Biology subjects

Degiacomi, M. T.

Publications and source records attributed to Degiacomi, M. T..

7 recordsLinked to original sources

bioRxiv2026

Carbonara: a SAXS-guided seeding framework for exploring protein solution-state dynamics

Proteins in solution often populate conformational ensembles that differ from the static states captured by crystallography or AI-based structure prediction. Conventional molecular dynamics (MD) simulations often fail to cross the energy barriers separating these states on accessible timescales, and statistical reweighting cannot recover conformations never sampled. Here we present Carbonara, a framework that uses experimental small-angle X-ray scattering (SAXS) data to predict alternative physically plausible protein conformations. Carbonara builds on Wiggle, a standalone C-based SAXS forward model validated against explicit-solvent calculations and experimental benchmarks. Using two case studies, an AI-predicted multi-domain helicase (SMAR-CAL1) and a crystallographic antibody fragment (ChiLob7/4 IgG2), we show how seeding MD simulations from Carbonara conformations enables efficient exploration of solution-state conformational landscapes. In both cases, MD ensembles initiated from available models either fail to match the SAXS data or do so only after discarding nearly all sampled conformations, whereas Carbonara-seeded ensembles reach agreement while retaining the majority of conformations. Our modelling framework provides a route from static structural models of flexible multi-domain proteins and multimeric assemblies to solution-state ensembles.

biophysics↗

bioRxiv2026

Tyrosine kinases sample unique activation ensembles

Protein kinase activation is driven by conformational changes across multiple structural components, including the conserved Asp-Phe-Gly (DFG) motif, but whether these transitions follow a universal mechanism remains unclear. Here we combine over 8.3 milliseconds of distributed unbiased molecular dynamics simulations with Markov state models (MSMs) to compare the conformational landscapes of the ABL1, EGFR and MET kinase domains. To maximize unbiased sampling of functionally relevant conformational space, we use a transfer seeding strategy that steers AlphaFold2 models derived from homologous templates to sample MET conformational states absent from available experimental databases. We find that related DFG-motif geometries separate into distinct kinetic networks. These shared structural states are connected by kinase-specific activation pathways with different regulatory elements controlling the slowest step of activation. Our findings reveal that the shared nomenclature masks distinct transition mechanisms between kinase domains, revealing new regions critical for activity and targetable conformations for inhibitor design.

biophysics↗

bioRxiv2024

Cardiac stress leads to regulation of Filamin C dimerisation via an ancient phosphorylation-modulated interaction with HSPB7

The biomechanical properties and responses of tissues underpin a variety of physiological functions and pathologies. In striated muscle, the actin-binding protein filamin C (FLNC) is a key protein whose variants causative for a wide range of cardiomyopathies and musculoskeletal pathologies. Seemingly a multi-functional protein that interacts with a variety of partners, how FLNC is regulated at the molecular level is not well understood. Here we have investigated its interaction with HSPB7, a cardiac-specific molecular chaperone whose absence is embryonically lethal. We found that FLNC and HSPB7 interact in cardiac tissue under biomechanical stress, forming a strong hetero-dimer whose structure we have solved by means of X-ray crystallography. Our quantitative analyses show that the hetero-dimer out-competes the FLNC homo-dimer interface, potentially acting to abrogate the ability of the protein to cross-link the actin cytoskeleton, and to enhance its diffusive mobility. We show that phosphorylation of FLNC at threonine 2677, located at the dimer interface and associated with cardiac stress, acts to favour the homo-dimer. Conversely, phosphorylation at tyrosine 2683, also at the dimer interface, has the opposite effect and shifts the equilibrium towards the hetero-dimer. Evolutionary analysis and ancestral sequence reconstruction reveals this interaction and its mechanisms of regulation to date around the time primitive hearts evolved in chordates. Our work rationalises on the molecular level how FLNC might switch between stabilising functions in the cell, and reveals how HSPB7 acts as a specific molecular chaperone that regulates FLNC.

molecular biology↗

bioRxiv2022

Complementing machine learning-based structure predictions with native mass spectrometry

The advent of machine learning-based structure prediction algorithms such as AlphaFold2 (AF2) has moved the generation of accurate structural models for the entire cellular protein machinery into the reach of the scientific community. However, structure predictions of protein complexes are based on user-provided input and may therefore require experimental validation. Mass spectrometry (MS) is a versatile, time-effective tool that provides information on post-translational modifications, ligand interactions, conformational changes, and higher-order oligomerization. Using three protein systems, we show that native MS experiments can uncover structural features of ligand interactions, homology models, and point mutations, that are undetectable by AF2 alone. We conclude that machine learning can be complemented with MS to yield more accurate structural models on the small and the large scale.

biophysics↗

bioRxiv2021

Cryo-EM structure of the octameric pore of Clostridium perfringens β-toxin

Clostridium perfringens is one of the most widely distributed and successful pathogens. It causes multiple severe diseases in animals and humans and produces an impressive arsenal of toxins with pore-forming properties, most of them belonging to the hemolysin-like family of {beta}-pore forming toxins ({beta}-PFTs). One of the most potent toxins produced by C. perfringens is {beta}-toxin (CPB). This toxin is the main virulence factor of type C strains and essential for the development of a fatal necrotic enteritis in humans and newborn animals. In the present study, we describe the cryo-electron microscopy (cryo-EM) structure of CPB in styrene maleic acid (SMA) discs, which represents the membrane-inserted pore form, at near atomic resolution. We show that CPB forms a homo-oligomeric pore with eightfold symmetry and similar conformation to the hetero-oligomeric pores of the bi-component leukocidins, with important differences in the receptor binding region and the N-terminal latch domain. Intriguingly, the octameric CPB pore complex contains a second 16-stranded {beta}-barrel protrusion atop of the cap domain that is formed by the N-termini of the eight protomers. We propose that CPB defines a new subclass of the hemolysin-like family of {beta}-PFTs s. In addition, we show that the {beta}-barrel protrusion domain can be changed or modified without affecting the pore forming ability, thus making the pore particularly attractive for macromolecule sensing and nanotechnology. The cryo-EM structure of the octameric pore of CPB will facilitate future developments in both nanotechnology and basic research.

biophysics↗

bioRxiv2020

Mass Photometry of Membrane Proteins

Integral membrane proteins (IMPs) are biologically highly significant but challenging to study because they require maintaining a cellular lipid-like environment. Here, we explore the application of mass photometry (MP) to IMPs and membrane mimetic systems at the single particle level. We apply MP to amphipathic vehicles, such as detergents and amphipols, as well as to lipid and native nanodiscs, characterising the particle size, sample purity and heterogeneity. Using methods established for cryogenic electron microscopy, we eliminate detergent background, enabling high-resolution studies of membrane protein structure and interactions. We find evidence that, when extracted from native membranes using native styrene-maleic acid nanodiscs, the potassium channel KcsA is present as a dimer of tetramers - in contrast to results obtained using detergent purification. Finally, using lipid nanodiscs, we show that MP can help distinguish between functional and non-functional nanodisc assemblies, as well as determine the critical factors for lipid nanodisc formation.

biophysics↗

bioRxiv2018

It takes a dimer to tango: Oligomeric small heat-shock proteins dissociate to capture substrate

Small heat-shock proteins (sHsps) are ubiquitous molecular chaperones, and their mutations or altered expression are linked to multiple human disease states. sHsp monomers assemble into large oligomers with dimeric substructure, and the dynamics of sHsp oligomers has led to major questions about the form that captures substrate, a critical aspect of their mechanism of action. We show that substructural dimers of plant dodecameric sHsps, Ta16.9 and homologous Ps18.1, are functional units in the initial encounter with unfolding substrate. We introduced inter-polypeptide disulfide bonds at the two dodecameric interfaces, dimeric and non-dimeric, to restrict how their assemblies can dissociate. When disulfide bonded at the non-dimeric interface, mutants of Ta16.9 and Ps18.1 (TaCT-ACD and PsCT-ACD) were inactive, but when reduced had wild-type-like chaperone activity, demonstrating that dissociation at non-dimeric interfaces is essential for activity. In addition, the size of the TaCT-ACD and PsCT-ACD covalent unit defined a new tetrahedral geometry for these sHsps, different than the Ta16.9 x-ray structure. Importantly, oxidized Tadimer (disulfide bonded at the dimeric interface) showed greatly enhanced ability to protect substrate, indicating that strengthening the dimeric interface increases chaperone efficiency. Size and secondary structure changes with temperature revealed that folded sHsp dimers interact with substrate, and support dimer stability as a determinant of chaperone efficiency. These data yield a model in which sHsp dimers capture substrate prior to assembly into larger, heterogeneous sHSP-substrate complexes for subsequent substrate refolding or degradation, and suggest that tuning the strength of the dimer interface can be used to engineer sHsp chaperone efficiency.

molecular biology↗