Biology subjects

Stoddard, B. L.

Publications and source records attributed to Stoddard, B. L..

15 recordsLinked to original sources

bioRxiv2026

The activity, divergence, and evolutionary degradation of modern-day homing endonucleases and their reconstructed ancestors

Homing endonucleases (HEs) are selfish genetic elements that drive the mobilization of their own coding sequences, often in concert with surrounding introns. Homing endonuclease genes (HEGs) usually display life cycles in which they accumulate inactivating mutations after invading a host genomic target site, leading to eventual removal from the genome. We identified several hundred novel HEGs and determined the distribution of their proteins behaviors and activities. Approximately 10% are expressed as properly folded functional proteins that cleave predictable DNA target sites. Another [~]20% display significant expression but little to no cleavage activity; the remainder display severely reduced expression. Despite the presence of debilitating mutations throughout most HEGs, ancestral reconstructions yielded endonucleases with improved expression and stability. One such reconstruction, at a hypothetical node preceding highly diverged HEs that cleave unique target sites, binds (but does not cleave) their individual targets. It instead cleaves a DNA sequence that represents a hybrid of those modern-day DNA targets, while displaying a specificity profile that resembled those of previously characterized HEs. Its DNA-bound crystal structure adds detail to our understanding of how homing endonuclease DNA contacting surfaces and residues shift and rearrange during evolution, ultimately leading to their action at new target sites.

biochemistry↗

bioRxiv2026

De novo design of a macrocycle induced dimerization system for cellular control

Investigating and manipulating cellular events requires precise control of protein function. To enable control over cellular processes, we set out to design a chemically induced dimerization (CID) system consisting of a de novo designed ligand and protein pair. Here we describe the design of a C2 symmetric membrane permeable macrocyclic peptide and a cognate protein homodimer which binds the macrocycle through a large interface with both chains. The designed homodimer binds the macrocycle with a KD of 36 nM, and the x-ray crystal structure of the protein homodimer-macrocycle complex is very close to the computational design model, with the C2 axis of the macrocycle aligned with the homodimer C2 axis. Transcriptional and split luciferase assays in mammalian cells demonstrates conditional control over both a reporter gene expression and luciferase reconstitution.

biochemistry↗

bioRxiv2026

Competing forms of protein-protein association and DNA binding exhibited by BrxC from the BREX phage restriction system

Bacteriophage exclusion (BREX) defense systems restrict phage infection via inhibition of phage DNA replication, while also modifying and protecting the bacterial genome. Type I BREX systems encode six conserved proteins, including a site-specific DNA methyltransferase. Host methylation requires a subset of BREX proteins, whereas phage restriction generally requires them all, suggesting that distinct but overlapping complexes mediate these activities. Full details of the mechanism and regulation of BREX remains to be understood. Here, we characterize the behavior and structures of the conserved BrxC AAA+ ATPase protein. BrxC forms multiple competing assemblages - various self-associating multimers, as well as a complex with BrxB-PglZ - that can be uncoupled via distinct point mutations, leading to differing effects on host methylation versus phage restriction. BrxCs self-association, as well as its ability to bind DNA, is regulated by ATP binding and hydrolysis; BrxA and BrxB appear to also regulate those behaviors. These collective results suggest that BrxC may play a key role in controlling the two activities of BREX, with BrxB, BrxC and PglZ forming a core complex, and the equilibrium among competing assemblies containing those proteins modulating the balance between idling and activated restrictive states.

biochemistry↗

bioRxiv2025

De novo design of RNA and nucleoprotein complexes

Nucleic acids fold into sequence-dependent tertiary structures and carry out diverse biological functions, much like proteins. However, while considerable advances have been made in the de novo design of protein structure and function, the same has not yet been achieved for RNA tertiary structures of similar intricacy. Here, we describe a generative diffusion framework, RFDpoly, for generalized de novo biopolymer (RNA, DNA and protein) design, and use it to create diverse and designable RNA structures. We design RNA structures with novel folds and experimentally validate them using a combination of chemical footprinting (SHAPE-seq) and electron microscopy. We further use this approach to design protein-nucleic acid assemblies; the crystal structure of one such design is nearly identical to the design model. This work demonstrates that the principles of structure-based de novo protein design can be extended to nucleic acids, opening the door to creating a wide range of new RNA structures and protein-nucleic acid complexes.

biochemistry↗

bioRxiv2025

PglZ from Type I BREX phage defence systems is a metal-dependent nuclease that forms a sub-complex with BrxB

BREX (Bacteriophage Exclusion) systems, identified through shared identity with Pgl (Phage Growth Limitation) systems, are a widespread, highly diverse group of phage defence systems found throughout bacteria and archaea. The varied BREX Types harbour multiple protein subunits (between four and eight) and all encode a conserved putative phosphatase (PglZ aka BrxZ) and an equally conserved, putative ATPase (BrxC). Almost all BREX systems also contain a site-specific methyltransferase (PglX aka BrxX). Despite having determined the structure and fundamental biophysical and biochemical behaviours for the PglX methyltransferase, the BrxL effector, the BrxA DNA-binding protein and the BrxR transcriptional regulator, the mechanism by which BREX impedes phage replication remains largely undetermined. In this study, we identify a stable BREX sub-complex of PglZ:BrxB, validate the structure and dynamic behaviour of that sub-complex, and assess the biochemical activity of PglZ, revealing it to be a metal-dependent nuclease. PglZ can cleave cyclic oligonucleotides, linear oligonucleotides, plasmid DNA and both non-modified and modified linear phage genomes. PglZ nuclease activity has no obvious role in BREX-dependent methylation, but does contribute to BREX phage defence. BrxB binding does not impact PglZ nuclease activity. These data contribute to our growing understanding of the BREX phage defence mechanism.

biochemistry↗

bioRxiv2023

Computational design of sequence-specific DNA-binding proteins

Sequence-specific DNA-binding proteins (DBPs) play critical roles in biology and biotechnology, and there has been considerable interest in the engineering of DBPs with new or altered specificities for genome editing and other applications. While there has been some success in reprogramming naturally occurring DBPs using selection methods, the computational design of new DBPs that recognize arbitrary target sites remains an outstanding challenge. We describe a computational method for the design of small DBPs that recognize specific target sequences through interactions with bases in the major groove, and employ this method in conjunction with experimental screening to generate binders for 5 distinct DNA targets. These binders exhibit specificity closely matching the computational models for the target DNA sequences at as many as 6 base positions and affinities as low as 30-100 nM. The crystal structure of a designed DBP-target site complex is in close agreement with the design model, highlighting the accuracy of the design method. The designed DBPs function in both Escherichia coli and mammalian cells to repress and activate transcription of neighboring genes. Our method is a substantial step towards a general route to small and hence readily deliverable sequence-specific DBPs for gene regulation and editing.

synthetic biology↗

bioRxiv2023

Design of four component T=4 tetrahedral, octahedral, and icosahedral protein nanocages through programmed symmetry breaking

Four, eight or twenty C3 symmetric protein trimers can be arranged with tetrahedral (T-sym), octahedral (O-sym) or icosahedral (I-sym) point group symmetry to generate closed cage-like structures1,2. Generating more complex closed structures requires breaking perfect point group symmetry. Viruses do this in the icosahedral case using quasi-symmetry or pseudo-symmetry to access higher triangulation number architectures3-9, but nature appears not to have explored higher triangulation number tetrahedral or octahedral symmetries. Here, we describe a general design strategy for building T = 4 architectures starting from simpler T = 1 structures through pseudo-symmetrization of trimeric building blocks. Electron microscopy confirms the structures of T = 4 cages with 48 (T-sym), 96 (O-sym), and 240 (I-sym) subunits, each with four distinct chains and six different protein-protein interfaces, and diameters of 33nm, 43nm, and 75nm, respectively. Higher triangulation number viruses possess very sophisticated functionalities; our general route to higher triangulation number nanocages should similarly enable a next generation of multiple antigen displaying vaccine candidates10,11 and targeted delivery vehicles12,13.

biochemistry↗

bioRxiv2023

Stepwise design of pseudosymmetric protein hetero-oligomers

Pseudosymmetric hetero-oligomers with three or more unique subunits with overall structural (but not sequence) symmetry play key roles in biology, and systematic approaches for generating such proteins de novo would provide new routes to controlling cell signaling and designing complex protein materials. However, the de novo design of protein hetero-oligomers with three or more distinct chains with nearly identical structures is a challenging problem because it requires the accurate design of multiple protein-protein interfaces simultaneously. Here, we describe a divide-and-conquer approach that breaks the multiple-interface design challenge into a set of more tractable symmetric single-interface redesign problems, followed by structural recombination of the validated homo-oligomers into pseudosymmetric hetero-oligomers. Starting from de novo designed circular homo-oligomers composed of 9 or 24 tandemly repeated units, we redesigned the inter-subunit interfaces to generate 15 new homo-oligomers and recombined them to make 17 new hetero-oligomers, including ABC heterotrimers, A2B2 heterotetramers, and A3B3 and A2B2C2 heterohexamers which assemble with high structural specificity. The symmetric homo-oligomers and pseudosymmetric hetero-oligomers generated for each system share a common backbone, and hence are ideal building blocks for generating and functionalizing larger symmetric assemblies. Significance StatementProtein oligomers composed of multiple unique subunits are versatile building blocks for creating functional materials and controlling biological processes. However, designing robust hetero-oligomers with distinct subunits and precise structural symmetry remains a major challenge. Here, we present a general strategy for designing such complexes by breaking down the problem into simpler steps by first symmetrically re-designing the interfaces of homo-oligomeric proteins, and then recombining validated variants to form pseudosymmetric hetero-oligomers. Using this method, we generated 17 hetero-oligomers with up to three unique subunits that assemble with high specificity. Our approach can be extended to create a wide range of pseudosymmetric assemblies for manipulating cellular signaling and as building blocks for advanced protein materials. These pseudosymmeteric heterotrimers have already enabled the construction of a set of massive nanocages, including a T=4 icosahedral nanocage with a 70 nm diameter and 240 subunits.1

biochemistry↗

bioRxiv2022

Immunization with a self-assembling nanoparticle vaccine displaying EBV gH/gL protects humanized mice against lethal viral challenge

Summary/AbstractEpstein-Barr virus (EBV) is a cancer-associated pathogen responsible for 140,000 deaths per year. EBV is also the etiological agent of infectious mononucleosis and is associated with multiple sclerosis and rheumatoid arthritis. Thus, an EBV vaccine could alleviate significant morbidity and mortality. EBV is orally transmitted and has tropism for both epithelial cells and B cells which are present in the oral cavity. Therefore, a prophylactic vaccine would need to prevent infection of both cell types. Passive transfer neutralizing monoclonal antibodies targeting the viral gH/gL glycoprotein complex prevent experimental EBV infection in humanized mice and rhesus macaques, suggesting that gH/gL is an attractive vaccine candidate. Here, we produced and evaluated the immunogenicity of several nanoparticle immunogens displaying gH/gL with distinct valencies and geometries. After one or two immunizations, all nanoparticles elicited superior binding and neutralizing titers relative to monomeric gH/gL. Antibodies elicited by a computationally designed self-assembling nanoparticle that displays 60 copies of the gH/gL protein conferred protection against a lethal dose of EBV in a humanized mouse challenge model, whereas antibodies elicited by monomeric gH/gL did not. Taken together, these data motivate further development of gH/gL nanoparticle vaccine candidates for EBV.

immunology↗

bioRxiv2021

Identification and characterization of BrxR as a regulatory gene in the BREX phage restriction system

Bacteriophage exclusion ( BREX) phage restriction systems are found in a wide range of bacteria. Various BREX systems encode unique combinations of proteins that usually include a site-specific methyltransferase; none appear to contain a nuclease. Here we describe the identification and characterization of a Type I BREX system from Acinetobacter and the effect of deleting each BREX ORF on growth, methylation, and restriction. We identified a previously uncharacterized gene in the BREX operon that is dispensable for methylation but involved in restriction. Biochemical and crystallographic analyses of this factor, which we term BrxR ( BREX Regulator), demonstrate that it forms a homodimer and specifically binds a DNA target site upstream of its transcription start site. Deletion of the BrxR gene causes cell toxicity, reduces restriction, and significantly increases the expression of BrxC. In contrast, the introduction of a premature stop codon into the BrxR gene, or a point mutation blocking its DNA binding ability, has little effect on restriction, implying that the BrxR coding sequence and BrxR protein play independent functional roles. We speculate that the BrxR coding sequence is involved in cis regulation of anti-phage activity, while the BrxR protein plays an additional regulatory role, perhaps during horizontal transfer.

molecular biology↗

bioRxiv2021

The structural basis of mRNA recognition and binding by eukaryotic pseudouridine synthase PUS1

The chemical modification of RNA bases represents a ubiquitous activity that spans all domains of life. Pseudouridylation is the most common RNA modification and is observed within tRNA, rRNA, ncRNA and mRNAs. Pseudouridine synthase or PUS enzymes include those that rely on guide RNA molecules and others that function as stand-alone enzymes. Among the latter, several have been shown to modify mRNA transcripts. Although recent studies have defined the structural requirements for RNA to act as a PUS target, the mechanisms by which PUS1 recognizes these target sequences in mRNA are not well understood. Here we describe the crystal structure of yeast PUS1 bound to an RNA target that we identified as being a hot spot for PUS1-interaction within a model mRNA at 2.4 [A] resolution. The enzyme recognizes and binds both strands in a helical base-paired RNA duplex, and thus guides the RNA containing the target uridine to the active site for subsequent modification of the transcript. The study also allows us to show the divergence of related PUS1 enzymes and their corresponding RNA target specificities, and to speculate on the basis by which PUS1 binds and modifies mRNA or tRNA substrates.

biophysics↗

bioRxiv2021

Coordination of phage genome degradation versus host genome protection by a bifunctional restriction-modification enzyme visualized by CryoEM

Restriction enzymes that combine DNA methylation and cleavage activities into a single polypeptide or protein assemblage and that modify just one DNA strand for host protection are capable of more efficient adaptation towards novel target sites. However, they must solve the problem of discrimination between newly replicated and unmodified host sites (needing methylation) and invasive foreign site (needing to lead to cleavage). One solution to this problem might be that the activity that occurs at any given site is dictated by the oligomeric state of the bound enzyme. Methylation requires just a single bound site and is relatively slow, while cleavage requires that multiple unmethylated target sites (often found in incoming, foreign DNA) be brought together into an enzyme-DNA complex to license rapid cleavage. To validate and visualize the basis for such a mechanism, we have determined the catalytic behavior of a bifunctional Type IIL restriction-modification ( RM) enzyme (DrdV) and determined its high-resolution structure at several different stages of assembly and coordination with multiple bound DNA targets using CryoEM. The structures demonstrate a mechanism of cleavage by which an initial dimer is formed between two DNA-bound enzyme molecules, positioning the single endonuclease domain from each enzyme against the others DNA and requiring further oligomerization through differing protein-protein contacts of additional DNA-bound enzyme molecules to enable cleavage. The analysis explains how endonuclease activity is licensed by the presence of multiple target-containing DNA duplexes and provides a clear view of the assembly through 3D space of a DNA-bound RM enzyme synapse that leads to rapid cleavage of foreign DNA.

biochemistry↗

bioRxiv2020

AcrIIA22 is a novel anti-CRISPR that impairs SpyCas9 activity by relieving DNA torsion of target plasmids

To overcome CRISPR-Cas defense systems, many phages and mobile genetic elements encode CRISPR-Cas inhibitors called anti-CRISPRs (Acrs). Nearly all characterized Acrs directly bind Cas proteins to inactivate CRISPR immunity. Here, using functional metagenomic selection, we describe AcrIIA22, an unconventional Acr found in hypervariable genomic regions of clostridial bacteria and their prophages from human gut microbiomes. AcrIIA22 does not bind strongly to SpyCas9 but nonetheless potently inhibits its activity against plasmids. To gain insight into its mechanism, we obtained an X-ray crystal structure of AcrIIA22, which revealed homology to PC4-like nucleic-acid binding proteins. Based on mutational analyses and functional assays, we deduced that acrIIA22 encodes a DNA nickase that relieves torsional stress in supercoiled plasmids. This may render them less susceptible to SpyCas9, which uses free energy from negative supercoils to form stable R-loops. Modifying DNA topology may provide an additional route to CRISPR-Cas resistance in phages and mobile genetic elements.

microbiology↗

bioRxiv2019

Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome

CRISPR-Cas systems protect bacteria and archaea from phages and other mobile genetic elements, which use small anti-CRISPR (Acr) proteins to overcome CRISPR-Cas immunity. Because they are difficult to identify, the natural diversity and impact of Acrs on microbial ecosystems is underappreciated. To overcome this discovery bottleneck, we developed a high-throughput functional selection that isolates acr genes based on their ability to inhibit CRISPR-Cas function. Using this selection, we discovered ten DNA fragments from human oral and fecal metagenomes that antagonize Streptococcus pyogenes Cas9 (SpyCas9). The most potent acr discovered, acrIIA11, was recovered from a Lachnospiraceae phage and is among the strongest known SpyCas9 inhibitors. AcrIIA11 homologs are distributed across multiple bacterial phyla and many divergent homologs inhibit SpyCas9. We show that AcrIIA11 antagonizes SpyCas9 using a different mechanism than that of previously characterized inhibitors. Our study highlights the power of functional selections to uncover widespread Cas9 inhibitors within diverse microbiomes.

microbiology↗

bioRxiv2016

Retargeting: an unrecognized consideration in endonuclease-based gene drive biology

There is intense interest surrounding the use of gene editing nucleases in gene drive systems to control agricultural insect pests and insect vectors of infectious diseases such as malaria, dengue and Zika virus. While gene drive systems offer immense promise for the beneficial modification of deleterious insect populations, their unique mechanism of action also raises novel safety concerns and regulatory issues. A recent US National Academies of Science report provides a list of potential regulatory issues associated with implementation of homologous recombination (HR)-mediated gene drive systems, based on the premise that all such systems would exhibit similar biological behaviors. Here we examine how HR-mediated gene drive systems based on different gene editing nuclease platforms could be affected by mutations that occur during host cell transcription, genome replication, and, in conjunction with gene editing nuclease activity, during HR-mediated gene drive. Our analysis suggests that the same feature that makes RNA-guided nucleases such attractive research tools--their ease of reprogramming by alterations to their guide RNA components--might also contribute to increased rates of retargeting that could influence the long term behavior of RNA-guided gene drive systems. Predictability of behavior over time is an issue that should be addressed by in-depth risk assessment before field testing of organisms incorporating nuclease-mediated gene drives.

synthetic biology↗