Biology subjects

Spyrakis, F.

Publications and source records attributed to Spyrakis, F..

4 recordsLinked to original sources

bioRxiv2025

Dissecting Annexin-A11 into its functional domains revealed calcium as a key regulator for RNA transport and its association with ALS

Recent studies reveal a "hitchhiking" mechanism in neurons, where organelles transported along microtubules carry other cargos via tethering molecules. Annexin A11 (ANXA11), a calcium-dependent phospholipid-binding protein, functions as a tether linking RNA granules to lysosomes, aiding mRNA transport for rapid neuronal responses. Structurally, its N-terminal (Nt) binds RNA, while the C-terminal (Ct) associates with lysosomal membranes. Mutations in ANXA11 linked to Amyotrophic lateral sclerosis (ALS) may disrupt this function. Here, applying a multidisciplinary approach, we revealed that Ca2+ acts as a master regulator of ANXAlls physiological function by modulating its conformational states. Specifically, Ca2+ influences a switch between two conformations: a close state, in which the Nt and Ct interact with each other, and an open state, which occurs in the presence of Ca2+ ions, where this self-interaction is disrupted, allowing the two domains to interact freely with RNA and liposomes. Surprisingly, we observed that both the Ct and Nt are capable of interacting with liposomes and RNA in a Ca2+-dependent manner, and these interactions can occur simultaneously. This dual binding and its calcium-regulated hierarchy finely tunes ANXAlls binding to RNA and lysosomes, promoting a large complex essential for overcoming transport steric hindrance. Moreover, our result showed that the p.D40G mutation, in the Nt domain, associated with ALS, displays destabilized interdomain interactions and bypass Ca2+ regulation, leading to aberrant aggregation. These insights advance our understanding of ANXAlls role in neuronal RNA transport and its disruption in neurodegeneration, highlighting potential targets for therapeutic intervention.

biochemistry↗

bioRxiv2025

Two birds with one stone: a novel potential antibiotic blocking IsdB-mediated heme extraction by Staphylococcus aureus with serendipitous hemoglobin left-shifting activity

Infections caused by Staphylococcus aureus are closely linked to its ability to secure essential nutrients, including iron, which is extracted from the heme of human hemoglobin (Hb) through the iron-regulated surface determinant (Isd) system. The compound 4-[[2-[[5- (1H-indol-3-yl)-1,3,4-oxadiazol-2-yl]sulfanyl]acetyl]amino]benzoate (C35) was recently identified as a new potential antimicrobial agent for its ability to bind Hb and hamper its interaction with the staphylococcal hemophore IsdB in vitro. Here, we show that C35 inhibits S. aureus growth by specifically targeting the hemophore-driven iron acquisition system. Our findings confirm both the potential of C35 as a first-in-class protein-protein interaction inhibitor with antimicrobial activity, and the effectiveness of targeting hemophores as a strategy to inhibit S. aureus growth. To gain information for drug discovery purposes, the X-ray structure of Hb in the presence of the compound was solved. Unexpectedly, we discovered that, rather than the predicted binding pose, the molecule binds to tetrameric Hb in a cleft between the alpha subunits, stabilizing an R2 relaxed Hb conformation. This triggered further investigation of the effect of C35 on Hb functional properties, which showed a pronounced left-shift activity on oxygen binding curve (i.e., it strongly increases the Hb oxygen affinity). These results highlight C35 as a promising dual-acting compound with both antimicrobial activity and the ability to modulate Hb function through non-covalent stabilization of a high-affinity state. Author SummaryStaphylococcus aureus is a dangerous bacterium that can cause severe infections in humans. To grow and survive it needs iron, which it steals from our red blood cells by taking it from hemoglobin, the protein that carries oxygen in the blood. In this study, we focused on a small molecule, called C35, that blocks the interaction between hemoglobin and a key bacterial protein involved in heme acquisition. We found that C35 strongly inhibits the growth of S. aureus when hemoglobin is the only available source of iron, showing a potential new method to starve the pathogen and consequently fight the infection. Surprisingly, we also found that C35 increases the affinity of hemoglobin for oxygen. This dual action makes C35 a unique molecule for future therapeutic development, with potential applications both as a new antimicrobial agent and in the treatment of diseases related to hemoglobin function.

microbiology↗

bioRxiv2023

Time-resolved X-ray solution scattering unveils the sequence of events leading to human Hb heme capture by Staphylococcus aureus IsdB

Infections caused by Staphylococcus aureus depend on its ability to acquire nutrients. One essential nutrient is iron, which is obtained from the heme of the human host hemoglobin (Hb) through a protein machinery called Iron-regulated Surface Determinant (Isd). IsdB is the protein in charge of heme extraction from Hb, which is the first step of the chain of events leading to iron transfer to the bacterium cell interior. In order to elucidate the molecular events leading from the formation of the initial IsdB:Hb complex to heme extraction, we have performed a time-resolved X-ray solution scattering (TR-XSS) investigation combined with a rapid mixing triggering approach. We succeeded in defining the stoichiometry of IsdB:Hb binding and in describing the kinetics of the subsequent structural changes. The presented approach is potentially applicable to unveil the complex kinetic pathways generated by protein-protein interaction in different biological systems.

biochemistry↗

bioRxiv2020

A blueprint for high affinity SARS-CoV-2 Mpro inhibitors from activity-based compound library screening guided by analysis of protein dynamics

The SARS-CoV-2 coronavirus outbreak continues to spread at a rapid rate worldwide. The main protease (Mpro) is an attractive target for anti-COVID-19 agents. Unfortunately, unexpected difficulties have been encountered in the design of specific inhibitors. Here, by analyzing an ensemble of ~30,000 SARS-CoV-2 Mpro conformations from crystallographic studies and molecular simulations, we show that small structural variations in the binding site dramatically impact ligand binding properties. Hence, traditional druggability indices fail to adequately discriminate between highly and poorly druggable conformations of the binding site. By performing ~200 virtual screenings of compound libraries on selected protein structures, we redefine the proteins druggability as the consensus chemical space arising from the multiple conformations of the binding site formed upon ligand binding. This procedure revealed a unique SARS-CoV-2 Mpro blueprint that led to a definition of a specific structure-based pharmacophore. The latter explains the poor transferability of potent SARS-CoV Mpro inhibitors to SARS-CoV-2 Mpro, despite the identical sequences of the active sites. Importantly, application of the pharmacophore predicted novel high affinity inhibitors of SARS-CoV-2 Mpro, that were validated by in vitro assays performed here and by a newly solved X-ray crystal structure. These results provide a strong basis for effective rational drug design campaigns against SARS-CoV-2 Mpro and a new computational approach to screen protein targets with malleable binding sites.

biophysics↗