Biology subjects

Cassidy, C. K.

Publications and source records attributed to Cassidy, C. K..

13 recordsLinked to original sources

bioRxiv2026

Full length TECPR1 displays cis Dysferlin domain architecture

Tectonin Beta-Propeller Repeat containing 1 (TECPR1) is an essential regulator of a noncanonical autophagy pathway known as Sphingomyelin TECPR1 induced LC3 lipidation (STIL). TECPR1 forms an E3-like ligase complex and recognizes exposed sphingomyelin on damaged membranes. TECPR1 contains five folded domains however the structural basis for TECPR1 function has remained unresolved. Here, we report the first structure of full length TECPR1 resolved using cryo electron microscopy. TECPR1 forms an elongated hook shaped architecture that positions Dysferlin domains in a cis arrangement. Our structure uncovers an uncharacterized intramolecular interface between tectonin repeat 1 and PH domains. This interaction forms a stabilizing bridge that contributes to the orientation of the DysF domains. Molecular dynamics simulations further demonstrate that TECPR1 maintains the overall structural arrangement during membrane association. Our data provide a structural framework for how TECPR1 domain arrangement corresponds with membrane binding.

biochemistry↗

bioRxiv2026

Structure and signaling mechanism of Helicobacter pylori transducer-like protein D

Chemoreceptors, or methyl-accepting chemotaxis proteins (MCPs), are ancient and widespread prokaryotic sensors that direct taxis in response to stimuli and are attractive targets for therapeutic control of bacteria 1-4. Decades of study have yielded substantial mechanistic insight into chemoreceptor function, but the absence of high-resolution full-length structures containing ligand-binding domains (LBD) has limited understanding of how effector sensing is structurally coupled to long-range signal transduction. Here, we present the intact structure of the chemoreceptor transducer-like protein D (TlpD) from the gastric pathogen Helicobacter pylori, in complex with its ligand Zn2+, determined by X-ray crystallography in two crystal forms at 2.4-3.0 [A]. Three different conformations are captured, revealing how interactions in the ligand-binding site of the chemoreceptor zinc-binding (CZB) domain are interconnected with the distal kinase interface. Small changes at the ligand-binding site coincide with cascades of side-chain rearrangements across the dimer, distortion of the receptor coiled-coil, and conformational and dynamic shifts at the kinase interface over 140 [A] away. These near-atomic resolution structures provide a framework for understanding cooperativity and allosteric communication in chemoreceptors, and establish a representative model for a widespread class of soluble chemoreceptors important in bacterial pathogenesis 2,5.

molecular biology↗

bioRxiv2025

Uncovering genetic architecture of the heart via genetic association studies of unsupervised deep learning derived endophenotypes

Recent genome-wide association studies (GWAS) have effectively linked genetic variants to quantitative traits derived from time-series cardiac magnetic resonance imaging, revealing insights into cardiac morphology and function. Deep learning approach generally requires extensive supervised training on manually annotated data. In this study, we developed a novel framework using a 3D U-architecture autoencoder (cineMAE) to learn deep image phenotypes from cardiac magnetic resonance (CMR) imaging for genetic discovery, focusing on long-axis two-chamber and four-chamber views. We trained a masked autoencoder to develop Unsupervised Derived Image Phenotypes for heart (Heart-UDIPs). These representations were found to be informative to indicate various heart-specific phenotypes (e.g., left ventricular hypertrophy) and diseases (e.g., hypertrophic cardiomyopathy). GWAS on Heart UDIP identified 323 lead SNP and 628 SNP-prioritized genes, which exceeded previous methods. The genes identified by method described herein, exhibited significant associations with cardiac function and showed substantial enrichment in pathways related to cardiac disorders. These results underscore the utility of our Heart-UDIP approach in enhancing the discovery potential for genetic associations, without the need for clinically defined phenotypes or manual annotations.

bioinformatics↗

bioRxiv2024

The Structure of ApoB100 from Human Low-density Lipoprotein

Low-density lipoprotein (LDL) plays a central role in lipid and cholesterol metabolism and is a key molecular agent involved in the development and progression of atherosclerosis, a leading cause of mortality worldwide. Apolipoprotein B100 (apoB100), one of the largest proteins in the genome, is the primary structural and functional component of LDL, yet its size and complex lipid associations have posed major challenges for structural studies. Here we overcome those challenges and present the first structure of apoB100 from human LDL using an integrative approach of cryo-electron microscopy, AlphaFold2, and molecular dynamics-based refinement. The structure consists of a large globular N-terminal domain that leads into a [~]58 nm long x 4 nm wide continuous amphipathic {beta}-sheet that wraps completely around the circumference of the particle, holding it together like a belt. Distributed symmetrically across the two sides of the {beta}-belt are 9 strategically located inserts that vary in size from [~]30-700 residues and appear to have diverse functions. The largest two form long flexible strings of paired amphipathic helices that extend across the lipid surface to provide additional structural support through specific long-range interactions. These results suggest a mechanism for how the various domains of apoB100 act in concert to maintain LDL shape and cohesion across a wide range of particle sizes. More generally, they advance our fundamental understanding of LDL form and function and will help accelerate the design of potential new therapeutics.

biophysics↗

bioRxiv2023

Structure of the Native Chemotaxis Core Signalling Unit from E-gene lysed E. coli cells

Motile bacteria detect ambient chemical gradients and control their locomotion via conserved chemotaxis signaling networks, enabling cells to locate nutrients, potential hosts and other important biological niches. The sensory apparatus of the chemotaxis pathway is an array of core-signaling units (CSU) composed of transmembrane chemoreceptors, the histidine kinase CheA and an adaptor protein CheW. Although chemotaxis pathways represent the best understood signaling systems, a detailed mechanistic understanding of signal transduction has been hindered by the lack of a complete structural picture of the CSU and extended array. In this study, we present the structure of the complete CSU from phage E-gene lysed E. coli cells, determined using cryo-electron tomography and sub-tomogram averaging to 12 [A] resolution. Using AlphaFold2, we further predict the atomic structures of the CSUs constituent proteins as well as key protein-protein interfaces, enabling the assembly an all-atom CSU model, which we conformationally refine using our cryoET map. Molecular dynamics simulations of the resulting model provide new insight into the periplasmic organization of the complex, including novel interactions between neighboring receptor ligand binding domains. Our results further elucidate previously unresolved interactions between individual CheA domains, including an anti-parallel P1 dimer and non-productive binding mode between P1 and P4, enhancing our understanding of the structural mechanisms underlying CheA signaling and regulation.

microbiology↗

bioRxiv2022

Structural basis for antagonism of the ubiquitin ligase BIRC6 by SMAC

Apoptosis, a form of genetically programmed cell death, can be triggered by either internal or external signals ultimately activating caspases, a family of proteases1. Certain members of the inhibitors of apoptosis (IAP) family are sentinel proteins preventing untimely cell death by inhibiting caspases. IAPs are in turn regulated by antagonists including second mitochondria-derived activator of caspase (SMAC). Baculoviral IAP repeat-containing protein 6 (BIRC6), a giant IAP, possesses dual E2/E3 ubiquitin ligase activity and is implicated in apoptosis via caspase inhibition2-7. How this is achieved remains unknown. Here we show BIRC6 directly restricts activated caspase-3, and ubiquitinates activated caspases-3, -7 and -9 working exclusively with the non-canonical E1, UBA6. Importantly, we show SMAC supresses both mechanisms. Cryo-electron microscopy (cryo-EM) structures of BIRC6 alone and in complex with SMAC reveal BIRC6 exists as an anti-parallel dimer with a substrate-binding module juxtaposed to the catalytic domain at each end, and we identify multiple highly conserved unannotated domains important for architecture and function. Through our structural, biochemical and biophysical findings, we discover SMAC engages BIRC6 at multiple sites resulting in a sub-nanomolar affinity enabling SMAC to competitively displace caspases, thus antagonising BIRC6-mediated caspase inhibition.

biochemistry↗

bioRxiv2022

LipIDens: Simulation assisted interpretation of lipid densities in cryo-EM structures of membrane proteins.

Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures. The pipeline integrates the implementation and analysis of multi-scale MD simulations for identification, ranking and refinement of lipid binding poses which superpose onto cryo-EM map densities. Thus, LipIDens enables direct integration of experimental and computational structural approaches to facilitate the interpretation of lipid-like cryo-EM densities and to reveal the molecular identities of protein-lipid interactions within a bilayer environment. The LipIDens code is open-source and embedded within a notebook format to assist automation and usability.

biophysics↗

bioRxiv2022

High resolution reconstruction of a Jumbo bacteriophage infecting capsulated bacteria using hyperbranched tail fibers

The Klebsiella jumbo myophage {phi}Kp24 displays an unusually complex arrangement of tail fibers interacting with a host cell. In this study, we combined cryo-electron microscopy methods, protein structure prediction methods, molecular simulations, and machine learning approaches to explore the capsid, tail, and tail fibers of this phage at high resolution. We determined the structure of the capsid and tail at 4.3[A] and 4.1[A] resolution. We observed that the tail fibers were highly branched and rearranged dramatically upon cell surface attachment. This complex configuration involves fourteen putative tail fibers with depolymerase activity that provide {phi}Kp24 with the ability to infect a broad panel of capsular polysaccharide (CPS) types of Klebsiella pneumoniae. Taken together, our study provides structural and functional insight into how {phi}Kp24 adapts to the highly variable surfaces of capsulated bacterial pathogens, which will be useful for the development of phage therapy approaches against pan-drug resistant K. pneumoniae strains.

microbiology↗

bioRxiv2021

UVC inactivation of pathogenic samples suitable for cryoEM analysis

Cryo-electron microscopy has become an essential tool to understand structure and function of biological samples, from individual proteins to whole cells. Especially for pathogens, such as disease-causing bacteria and viruses, insights gained by cryo-EM can aid in developing cures. However, due to the biosafety restrictions of human pathogens, samples are often treated by chemical fixation to render the pathogen inert, affecting the delicate ultrastructure of the sample. Alternatively, researchers use in vitro or ex vivo models, which are non-pathogenic but lack the complexity of the pathogen of interest. Here we show that ultraviolet-C (UVC) radiation at cryogenic temperatures can be used to eliminate or dramatically reduce the infectivity of two model organisms, a pathogenic bacterium (Vibrio cholerae) and a virus-like particle (the ICP1 bacteriophage). We show no discernable structural impact of this treatment of either sample using two cryo-EM methods: cryo-electron tomography (cryo-ET) followed by sub-tomogram averaging (STA), and single particle analysis (SPA). Additionally, we applied the UVC irradiation to the protein apoferritin (ApoF), which is a widely used test sample for high resolution SPA studies. The UVC-treated ApoF sample resulted in a 2.1 [A] structure that did not reveal any discernable structural damage. Together, these results show that the UVC irradiation dose that effectively inactivates cryo-EM samples does not negatively impact their structure. This research demonstrates that UVC treatment is an effective and inexpensive addition to the cryo-EM sample preparation toolbox.

molecular biology↗

bioRxiv2021

Alternative architecture of the E. coli chemosensory array

Chemotactic responses in motile bacteria are the result of sophisticated signal transduction by large, highly organized arrays of sensory proteins. Despite tremendous progress in the understanding of chemosensory array structure and function, a structural basis for the heightened sensitivity of networked chemoreceptors is not yet complete. Here we present cryo-electron tomography visualisations of native-state chemosensory arrays in E. coli minicells. Strikingly, these arrays exhibit a p2-symmetric array architecture that differs markedly from the p6-symmetric architecture previously described in E. coli. Based on this data, we propose molecular models of this alternative architecture and the canonical p6-symmetric assembly. We evaluate our observations and each model in the context of previously published data, assessing the functional implications of an alternative architecture and effects for future studies.

biochemistry↗

bioRxiv2020

A bacterial inflammation sensor regulates c-di-GMP signaling, adhesion, and biofilm formation.

The reactive oxygen species produced during inflammation through the neutrophilic respiratory burst play profound roles in combating bacterial pathogens and regulating the microbiota. Among these, the neutrophilic oxidant bleach, hypochlorous acid (HOCl), is the most prevalent and strongest oxidizer and kills bacteria through non-specific oxidation of proteins, lipids, and DNA. Thus, HOCl can be viewed as a host-specific cue that conveys important information about what bacterial physiology and lifestyle programs may be required for successful colonization. Nevertheless, bacteria that colonize animals face a molecular challenge in how to achieve highly selective detection of HOCl due to its reactive and transient nature and chemical similarity to more benign and non-host-specific oxidants like hydrogen peroxide (H2O2). Here, we report that in response to increasing HOCl levels E. coli regulates biofilm production via activation of the diguanylate cyclase DgcZ. We show the molecular mechanism of this activation to be specific oxidation of a conserved cysteine that coordinates the zinc of its regulatory chemoreceptor zinc-binding (CZB) domain, forming a zinc-cysteine redox switch 685-fold more sensitive to oxidation by HOCl over H2O2. Dissection of the signal transduction mechanism through quantum mechanics, molecular dynamics, and biochemical analyses reveal how the cysteine redox state alters the delicate equilibrium of competition for Zn++ between the CZB domain and other zinc binders to relay the presence of HOCl through activating the associated GGDEF domain to catalyze c-di-GMP. We find biofilm formation and HOCl-sensing in vivo to be regulated by the conserved cysteine, and point mutants that mimic oxidized CZB states increase production of the biofilm matrix polymer poly-N-acetylglucosamine and total biofilm. We observe CZB-regulated diguanylate cyclases and chemoreceptors in phyla in which host-associated bacteria are prevalent and are possessed by pathogens that manipulate host inflammation as part of their colonization strategy. A phylogenetic survey of all known CZB sequences shows these domains to be conserved and widespread across diverse phyla, suggesting CZB origin predates the bacterial last universal common ancestor. The ability of bacteria to use CZB protein domains to perceive and thwart the host neutrophilic respiratory burst has implications for understanding the mechanisms of diseases of chronic inflammation and gut dysbiosis.

microbiology↗

bioRxiv2020

The unconventional cytoplasmic sensing mechanism for ethanol chemotaxis in Bacillus subtilis

Motile bacteria sense chemical gradients using chemoreceptors, which consist of distinct sensing and signaling domains. The general model is that the sensing domain binds the chemical and the signaling domain induces the tactic response. Here, we investigated the unconventional sensing mechanism for ethanol taxis in Bacillus subtilis. Ethanol and other short-chain alcohols are attractants for B. subtilis. Two chemoreceptors, McpB and HemAT, sense these alcohols. In the case of McpB, the signaling domain directly binds ethanol. We were further able to identify a single amino-acid residue Ala431 on the cytoplasmic signaling domain of McpB, that when mutated to a serine, reduces taxis to ethanol. Molecular dynamics simulations suggest ethanol binds McpB near residue Ala431 and mutation of this residue to serine increases coiled-coil packing within the signaling domain, thereby reducing the ability of ethanol to bind between the helices of the signaling domain. In the case of HemAT, the myoglobin-like sensing domain binds ethanol, likely between the helices encapsulating the heme group. Aside from being sensed by an unconventional mechanism, ethanol also differs from many other chemoattractants because it is not metabolized by B. subtilis and is toxic. We propose that B. subtilis uses ethanol and other short-chain alcohols to locate prey, namely alcohol-producing microorganisms. ImportanceEthanol is a chemoattractant for Bacillus subtilis even though it is not metabolized and inhibits growth. B. subtilis likely uses ethanol to find ethanol-fermenting microorganisms for prey. Two chemoreceptors sense ethanol: HemAT and McpB. HemATs myoglobin-like sensing domain directly binds ethanol, but the heme group is not involved. McpB is a transmembrane receptor consisting of an extracellular sensing domain and a cytoplasmic signaling domain. While most attractants bind the extracellular sensing domain, we found that ethanol directly binds between inter-monomer helices of the cytoplasmic signaling domain of McpB, using a mechanism akin to those identified in many mammalian ethanol-binding proteins. Our results indicate that the sensory repertoire of chemoreceptors extends beyond the sensing domain and can directly involve the signaling domain.

microbiology↗

bioRxiv2019

Complete structure of the core signalling unit of the E. coli chemosensory array in an optimised minicell strain

Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays.1,2 Understanding stimulus detection and transmission at the molecular level requires precise structural characterisation of the array building block known as a core signalling unit (CSU). Here we introduce a novel E. coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays. We provide a three-dimensional (3D) map of a complete CSU at ~16 [A] resolution by cryo-electron tomography (cryo-ET) and subtomogram averaging. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields an atomistic model of the membrane-bound CSU and enables spatial localisation of its signalling domains. Our work thus offers a solid structural basis for interpretation of existing data and design of new experiments to elucidate signalling mechanisms within the CSU and larger array.

cell biology↗