Biology subjects

Gragera, M.

Publications and source records attributed to Gragera, M..

4 recordsLinked to original sources

bioRxiv2026

High-resolution cryo-EM structure of integrin αIIbβ3 bound to disease-causing maternal HPA-1a antibody that blocks integrin activation

Integrins promote immunity, embryonic development, wound healing, and hemostasis, and are activated by bent/closed to extended/open conformational changes. Integrin IIb{beta}3, being crucial for platelet activation and aggregation, is a therapeutic target for bleeding disorders and thrombosis. Human Platelet Antigen-1a (HPA-1a) on {beta}3 is recognized by pregnancy-associated maternal alloantibodies, potentially causing fetal/neonatal alloimmune thrombocytopenia (FNAIT) and even intracranial hemorrhage or perinatal death. However, severe disease determinants are largely unknown. We report the first structure of an anti-HPA-1a antibody fragment (Fab 26.4) in complex with integrin IIb{beta}3 at high resolution by cryo-electron microscopy. Fab 26.4 binding traps IIb{beta}3 in the inactive, bent/closed conformation, is incompatible with integrin extension, and inhibits IIb{beta}3-dependent fibrinogen binding and platelet aggregation. Thus, anti-HPA-1a antibodies directly impair integrin activation by preventing required conformational changes. These insights will improve FNAIT diagnostics and treatment, and spark the development of novel allosteric inhibitors against {beta}3 integrins for future therapeutic applications.

biophysics↗

bioRxiv2025

How many (distinguishable) classes can we identify in Single Particle Analysis?

Heterogeneity in cryoEM is essential for capturing macromolecule structural variability, reflecting their functional states and biological significance. However, estimating heterogeneity remains challenging due to particle misclassification and algorithmic biases, which can lead to reconstructions that blend distinct conformations or fail to resolve subtle differences. Furthermore, the low signal-to-noise ratio (SNR) inherent in cryo-EM data makes it nearly impossible to detect minute structural changes, as noise often obscures subtle variations in macromolecular projections. In this paper, we investigate the use of p-values associated with the null hypothesis that the observed classification differs from a random partition of the input dataset, thereby providing a statistical framework for determining the number of distinguishable classes present in a given dataset.

molecular biology↗

bioRxiv2025

Insights from aquaporin structures into drug-resistant sleeping sickness

Trypanosoma brucei is the causal agent of African trypanosomiasis in humans and animals, the latter resulting in significant negative economic impacts in afflicted areas of the world. Resistance has arisen to the trypanocidal drugs pentamidine and melarsoprol through mutations in the aquaglyceroporin TbAQP2 that prevent their uptake. Here we use cryogenic electron microscopy to determine the structure of TbAQP2 from Trypanosoma brucei, bound to either the substrate glycerol or to the sleeping sickness drugs, pentamidine or melarsoprol. The drugs bind within the AQP2 channel at a site completely overlapping that of glycerol. Mutations leading to a drug-resistant phenotype were found in the channel lining. Molecular dynamics simulations showed the channel can be traversed by pentamidine, with a low energy binding site at the centre of the channel, flanked by regions of high energy association at the extracellular and intracellular ends. Drug-resistant TbAQP2 mutants are still predicted to bind pentamidine, but the much weaker binding in the centre of the channel observed in the MD simulations would be insufficient to compensate for the high energy processes of ingress and egress, hence impairing transport at pharmacologically relevant concentrations. The structures of drug-bound TbAQP2 represent a novel paradigm for drug-transporter interactions that could provide new mechanisms for targeting drugs into pathogens and human cells.

molecular biology↗

bioRxiv2025

Activation Mechanism and Structural Assembly of the Mycobacterium tuberculosis ClpP1P2 Protease and Its Associated ATPases.

Supramolecular assemblies are integral to cellular biochemical processes, relying on their dynamic nature to fulfill essential functions. The protease ClpP1P2, paired with ATPase partners ClpC1 or ClpX, is vital for the survival of Mycobacterium tuberculosis (Mtb). While the ClpP1P2 complex requires activation by specific N-blocked dipeptides (e.g., Z-Leu-Leu) to exhibit proteolytic activity in vitro, the mechanism of in vivo activation remains unclear. In this study, we identified novel activators that enabled the structural determination of the ClpC1P1P2 complex, providing insights into its assembly. Furthermore, we discovered that trehalose - a key metabolite and molecular crowding agent in Mtb, significantly enhances the activity of both ClpC1P1P2 and ClpXP1P2 complexes without the need for activating peptides. Analytical ultracentrifugation revealed that trehalose promotes the formation of these active complexes, mimicking intracellular conditions. These findings propose a new model of Clp system activation in vivo, offering promising avenues for therapeutic targeting in tuberculosis treatment. Significance StatementThe proteolytic complex formed by the essential proteins ClpP1 and ClpP2, along with their specific ATP-dependent activators ClpX and ClpC1, has emerged as a highly attractive target for anti-tuberculosis drug development. While previous studies have shown that ClpP1P2 can be activated in vitro by small peptide activators, its in vivo activation mechanism remains unclear. In this study, we identify novel activators and demonstrate that trehalose, a key metabolite in Mycobacterium tuberculosis, enhances ClpC1P1P2 and ClpXP1P2 activity without the need for activating peptides. These findings propose a new model for Clp system activation in Mycobacterium tuberculosis, advancing our understanding of its regulation and potential as a therapeutic target.

biochemistry↗