Biology subjects

Magistrato, A.

Publications and source records attributed to Magistrato, A..

12 recordsLinked to original sources

bioRxiv2026

Charting the Structure and Functional Landscape of the SF3B1 Intrinsically Disordered Region

Despite its essential role in pre-mRNA splicing, the structure and functional significance of the intrinsically disordered N-terminal domain (NTD) of SF3B1, a core component of the SF3b spliceosome complex, remain poorly understood. Here, we combine nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), biochemistry, bioinformatics approaches, with molecular dynamics simulations to characterize the conformational landscape of the SF3B1-NTD and its phosphorylation-dependent remodelling. Specifically, we focus on the 186-341 region (186-341SF3B1), which contains at least four regulatory phosphorylation sites. We reveal that 186-341SF3B1 is highly dynamic, lacking a stable secondary structure, and that phosphorylation of T211, T235, T313, and T328 induces a shift toward more compact states driven by the formation of intramolecular interactions. Additionally, we unveil that 186-341SF3B1 interacts with RNA primarily via the arginine residues flaking one of the conserved tryptophan-centred motifs present within SF3B1-NTD. These interactions are transient and heterogeneous, reflecting a nonspecific yet sequence-dependent recognition mechanism. Collectively, this study provides a comprehensive description of the conformational landscape and functional features of the SF3B1-NTD, and expands the portfolio of its possible interactors, thus broadening our understanding of how intrinsic disorder, post-translational modifications, and sequence-encoded motifs cooperate to regulate pre-mRNA splicing.

biochemistry↗

bioRxiv2026

Glutamine Tautomerization Drives RhoGAP-Aided GTP Hydrolysis in Small Rho GTPases

Rho GTPases promote GTP hydrolysis aided by specific GTPase-activating proteins (GAPs). By alternating between an active GTP-bound and an inactive GDP-bound state, Rho GTPases function as molecular switches regulating cytoskeletal dynamics and cell motility. Despite their biological relevance, the detailed molecular mechanism underlying Rho GTPases catalysis remains contentious. Here, using classical and hybrid quantum-classical molecular dynamics, we resolve the mechanism of GTP hydrolysis in the RhoGAP-RhoA complex. We reveal that GTP hydrolysis proceeds through a dissociative nucleophilic substitution mechanism, driven by an amide [->] imide tautomerization of Gln63, which aids in delivering a proton from the nucleophilic water to the leaving phosphate group. The Gln63 imide tautomer also loosens RhoGAP-RhoA interfacial contacts, allowing solvent molecules to enter and drive a water-mediated reverse tautomerization of Gln63 that restores the catalytically-competent configuration of the RhoA active site. Conservation of key interface residues across Rho/Rho GAP family members suggests that this mechanism may be shared by most Rho GTPases.

biophysics↗

bioRxiv2026

Exploring Disordered Regions of Human Spliceosome Proteins

Introns are removed from messenger RNAs by the spliceosome, a protein-RNA machinery enriched with intrinsically disordered regions (IDRs). Lacking stable 3D structures, IDRs can adopt diverse conformations interlacing proteins and RNAs components of the spliceosome and regulating splicing. Here we performed a comprehensive bioinformatics analysis of the human spliceosome proteome revealing that many proteins contain over 40% of disordered residues. Spliceosome IDRs are mainly driven by compositional bias due to an excess of charged and RS-like sequences, with both the nature and extent of this disorder being broadly conserved evolutionarily. Additionally, these IDRs are frequent targets of post-translational modifications, especially phosphorylation, and are hotspots for cancer-associated mutations, implicated in different cancer types. Our results collectively underscore central role of IDRs in splicing regulation and disease. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/698225v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@a6ed0dorg.highwire.dtl.DTLVardef@11514b7org.highwire.dtl.DTLVardef@103387borg.highwire.dtl.DTLVardef@713fde_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

U2 snRNP recognizes the branch site through a loaded-spring strand-invasion mechanism

Recognition of the branch sequence (BS) by U2 snRNP is a pivotal step in pre-mRNA splicing and spliceosome assembly. Structural studies suggest that BS recognition occurs through a toe-hold-mediated strand-invasion mechanism, in which U2 snRNA progressively base-pairs with the intron to form the branch helix. However, given the limited complementarity between U2 snRNA and the intronic BS, it remains unclear how such strand invasion can occur spontaneously. Here, using all-atom and coarse-grained molecular dynamics simulations, we show that strand invasion proceeds spontaneously once the toehold region is engaged and the TAT-SF1 factor is released. The key finding is that the branch-stem loop (BSL) of U2 snRNA is maintained in a supercoiled, high-energy conformation by TAT-SF1, which acts as a molecular latch holding the BSL in a poised "loaded-spring" state. Displacement of TAT-SF1 allows the BSL to relax, releasing the stored conformational energy that drives strand invasion through local strand-slip and base-pair exchange. Moreover, the simulations reveal that strand invasion can proceed bidirectionally, refining previous models of U2-BS pairing. This work establishes a loaded-spring mechanism as a key physical driver of toehold-mediated strand invasion underlying branch-site recognition within early spliceosome.

molecular biology↗

bioRxiv2025

The Covalent Binding of Splicing Inhibitors occurs via a Zinc- Assisted Enzymatic-like mechanism

Branch-site recognition is a pivotal event in spliceosome assembly, and branch-site antagonists disrupt this process, inducing a splicing rewiring that underlies much of their antitumor activity. Antagonists from the spliceostatin family covalently bind the SF3b complex nearby the branch-site pocket, forming an adduct with Cys26 of the PHF5A zinc finger while remarkably preserving Zn2+ coordination - an unconventional mode of covalent inhibition. Yet the molecular features that activate this normally protected ZnCys4 cysteine and initiate spliceostatins epoxide ring opening have remained obscure. Here, we combine extensive classical and QM/MM molecular dynamics simulations to map the full reactive trajectory of spliceostatin attachment to PHF5A. We identify two alternative noncovalent spliceostatin binding poses and show that only its thermodynamically favoured conformation pre-organizes the epoxide for nucleophilic attack. The local distortion of the ZnCys4 coordination sphere weakens the Zn-Cys26 bond, enabling water-Cys26 exchange and generating a highly reactive nucleophilic thiolate. Covalent bond formation is subsequently accelerated by an Asp34-Lys29 proton relay that activates the epoxide leaving group. Free-energy calculations confirm that the overall reaction is fast and strongly exergonic. Together, these findings provide a complete mechanistic framework for zinc-assisted inhibition of early spliceosomes by branch-site antagonists and advance our fundamental understanding of zinc finger reactivity.

biochemistry↗

bioRxiv2025

Structural flexibility of the human vault protein revealed by high-resolution cryo-EM and molecular dynamics simulations

Vaults are massive ribonucleoprotein complexes, highly conserved and abundant in eukaryotic cells, yet with unclear function. Their thin-walled barrel-shape architecture is composed of two symmetrical, antiparallel half-shells, each containing 39 copies of the major vault protein (MVP). The spacious lumen of the vault suggests a role in cellular transport. To facilitate cargo encapsulation and release, the vault is thought to open into two halves, yet the molecular mechanism governing vault opening remains elusive. Here, we combine cryogenic electron microscopy (cryo-EM) and multi-scale molecular dynamics (MD) simulations to reveal the structural factors giving flexibility to the human vault protein. Using cryo-EM, we identified two alternative conformational states of the human vault, along with the half-vault shell. MD simulations of these structures show extensive, breathing-like motions, porous solvent-exposed surfaces, and distinct structural variability between conformational states. The stable intermediates and the flexibility at the interface of the half vaults together suggest a possible mechanism for the dynamic assembly and disassembly of the vault.

biochemistry↗

bioRxiv2025

Recognition and catalytic mechanism of tRNA m3C methyltransferase METTL6

Post-transcriptional RNA modifications regulate the RNA structural and functional landscape, and thus play a central role in many cellular processes. Accordingly, aberrant RNA modification pathways contribute to a variety of human diseases, including cancer and neurological disorders. However, recognition and catalytic mechanisms of most RNA-modifying proteins remain obscure, due to sparse structural data. Recently, the cryogenic electron microscopy structure of N3-methyl-cytosine (m3C) methyltransferase METTL6 revealed seryl-tRNA synthetase (SerRS) as a cofactor for tRNASer selection, but left the cooperative role of SerRS and the reaction process undefined. Here, combining computer simulations and in vitro experiments, we define the mechanism of concerted substrate identification by METTL6 and SerRS, demonstrating that SerRS pre-organizes the tRNASer variable arm for METTL6 recruitment. We show that once METTL6 is stably docked onto the SerRS-tRNASer complex, thereby ensuring the precise alignment of the reactants, the methylation reaction proceeds spontaneously. Our results establish the catalytic mechanism of m3C methyltransferases and provide the foundation for rational design of targeted therapeutics.

biochemistry↗

bioRxiv2024

All-atom simulations elucidate the molecular mechanismunderlying RNA-membrane interactions

RNA-membrane interactions are starting to emerge as an important organizing force in both natural and synthetic biological systems. Notably, RNA molecules were recently discovered to be present on the extracellular surface of living cells, where they mediate intercellular signalling. Furthermore, RNA-membrane interactions influence the efficacy of lipid-based RNA delivery systems. However, the molecular terms driving RNA localisation at the membrane remain poorly understood. In this work, we investigate how RNA-phospholipid membrane interactions occur, by means of all-atom simulations. We find that among the four RNA nucleobases guanine exhibits the strongest interaction with the membrane due to extensive hydrogen bond formation. Additionally, we show that intra-RNA base pairing present in organised RNA structures significantly hinders RNA binding to the membrane. Elucidating the molecular details of RNA-membrane association will importantly contribute to improving the design of RNA-based drugs as well as lipid-based RNA delivery systems and to parsing out RNA transport and localisation mechanisms.

biophysics↗

bioRxiv2024

All atom molecular dynamics simulations enable ensemble refinement of flexible and mismodelled cryo-EM derived RNA structures

The advent of single-particle cryogenic electron microscopy (cryo-EM) has enabled near-atomic resolution imaging of large macromolecules, enhancing functional insights. However, current cryo-EM refinement tools condense all single-particle images into a single structure, which can misrepresent highly flexible molecules like RNAs. Here, we combine molecular dynamics simulations with cryo-EM density maps to better account for the structural dynamics of a complex and biologically relevant RNA macromolecule. Namely, using metainference, a Bayesian method, we reconstruct an ensemble of structures of the group II intron ribozyme, which better match experimental data, and we reveal inaccuracies of single-structure approaches in modeling flexible regions. An analysis of all RNA-containing structures deposited in the PDB reveal that this issue affects most cryo-EM structures in the 2.5--4 [A] range. Thus, RNA structures determined by cryo-EM require careful handling, and our method may be broadly applicable to other RNA systems.

molecular biology↗

bioRxiv2021

The Conformational Plasticity of the Selectivity Filter Methionines Controls the In-Cell Cu(I) Uptake through the CTR1 transporter

Copper is a trace element vital to many cellular functions. Yet its abnormal levels are toxic to cells, provoking a variety of severe diseases. The high affinity Copper Transporter 1 (CTR1), being the main in-cell copper (Cu(I)) entry route, tightly regulates its cellular uptake via a still elusive mechanism. Here, all-atoms simulations unlock the molecular terms of Cu(I) transport in eukaryotes disclosing that the two Methionine triads, forming the selectivity filter, play an unprecedented dual role both enabling selective Cu(I) transport and regulating its uptake-rate thanks to an intimate coupling between the conformational plasticity of their bulky side chains and the number of bound Cu(I) ions. Namely, the Met residues act as a gate reducing the Cu(I) import-rate when two ions simultaneously bind to CTR1. This may represent an elegant autoregulatory mechanism through which CTR1 protects the cells from excessively high, and hence toxic, in-cell Cu(I) levels. Overall, these outcomes resolve fundamental questions in CTR1 biology and open new windows of opportunity to tackle diseases associated with an imbalanced copper uptake. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/467269v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@75fbe6org.highwire.dtl.DTLVardef@1ece24eorg.highwire.dtl.DTLVardef@fda950org.highwire.dtl.DTLVardef@1770038_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

All-atom Simulations Uncover the Molecular Terms of NKCC1 Transport Mechanism

The secondary-active Na-K-Cl Cotransporter 1 (NKCC1), member of the Cation Chloride Cotransporters (CCC) family, ensures the electroneutral movement of Cl-, Na+, K+ ions across cellular membranes. NKCC1 regulates Cl- homeostasis and cell volume, handling a pivotal role in transepithelial water transport and neuronal excitability. Aberrant NKCC1 transport is hence implicated in a variety of human diseases (hypertension, renal disorders, neuropathies, cancer). Building on the newly-resolved NKCC1 cryo-EM structure, all-atom enhanced sampling simulations unprecedentedly unlock the mechanism of NKCC1-mediated ions transport, assessing the order and the molecular basis of its interdependent ions translocation. Our outcomes strikingly advance the understanding of the physiological mechanism of CCCs transporters and disclose a key role of CCC-conserved asparagine residues, whose side-chain promiscuity ensures the transport of both negatively and positively charged ions along the same translocation route. This study sets a conceptual basis to devise NKCC-selective inhibitors to treat diseases linked to Cl- dishomeostasis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/443869v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@182ddaforg.highwire.dtl.DTLVardef@a83f33org.highwire.dtl.DTLVardef@1cd4012org.highwire.dtl.DTLVardef@296f14_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Allosteric Cross-Talk Among SARS-CoV-2 Spike's Receptor-Binding Domain Mutations Triggers an Effective Hijacking of Human Cell Receptor

The rapid and relentless emergence of novel highly transmissible SARS-CoV-2 variants, possibly decreasing vaccine efficacy, currently represents a formidable medical and societal challenge. These variants frequently hold mutations on the Spike proteins Receptor-Binding Domain (RBD), which, binding to the Angiotensin-Converting Enzyme 2 (ACE2) receptor, mediates viral entry into the host cells. Here, all-atom Molecular Dynamics simulations and Dynamical Network Theory of the wild-type and mutant RBD/ACE2 adducts disclose that while the N501Y mutation (UK variant) enhances the Spikes binding affinity towards ACE2, the N501Y, E484K and K417N mutations (South African variant) aptly adapt to increase SARS-CoV-2 propagation via a two-pronged strategy: (i) effectively grasping ACE2 through an allosteric signaling between pivotal RBD structural elements; and (ii) impairing the binding of antibodies elicited by infected/vaccinated patients. This information, unlocking the molecular terms and evolutionary strategies underlying the increased virulence of emerging SARS-CoV-2 variants, set the basis for developing the next-generation anti-COVID-19 therapeutics. TOC GRAPHICS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/441093v1_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@107419aorg.highwire.dtl.DTLVardef@163fc8eorg.highwire.dtl.DTLVardef@159eb0borg.highwire.dtl.DTLVardef@5621eb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗