Biology subjects

des Georges, A.

Publications and source records attributed to des Georges, A..

10 recordsLinked to original sources

bioRxiv2026

Structural basis for direct NGF/TrkA blockade by an analgesic antibody

The NGF/TrkA signaling axis is a central mediator of inflammatory and chronic pain, where injury-induced NGF binds and activates TrkA on nociceptive neurons to drive peripheral sensitization and persistent pain states. Despite its therapeutic promise, targeting this pathway is limited by adverse effects of systemic NGF sequestration such as rapidly progressive osteoarthritis and poor isoform selectivity of Trk kinase inhibitors leading to off-target neurological effects. Targeting the TrkA extracellular domain (TrkAECD) offers a pathway to achieve high isoform selectivity while avoiding these complications. However, the precise structural basis for selective TrkA neutralization remains poorly understood. Monoclonal antibody (mAb) 42F5-15 inhibits TrkA-mediated signaling and increases pain threshold. Here, we report the high-resolution (2.60 [A]) cryo-EM structure of the TrkAECD in complex with the Fab region of the TrkA-neutralizing mAb 42F5-15. Structural analysis reveals that the antibody epitope overlaps the NGF-binding interface, consistent with orthosteric inhibition and distinct from previously proposed allosteric mechanisms. The epitope includes residues conserved in TrkA but divergent in TrkB and TrkC, providing a structural basis for receptor isoform selectivity. Furthermore, we demonstrate in vivo that the mAb 42F5-15 potently mitigates mechanical allodynia and nociceptive sensitization. These findings establish a structural framework for the development of selective extracellular TrkA-targeted therapies for safer, non-opioid chronic pain management.

neuroscience↗

bioRxiv2025

Connecting the dots: deep learning-based automated model building methods in cryo-EM

The resolution revolution in single particle cryo-electron microscopy (cryo-EM) has dramatically expanded our structural knowledge of large biomolecular complexes. While high-resolution cryo-EM density maps enable atomic model building, lower-resolution maps can still reveal secondary structures, folds, and domains. When combined with integrative modeling approaches, such data can provide meaningful insights into biomolecular structure and function. Constructing accurate models, however, remains challenging: at low resolutions it is difficult to interpret density maps features reliably, and at high resolutions traditional model-building workflows can become a time-consuming bottleneck. Deep learning, which is transforming problem-solving across scientific domains, offers powerful new tools to automate and accelerate this process. In this review, we discuss deep learning-based methods developed to automate model building in cryo-EM density maps, assessing their impact on streamlining structure determination. Recognizing that biomacromolecular structures exhibit hierarchical organization, we classify these methods according to their ability to model primary, secondary, tertiary, and quaternary structures of biomolecules. Deep learning tools for building atomic models in cryo-EM density maps are further grouped as de novo, where the model is predicted directly from features learned from the cryo-EM density, or hybrid, where it is derived by integrating structural templates with these features. We outline current limitations, including the challenge of obtaining sufficiently large and diverse datasets for training networks to model different types of biomolecules in the cryo-EM density maps, and the open challenge of constructing training sets that capture the conformational heterogeneity often observed in the cryo-EM maps. We conclude by highlighting emerging directions for this rapidly advancing field, which promise to make automated, data-driven model building an integral part of structural biology.

bioinformatics↗

bioRxiv2025

The translation initiation factor DHX29 appears to pull on mRNA in a direction opposite to scanning.

The DExH-box helicase DHX29 plays a critical role in mammalian translation initiation. It is required for the scanning of mRNAs with complex 5UTRs. Despite its importance, the detailed mechanism of DHX29s action has remained debated. Using structural models derived from AlphaFold and cryo-EM structure of DHX29 bound to the ribosomal 43S pre-initiation complex, we provide a revised structural framework that clarifies the interplay between DHX29, the 40S ribosomal subunit, and eIF3. Our findings suggest that the 40S subunit regulates DHX29s NTPase activity through an activation mechanism resembling the G-patch protein regulation of DEAH helicases. Moreover, our model supports a 3 to 5 translocase mechanism, in which DHX29 transiently pulls the mRNA opposite to the scanning direction, destabilizing stable stem-loops trapped in the mRNA channel and allowing scanning to resume. This structural analysis refines our understanding of DHX29s function and provides new hypotheses regarding its role in mRNA unwinding during scanning and start codon selection.

biophysics↗

bioRxiv2024

Beta-arrestin 1 mediated Src activation via Src SH3 domain revealed by cryo-electron microscopy

Beta-arrestins ({beta}arrs) are key regulators and transducers of G-protein coupled receptor signaling; however, little is known of how {beta}arrs communicate with their downstream effectors. Here, we report the first structural insights into the fundamental mechanisms driving {beta}arr-mediated signal transduction. Using cryo-electron microscopy, we elucidate how {beta}arr1 recruits and activates the non-receptor tyrosine kinase Src, the first identified signaling partner of {beta}arrs. {beta}arr1 engages Src SH3 through two distinct sites, each employing a different recognition mechanism: a polyproline motif in the N-domain and a non-proline-based interaction in the central crest region. At both sites {beta}arr1 interacts with the aromatic surface of SH3, disrupting the autoinhibited conformation of Src and directly triggering its allosteric activation. This structural evidence establishes {beta}arr1 as an active regulatory protein rather than a passive scaffold and suggests a potentially general mechanism for {beta}arr-mediated signaling across diverse effectors.

biochemistry↗

bioRxiv2023

Endosomal activation of the Clostridioides difficile binary toxin is Ca2+-dependent

The C. difficile binary toxin (CDT) enters host cells via endosomal delivery like many other AB-type binary toxins. In this study, the cell-binding component of CDT, termed CDTb, was found to bind and form pores in lipid bilayers upon depleting free Ca2+ ion concentrations, and not by lowering pH, as found for other binary toxins (i.e., anthrax). Cryoelectron microscopy, nuclear magnetic resonance spectroscopy, surface plasmon resonance, electrochemical impedance spectroscopy, CDT toxicity studies, and site directed mutagenesis show that dissociation of Ca2+ from a single site in receptor binding domain 1 (RBD1) of CDTb is consistent with a molecular mechanism in which Ca2+ dissociation from RBD1 induces a "trigger" via conformational exchange that enables CDTb to bind and form pores in endosomal membrane bilayers as free Ca2+ concentrations decrease during CDT endosomal delivery. One-Sentence SummaryCa2+ dissociation and unfolding of a receptor binding domain in CDTb activates pore-formation by the C. difficile binary toxin.

biophysics↗

bioRxiv2023

Visualization of translation reorganization upon persistent collision stress in mammalian cells

Aberrantly slow mRNA translation leads to ribosome stalling and subsequent collision with the trailing neighbor. Ribosome collisions have recently been shown to act as stress sensors in the cell, with the ability to trigger stress responses balancing survival and apoptotic cell-fate decisions depending on the stress level. However, we lack a molecular understanding of the reorganization of translation processes over time in mammalian cells exposed to an unresolved collision stress. Here we visualize the effect of a persistent collision stress on translation using in situ cryo electron tomography. We observe that low dose anisomycin collision stress leads to the stabilization of Z-site bound tRNA on elongating 80S ribosomes, as well as to the accumulation of an off-pathway 80S complex possibly resulting from collision splitting events. We visualize collided disomes in situ, occurring on compressed polysomes and revealing a stabilized geometry involving the Z-tRNA and L1 stalk on the stalled ribosome, and eEF2 bound to its collided rotated-2 neighbor. In addition, non-functional post-splitting 60S complexes accumulate in the stressed cells, indicating a limiting Ribosome associated Quality Control clearing rate. Finally, we observe the apparition of tRNA-bound aberrant 40S complexes shifting with the stress timepoint, suggesting a succession of different initiation inhibition mechanisms over time. Altogether, our work visualizes the changes of translation complexes under persistent collision stress in mammalian cells, indicating how perturbations in initiation, elongation and quality control processes contribute to an overall reduced protein synthesis. SummaryUsing in situ cryo electron tomography we visualized the reorganization of mammalian translation processes during a persistent collision stress.

cell biology↗

bioRxiv2019

Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a novel macromolecular assembly

Targeting Clostridium difficile infection (CDI) is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent CDI often has a binary toxin termed the C. difficile toxin (CDT), in addition to the enterotoxins TsdA and TsdB. CDT has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single particle cryoEM, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, two novel di-heptamer structures for activated CDTb (aCDTb; 1.0 MDa) were solved at atomic resolution including a symmetric (SymCDTb; 3.14 [A]) and an asymmetric form (AsymCDTb; 2.84 [A]). Roles played by two receptor-binding domains of aCDTb were of particular interest since RBD1 lacks sequence homology to any other known toxin, and the RBD2 domain is completely absent in other well-studied heptameric toxins (i.e. anthrax). For AsymCDTb, a novel Ca2+ binding site was discovered in RBD1 that is important for its stability, and RBD2 was found to be critical for host cell toxicity and the novel di-heptamer fold for both forms of aCDTb. Together, these studies represent a starting point for structure-based drug-discovery strategies to targeting CDT in the most severe strains of CDI. SIGNIFICANCE STATEMENTThere is a high burden from C. difficile infection (CDI) throughout the world, and the Center for Disease Control (CDC) reports more than 500,000 cases annually in the United States, resulting in an estimated 15,000 deaths. In addition to the large clostridial toxins, TcdA/TcdB, a third C. difficile binary toxin (CDT) is associated with the most serious outbreaks of drug resistant CDI in the 21st century. Here, structural biology and biophysical approaches were used to characterize the cell binding component of CDT, termed CDTb, at atomic resolution. Surprisingly, two novel structures were solved from a single sample that help to explain the molecular underpinnings of C. difficile toxicity. These structures will also be important for targeting this human pathogen via structure-based therapeutic design methods.

biophysics↗

bioRxiv2018

Functional Pathways of Biomolecules Retrieved from Single-particle Snapshots

We present a new approach to determining the conformational changes associated with biological function, and demonstrate its capabilities in the context of experimental single-particle cryo-EM snapshots of ryanodine receptor (RyR1), a Ca2+-channel involved in skeletal muscle excitation/contraction coupling. These results include the detailed conformational motions associated with functional paths including transitions between energy landscapes. The functional motions differ substantially from those inferred from discrete structures, shedding new light on the gating mechanism in RyR1. The differences include the conformationally active structural domains, the nature, sequence, and extent of conformational motions involved in function, and the way allosteric signals are transduced within and between domains. The approach is general, and applicable to a wide range of systems and processes.

biophysics↗

bioRxiv2017

Routine Single Particle CryoEM Sample and Grid Characterization by Tomography

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are freely floating away from the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1,000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

biophysics↗

bioRxiv2017

Conformational Dynamics and Energy Landscapes of Ligand Binding in RyR1

Using experimental single-particle cryo-EM snapshots of ryanodine receptor (RyR1), a Ca2+-channel involved in skeletal muscle excitation/contraction coupling, we present quantitative free-energy landscapes, reaction coordinates, and three-dimensional movies of the continuous conformational changes associated with the binding of activating ligands. Our results show multiple routes to ligand binding with comparable branching ratios. All high-probability routes involve significant conformational changes before and after the binding of ligands. We also present new insights into the local structural changes along the ligand-binding route, including accommodations at the calcium, ATP, and caffeine binding sites. These observations shed new light on the mechanisms and conformational routes to ligand binding.

biophysics↗