Biology subjects

Hammel, M.

Publications and source records attributed to Hammel, M..

11 recordsLinked to original sources

bioRxiv2026

Germline-encoded V(D)J gene usage does not impose strict constraints on the epitope-specificity of T cell receptors

The theoretical diversity of T cell receptors (TCRs), generated through V(D)J recombination, is enormous, yet the diversity of TCRs capable of recognizing the same epitope remains unknown. Defining this TCR solution space is essential for uncovering basic principles that govern TCR specificity. Using single-cell RNA and TCR sequencing, we generated ultra-deep (more than 4000 unique TCRs per epitope) epitope-specific TCR libraries derived from 560 immunized C57BL/6 mice, identifying over 27,000 unique epitope-reactive TCRs across three distinct CD8+ T cell epitopes presented by two major histocompatibility complex (MHC) class I alleles. Saturation analyses indicated that the solution space for all studied epitopes comprises many tens of thousands of unique TCRs. Despite highly skewed and peptide-dependent VJ-usage patterns, nearly the entire set of functional germline V/ and J/ segments was detected at least once within each epitope-specific repertoire. Therefore, diversity of epitope-specific TCRs is not limited by distinct germline combinations but rather can emerge from a near-to-complete combinatorial space of - and -chain, V and J segments paired with compatible CDR3 sequences.

immunology↗

bioRxiv2025

Diversity-driven biochemical survey reveals dimeric structural origin of rubisco

Rubisco is the entry point of nearly all organic carbon into the biosphere and is present in all domains of life. Despite its global importance, biochemical studies of this enzyme superfamily have been limited to a relatively narrow set of subclades. Recent advances in metagenomics have dramatically reshaped our understanding of both microbial and rubisco diversity; however, biochemical characterization of these sequences has not kept pace with the exponential growth in sequence data. To better survey the functional and structural diversity of rubisco, we systematically sampled and synthesized a library of diverse rubisco sequences with an emphasis on clades that have previously not been characterized. Our updated phylogenetic analysis reveals that many deep-branching rubiscos assemble as dimers, supporting a dimeric origin for the superfamily -- in contrast to the ecologically dominant hexadecameric form I. Additionally, we discover and structurally characterize the largest rubisco described to date, originating from a cryptic, early-branching subclade with novel structural folds that have previously not been observed in the rubisco superfamily. By integrating biochemical data with an updated phylogenetic framework, we propose a revised nomenclature for the rubisco protein family that reflects current insights and will better accommodate future discoveries.

biochemistry↗

bioRxiv2025

Predicting pose distribution of protein domains connected by flexible linkers is an unsolved problem

In CASP16, we assessed the ability of computational methods to predict the distribution of relative orientations of two domains tethered by a flexible linker. The range of interdomain distances and orientations (poses) of such domain-linker-domain (D-L-D) proteins can play an important role in protein function, allostery, aggregation, and the thermodynamics of binding. The CASP16 Conformational Ensembles Experiment included two challenges to predict the interdomain pose distribution of a Staphylococcal protein A (SpA) D-L-D construct, called ZLBT-C, in which two of SpAs five nearly identical domains are connected by either (1) a six-residue wild-type (WT) linker (kadnkf), or (2) an all-glycine (Gly6) linker. The wild-type linker has a highly conserved sequence and is thought to contribute to the energetic barrier for binding with host antibodies. Ground truth was provided by nuclear magnetic resonance (NMR) residual dipolar coupling (RDC) data on WT protein and small angle X-ray scattering (SAXS) data on both proteins in solution. Twenty-five predictor groups submitted 35 sets of predicted conformational distributions, in the form of population-weighted finite ensembles of discrete structures. Unlike traditional CASP assessments that compare predicted atomic models to experimental atomic models, the accuracy of these predictions was assessed by back-calculating NMR RDCs and SAXS curves from each ensemble of atomic models and comparing these results to respective experimental data. Accuracy was also assessed by using kernelization to compare ensembles to the continuous orientational distributions optimally fit to experimental data. In our assessment, predictions spanned a wide range of accuracy, but none were close fits to the combined NMR and SAXS data. In addition, none were able to recapitulate the observed difference between WT and Gly6 proteins, as observed in the SAXS data. These results, and our analysis, highlighted strengths and weaknesses, plus complementarity of NMR RDC and SAXS analysis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/650885v2_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@148f520org.highwire.dtl.DTLVardef@d5450eorg.highwire.dtl.DTLVardef@1a8c6aborg.highwire.dtl.DTLVardef@18b2b5b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Horizontal transfer of nuclear DNA in transmissible cancer

Although somatic cell genomes are usually entirely clonally inherited, nuclear DNA exchange between cells of an organism can occur sporadically by cell fusion, phagocytosis or other mechanisms1-3. This phenomenon has long been noted in the context of cancer, where it could be envisaged that DNA horizontal transfer plays a functional role in disease evolution4-13. However, an understanding of the frequency and significance of this process in naturally occurring tumours is lacking. The host-tumour genetic discordance of transmissible cancers, malignant clones which pass between animals as allogeneic grafts, provides an opportunity to investigate this. We screened for host-to-tumour horizontal transfer of nuclear DNA in 174 tumours from three transmissible cancers affecting dogs and Tasmanian devils, and detected a single instance in the canine transmissible venereal tumour (CTVT). This involved introduction of a 15-megabase dicentric genetic element, composed of 11 rearranged fragments of six chromosomes, to a CTVT sublineage occurring in Asia around 2,000 years ago. The element forms the short arm of a small submetacentric chromosome, and derives from a dog with ancestry associated with the ancient Middle East. The introduced DNA fragment is transcriptionally active and has adopted the expression profile of CTVT. Its 143 genes do not, however, confer any obvious advantage to its spatially restricted CTVT sublineage. Our findings indicate that nuclear DNA horizontal transfer, although likely a rare event in tumour evolution, provides a viable mechanism for the acquisition of genetic material in naturally occurring cancer genomes.

cancer biology↗

bioRxiv2024

Predicting RNA Structure and Dynamics with Deep Learning and Solution Scattering

Advanced deep learning and statistical methods can predict structural models for RNA molecules. However, RNAs are flexible, and it remains difficult to describe their macromolecular conformations in solutions where varying conditions can induce conformational changes. Small-angle X-ray scattering (SAXS) in solution is an efficient technique to validate structural predictions by comparing the experimental SAXS profile with those calculated from predicted structures. There are two main challenges in comparing SAXS profiles to RNA structures: the absence of cations essential for stability and charge neutralization in predicted structures and the inadequacy of a single structure to represent RNAs conformational plasticity. We introduce Solution Conformation Predictor for RNA (SCOPER) to address these challenges. This pipeline integrates kinematics-based conformational sampling with the innovative deep-learning model, IonNet, designed for predicting Mg2+ ion binding sites. Validated through benchmarking against fourteen experimental datasets, SCOPER significantly improved the quality of SAXS profile fits by including Mg2+ ions and sampling of conformational plasticity. We observe that an increased content of monovalent and bivalent ions leads to decreased RNA plasticity. Therefore, carefully adjusting the plasticity and ion density is crucial to avoid overfitting experimental SAXS data. SCOPER is an efficient tool for accurately validating the solution state of RNAs given an initial, sufficiently accurate structure and provides the corrected atomistic model, including ions. The method is available from: https://github.com/dina-lab3d/IonNet Our pipeline is available for use as a web server: https://bilbomd.bl1231.als.lbl.gov/ Statement of SignificanceUnderstanding the behavior of RNA in solution is critical for deciphering its biological functions, yet predicting its macromolecular conformation remains challenging. While advanced computational methods can predict RNA structures, their accuracy in solution is often limited by the absence of stabilizing ions and the failure to account for RNAs conformational flexibility. This study presents SCOPER, an innovative tool that addresses these challenges by integrating deep-learning-based ion binding site prediction with conformational sampling, offering a more reliable approach to validate and refine RNA structures against experimental SAXS data. We provide our source code and a web server that runs the pipeline.

molecular biology↗

bioRxiv2023

Marine transmissible cancer navigates urbanised waters, threatening to spillover

Inter-individual transmission of cancer cells represents a unique form of microparasites increasingly reported in marine bivalves. In this study, we sought to understand the ecology of the propagation of Mytilus trossulus Bivalve Transmissible Neoplasia 2 (MtrBTN2), a transmissible cancer affecting four Mytilus mussel species worldwide. We investigated the prevalence of MtrBTN2 in the mosaic hybrid zone of M. edulis and M. galloprovincialis along the French Atlantic coast, sampling contrasting natural and anthropogenic habitats. We observed a similar prevalence in both species, likely due to the spatial proximity of the two species in this region. Our results showed that ports had higher prevalence of MtrBTN2, with a possible hotspot observed at a shuttle landing dock. No cancer was found in natural beds except for two sites close to the hotspot, suggesting spillover. Ports may provide favourable conditions for the transmission of MtrBTN2, such as high mussel density, stressful conditions, sheltered and confined shores, or buffered temperatures. Ships may also spread the disease through biofouling. Our results suggest ports may serve as epidemiological hubs, with maritime routes providing artificial gateways for MtrBTN2 propagation. This highlights the importance of preventing biofouling on docks and ship hulls to limit the spread of marine pathogens hosted by fouling species.

ecology↗

bioRxiv2021

Prevalence and polymorphism of a mussel transmissible cancer in Europe

Transmissible cancers are parasitic malignant cell lineages that acquired the ability to infect new hosts from the same species, or sometimes related species. First described in dogs and Tasmanian devils, transmissible cancers were later discovered in some marine bivalves affected by a leukemia-like disease. In Mytilus mussels, two lineages of Bivalve Transmissible Neoplasia (BTN), both emerged in a M. trossulus founder individual, have been described to date (MtrBTN1 and MtrBTN2). Here, we performed an extensive screening of genetic chimerism, a hallmark of transmissible cancer, by genotyping hundred SNPs of thousands of European Mytilus mussels. The genetic analysis allowed us to simultaneously obtain the genotype of hosts -M. edulis, M. galloprovincialis or hybrids- and the genotype of tumors of heavily infected individuals. In addition, a subset of individuals were systematically genotyped and analysed by histology in order to screen for possible non-transmissible cancers. We detected MtrBTN2 at low prevalence in M. edulis, and also in M. galloprovincialis and hybrids although at a much lower prevalence. No MtrBTN1 or new BTN were found but a few individuals with non-transmissible neoplasia were observed at a single polluted site on the same sampling date. We observed a diversity of MtrBTN2 genotypes that appeared more introgressed or more ancestral than MtrBTN1 and reference healthy M. trossulus individuals. The observed polymorphism is most likely due to somatic null alleles caused by structural variations or point mutations in primer-binding sites leading to enhanced detection of the host alleles. Despite low prevalence, two divergent sublineages, confirmed by mtCOI sequences, are co-spreading in the same geographic area, suggesting a complex diversification of MtrBTN2 since its emergence and host species shift.

evolutionary biology↗

bioRxiv2021

A structural basis for inhibition of the complement initiator protease C1r by Lyme disease spirochetes

Complement evasion is a hallmark of extracellular microbial pathogens such as Borreliella burgdorferi, the causative agent of Lyme disease. Lyme disease spirochetes express nearly a dozen outer surface lipoproteins that bind complement components and interfere with their native activities. Among these, BBK32 is unique in its selective inhibition of the classical pathway. BBK32 blocks activation of this pathway by selectively binding and inhibiting the C1r serine protease of first component of complement, C1. To understand the structural basis for BBK32-mediated C1r inhibition, we performed co-crystallography and size exclusion chromatography-coupled small angle x-ray scattering experiments, which revealed a molecular model of BBK32-C in complex with activated human C1r. Structure-guided site-directed mutagenesis was combined with surface plasmon resonance binding experiments and assays of complement function to validate the predicted molecular interface. The studies reported here, for the first time, provide a structural basis for classical pathway-specific inhibition by a human pathogen.

microbiology↗

bioRxiv2021

Rigid monoclonal antibodies improve detection of SARS-CoV-2 nucleocapsid protein

Monoclonal antibodies (mAbs) are the basis of treatments and diagnostics for pathogens and other biological phenomena. We conducted a structural characterization of mAbs against the N-terminal domain of nucleocapsid protein (NPNTD) from SARS-CoV-2 using small angle X-ray scattering (SAXS). Our solution-based results distinguished the mAbs flexibility and how this flexibility impacts the assembly of multiple mAbs on an antigen. By pairing two mAbs that bind different epitopes on the NPNTD, we show that flexible mAbs form a closed sandwich-like complex. With rigid mAbs, a juxtaposition of the Fabs is prevented, enforcing a linear arrangement of the mAb pair, which facilitates further mAb polymerization. In a modified sandwich ELISA, we show the rigid mAb-pairings with linear polymerization led to increased NPNTD detection sensitivity. These enhancements can expedite the development of more sensitive and selective antigen-detecting point-of-care lateral flow devices (LFA), key for early diagnosis and epidemiological studies of SARS-CoV-2 and other pathogens.

biophysics↗

bioRxiv2020

Protein Corona Composition and Dynamics on Carbon Nanotubes in Blood Plasma and Cerebrospinal Fluid

When a nanoparticle enters a biological environment, the surface is rapidly coated with proteins to form a "protein corona". Presence of the protein corona surrounding the nanoparticle has significant implications for applying nanotechnologies within biological systems, affecting outcomes such as biodistribution and toxicity. Herein, we measure protein corona formation on single-stranded DNA wrapped single-walled carbon nanotubes (ssDNA-SWCNTs), a high-aspect ratio nanoparticle ideal for sensing and delivery applications, and polystyrene nanoparticles, a model nanoparticle system. The protein corona of each nanoparticle is studied in human blood plasma and cerebrospinal fluid. We characterize corona composition by proteomic mass spectrometry to determine abundant and differentially enriched vs. depleted corona proteins. High-binding corona proteins on ssDNA-SWCNTs include proteins involved in lipid binding and transport (clusterin and apolipoprotein A-I), complement activation (complement C3), and blood coagulation (fibrinogen). Of note, albumin is the most common blood protein (55% w/v), yet exhibits low-binding affinity towards ssDNA-SWCNTs, displaying 1300-fold lower bound concentration relative to native plasma. We investigate the role of electrostatic and entropic interactions driving selective protein corona formation, and find that hydrophobic interactions drive inner corona formation, while shielding of electrostatic interactions allows for outer corona formation. Lastly, we study real-time binding of proteins on ssDNA-SWCNTs and find relative agreement between proteins that are enriched and bind strongly, such as fibrinogen, and proteins that are depleted and bind marginally, such as albumin. Interestingly, certain proteins express contrary behavior in single-protein experiments than within the whole biofluid, highlighting the importance of cooperative mechanisms driving selective corona adsorption on the SWCNT surface. Knowledge of the protein corona composition, dynamics, and structure informs translation of engineered nanoparticles from in vitro design to effective in vivo application.

bioengineering↗

bioRxiv2019

Allosteric activation of the nitric oxide receptor soluble guanylate cyclase mapped by cryo-electron microscopy

Soluble guanylate cyclase (sGC) is the primary receptor for nitric oxide (NO) in mammalian nitric oxide signaling. We determined structures of full-length Manduca sexta sGC in both inactive and active states using cryo-electron microscopy. NO and the sGC-specific stimulator YC-1 induce a 71{degrees} rotation of the heme-binding {beta} H-NOX and PAS domains. Repositioning of the {beta} H-NOX domain leads to a straightening of the coiled-coil domains, which, in turn, use the motion to move the catalytic domains into an active conformation. YC-1 binds directly between the {beta} H-NOX domain and the two CC domains. The structural elongation of the particle observed in cryo-EM was corroborated in solution using small angle X-ray scattering (SAXS). These structures delineate the endpoints of the allosteric transition responsible for the major cyclic GMP-dependent physiological effects of NO.

biochemistry↗