory conditions. To investigate the role of ShyB, we conducted a transposon screen to identify mutations that activate shyB transcription. We found that shyB is induced as part of the Zur-mediated zinc starvation response, a mode of regulation not previously reported for cell wall lytic enzymes. In vivo, ShyB alone was sufficient to sustain cell growth in low-zinc environments. In vitro, ShyB retained its D,D-endopeptidase activity against purified sacculi in the presence of the metal chelator EDTA at a concentration that inhibits ShyA and ShyC. This suggests that ShyB can substitute for the other EPs during zinc starvation, a condition that pathogens encounter while infecting a human host. Our survey of transcriptomic data from diverse bacteria identified other candidate Zur-regulated endopeptidases, suggesting that this adaptation to zinc starvation is conserved in other Gram-negative bacteria.\n\nImportanceThe human host sequesters zinc and other essential metals in order to restrict growth of potentially harmful bacteria. In response, invading bacteria express a set of genes enabling them to cope with zinc starvation. In Vibrio cholerae, the causative agent of the diarrheal disease cholera, we have identified a novel member of this zinc starvation response: a cell wall hydrolase that retains function in low-zinc environments and is conditionally essential for cell growth. Other human pathogens contain homologs that appear to be under similar regulatory control. These findings are significant because they represent, to our knowledge, the first evidence that zinc homeostasis influences cell wall turnover. Anti-infective therapies commonly target the bacterial cell wall and, therefore, an improved understanding of how the cell wall adapts to host-induced zinc starvation could lead to new antibiotic development. Such therapeutic interventions are required to combat the rising threat of drug resistant infections.

microbiology↗