Biology subjects

Ballabio, F.

Publications and source records attributed to Ballabio, F..

6 recordsLinked to original sources

bioRxiv2026

Calcium-Driven Activation of Gelsolin: Mechanism and the Active-State Ensemble

Gelsolin is a master regulator of the actin cytoskeleton whose activity is controlled by large Ca2+ dependent conformational transitions of its 6 domains (G1-G6). Despite decades of biochemical and cellular studies, the structural basis of activation of the full-length protein remains poorly understood because active gelsolin is highly dynamic and has largely escaped structural characterization. Here, we combine small-angle X-ray scattering, DEER-EPR spectroscopy, and structure-based molecular dynamics simulations to investigate the transition from the compact inactive state to the actin-binding active form. This integrative approach reveals that activation proceeds through a sequential and domain-dependent mechanism, where different regions of the protein respond to Ca2+ with distinct kinetics and conformational trajectories. Rather than converging toward a single active structure, activated gelsolin populates a heterogeneous ensemble of plastic conformations characterized by asymmetric domain arrangements and extensive structural variability. The two major domain triplets (G1-G3 and G4-G6) display markedly different dynamic behaviors, while actin-binding surfaces are exposed according to a defined hierarchy and remain differentially accessible within the active ensemble, suggesting specialized functional roles for different domains during filament engagement. Together, these findings provide a time- and domain-resolved view of gelsolin activation, establish a structural framework for interpreting its diverse biological functions, and offer new opportunities for targeting gelsolin-associated diseases.

biophysics↗

bioRxiv2025

Constant-pH simulation of the human β2 adrenergic receptor inactivation

Understanding the molecular basis of pH-dependent G protein-coupled receptor (GPCR) signaling is crucial for comprehending physiological regulation and drug design. Here, we investigate the human {beta}2 adrenergic receptor ({beta}2AR), a prototypical GPCR whose function is sensitive to pH conditions. Employing extensive constant-pH molecular dynamics simulations, we provide a detailed atomistic characterization of {beta}2AR inactivation across physiologically relevant pH values (4-9). Our simulations reveal that {beta}2AR inactivation is closely linked to protonation events at critical residues, notably E2686x30 involved in the ionic lock formation. Furthermore, we find that inactivation occurs without direct sodium binding to the ion-binding pocket around residue D792x50. Instead, sodium ions predominantly interact with D1133x32, effectively blocking deeper entry toward the traditional binding site. These results challenge existing mechanistic models and highlight the necessity of accurately modeling electrostatics in GPCR simulations. Our findings underscore the potential of constant-pH methodologies to advance the understanding of GPCR dynamics, influencing both fundamental biology and therapeutic strategies.

biophysics↗

bioRxiv2024

A conformational fingerprint for amyloidogenic light chains.

Immunoglobulin light chain amyloidosis (AL) and multiple myeloma (MM) both share the overproduction of a clonal light chain (LC). However, while LCs in MM remain soluble in circulation, AL LCs misfold into toxic soluble species and amyloid fibrils that accumulate in organs, leading to distinct clinical manifestations. The significant sequence variability of LCs has hindered understanding of the mechanisms driving LC aggregation. Nevertheless, emerging biochemical properties, including dimer stability, conformational dynamics, and proteolysis susceptibility, distinguish AL LCs from those in MM under native conditions. This study aimed to identify a conformational fingerprint distinguishing AL from MM LCs. Using small-angle X-ray scattering (SAXS) under native conditions, we analyzed four AL and two MM LCs. We observed that AL LCs exhibited a slightly larger radius of gyration and greater deviations from X-ray crystallography-determined or predicted structures, reflecting enhanced conformational dynamics. SAXS data, integrated with molecular dynamics (MD) simulations, revealed a conformational ensemble where LCs adopt multiple states, with variable and constant domains either bent or straight. AL LCs displayed a distinct, low-populated, straight conformation (termed H state), which maximized solvent accessibility at the interface between constant and variable domains. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experimentally validated this H state. These findings reconcile diverse experimental observations and provide a precise structural target for future drug design efforts.

biophysics↗

bioRxiv2024

A missense mutation in the gene encoding the Mg-chelatase subunit I leads to a viable pale green line with phenotypic features of potential interest for barley breeding programs

The pale green trait, i.e. reduced chlorophyll content, has been shown to increase the efficiency of photosynthesis and biomass accumulation when photosynthetic microorganisms and tobacco plants are cultivated at high densities. Thus, the hus1 barley mutant is defective in photosystem antenna biogenesis, and exhibits a 50% reduction in leaf chlorophyll content. Nevertheless, its agronomical performance under standard field conditions is comparable to that of the wild-type. This supports the notion that crops can decrease their investment in antenna proteins and chlorophyll biosynthesis without detrimental effects on productivity. Here, we assess the effects of reducing leaf chlorophyll content in barley by altering the chlorophyll biosynthesis pathway (CBP). To this end, we have isolated and characterised the pale green barley mutant xan-h.chli-1, which carries a missense mutation in the Xan-h gene for subunit I of Mg-chelatase (HvCHLI), the first enzyme in the CBP. Intriguingly, xan-h.chli-1 is the only known viable homozygous mutant at the Xan-h locus in barley. The Arg298Lys amino-acid substitution in the ATP-binding cleft causes a slight decrease in HvCHLI protein abundance, and a marked reduction in Mg-chelatase activity. Under controlled growth conditions, mutant plants display reduced accumulation of antenna and photosystem core subunits, together with reduced photosystem II yield relative to wild type under moderate illumination, and consistently higher than wild-type levels at high light intensities. Moreover, the reduced content of leaf chlorophyll is associated with a stable reduction in daily transpiration rate, and slight decreases in total biomass accumulation and water-use efficiency. These traits are reminiscent of phenotypic features of wild barley accessions and landraces that thrive under arid climatic conditions. Overall, our findings make the xan-h.chli-1 allelic variant of potential interest for tailoring barley, and other crop plants, for growth in harsh environments.

plant biology↗

bioRxiv2024

Calcium-driven In Silico Inactivation of a Human Olfactory Receptor

Conformational changes as well as molecular determinants related to the activation and inactivation of olfactory receptors are still poorly understood due to the intrinsic difficulties in the structural determination of this GPCR family. Here, we perform, for the first time, the in silico inactivation of the human olfactory receptor OR51E2, highlighting the possible role of calcium in this receptor state transition. Using molecular dynamics simulations, we show that a divalent ion in the ion binding site, coordinated by two acidic residues at positions 2.50 and 3.39 conserved across most ORs, stabilizes the receptor in its inactive state. In contrast, protonation of the same two acidic residues is not sufficient to drive inactivation within the {micro}s timescale of our simulations. Our findings suggest a novel molecular mechanism for OR inactivation, potentially guiding experimental validation and offering insights into the possible broader role of divalent ions in GPCR signaling.

biophysics↗

bioRxiv2022

Spike mutation resilient scFv76 antibody counteracts SARS-CoV-2 lung damage upon aerosol delivery

Uneven worldwide vaccination coverage against SARS-CoV-2 and emergence of variants escaping immunity call for broadly-effective and easily-deployable therapeutics. We previously described the human single-chain scFv76 antibody, which recognizes SARS-CoV-2 Alfa, Beta, Gamma and Delta variants. We now show that scFv76 also neutralizes infectivity and fusogenic activity of Omicron BA.1 and BA.2 variants. Cryo-EM analysis reveals that scFv76 binds to a well-conserved SARS-CoV-2 spike epitope, providing the structural basis for its broad-spectrum activity. Moreover, we demonstrate that nebulized scFv76 exhibits therapeutic efficacy in a severe hACE2 transgenic mouse model of COVID-19 pneumonia, as shown by body weight and pulmonary viral load data. Counteraction of infection correlates with the inhibition of lung inflammation observed by histopathology and expression of inflammatory cytokines and chemokines. Biomarkers of pulmonary endothelial damage were also significantly reduced in scFv76-treated mice. Altogether the results support the use of nebulized scFv76 for COVID-19 induced by any SARS-CoV-2 variants emerged so far.

microbiology↗