Biology subjects

Iacobucci, C.

Publications and source records attributed to Iacobucci, C..

12 recordsLinked to original sources

bioRxiv2026

Structural studies of the C-terminal cysteine-rich domain of Aedes albopictus vitellogenin reveal an evolutionarily divergent, monomeric C-terminal cysteine knot (CTCK) architecture

Vitellogenins are essential transport lipoproteins and precursors to egg-yolk proteins in oviparous species. Several molecular structure studies have elucidated most of their multi-domain organization, yet the structure and function of their C-terminal cysteine knot (CTCK) domain have remained largely elusive. In this study, we present the 1.2 [A] resolution crystal structure of the recombinant CTCK domain from a vitellogenin isoform of the mosquito Aedes albopictus (Vg-CTCK). The molecular architecture reveals a CTCK fold defined by two antiparallel beta sheets stabilized by three intramolecular disulfide bonds, featuring a unique 12-amino acid insertion shaping an alpha helix positioned between the two main beta sheets. Analysis of the crystal packing and biophysical characterization in solution consistently confirms that recombinant Vg-CTCK is monomeric. To validate these findings in a native context, we employed ex vivo cross-linking mass spectrometry (XL-MS) on intact mosquito ovaries, which corroborated the molecular architecture of the Vg-CTCK observed in the crystal structure and highlighted the absence of inter-molecular cross-links. Collectively, our data highlight an evolutionarily divergent, monomeric assembly for the mosquito Vg-CTCK domain, challenging previous hypotheses that suggested this domain might facilitate vitellogenin oligomerization.

molecular biology↗

bioRxiv2026

Revisiting p53:Sirt1 interaction in the light of controlling p53 acetylation levels

The NAD+-dependent deacetylase sirtuin 1 (Sirt1) is known to regulate the tumor suppressor p53 via deacetylation, but the structural basis of the protein-protein interaction between full-length p53 and Sirt1 has so far remained elusive. We apply an integrated approach, combining structural mass spectrometry (MS) with data-driven molecular docking, to study the interaction between human p53 and Sirt1. Sirt1 was found to bind exclusively to acetylated p53 forming complexes with a 1:1 stoichiometry, irrespectively of p53s oligomeric state. The lysine residue at position 582 (K582) in p53 was identified as predominant acetylation site showing a selective Sirt1-dependent deacetylation at this position. Cross-linking mass spectrometry (XL-MS) provided valuable distance constraints between p53 and Sirt1. Specifically, cross-links created between p53-K382 / Sirt1-K427 and p53-K120 / Sirt1-K622 give hints on a highly flexible interface. Molecular docking was conducted based on the distance constraints imposed by the cross-links, positioning Sirt1 at the DNA-binding and tetramerization domains of p53. This gives a rationale for a steric exclusion of additional Sirt1 molecules binding to p53. We present the first structural model of the full-length p53:Sirt1 (1:1) complex, establishing a mechanistic framework that links p53 activity to its Sirt1-controlled acetylation status.

biochemistry↗

bioRxiv2025

Enantiopurity-Dependent Peptide Coacervates and Asymmetric Organocatalysis

Membraneless compartmentalization via liquid-liquid phase separation (LLPS) has emerged as a powerful strategy to organize biochemical reactions. Recently, peptide-based coacervates demonstrated the potential to function as microreactors by enhancing reaction kinetics through increased local concentrations and altered microenvironments. Here, we introduce an O-methylated diphenylalanine-based tripeptide LLLPFF-OCH3 containing an N-terminal proline, designed to undergo LLPS, and simultaneously function as an enantioselective organocatalyst. Comprehensive characterization via confocal microscopy, fluorescence recovery after photobleaching (FRAP), micro-Raman and attenuated total reflection infrared (ATR-IR) spectroscopy, diffusion-surface plasmon resonance (D-SPR), and molecular dynamics (MD) simulations revealed the formation of stable liquid droplets. In contrast, a racemic mixture of LLLPFF-OCH3 and DDDPFF-OCH3 failed to form liquid droplets and instead formed a solid precipitate, unveiling a critical role of enantiopurity in LLPS. Proof-of-concept catalytic studies proved enantioselective organocatalytic activity of the LLLPFF-OCH3 liquid coacervates. Beyond catalysis these results may have broader implications in understanding prebiotic chemistry and neurodegeneration.

biochemistry↗

bioRxiv2025

Protein Secondary Structure Patterns In Short-Range Cross-Link Atlas

Cross-linking mass spectrometry (XL-MS) has become a powerful tool in structural biology for investigating protein structure, dynamics, and interactomics. However, short-range cross-links, defined as those connecting residues fewer than 20 positions apart, have traditionally been considered less informative and largely overlooked, leaving significant data unexplored in a systematic manner. Here, we present a system-wide analysis of short-range cross-links, demonstrating their intrinsic correlation with protein secondary structure. We introduce the X-SPAN (Cross-link Structural Pattern Analyzer) software, which integrates publicly available XL-MS datasets from system-wide experiments with AlphaFold-predicted protein structures. Our analysis reveals distinct cross-linking patterns that reflect the spatial constraints imposed by secondary structural elements. Specifically, -helices exhibit periodic cross-linking patterns consistent with their characteristic helical pitch, whereas coils and {beta}-strands display nearly monotonic distributions. A context-dependent protein grammar reinforces short-range cross-link specificity. Short-range cross-links can enhance the statistical inference of secondary structures within integrative modeling workflows. Additionally, our work establishes a framework for benchmarking AlphaFolds local prediction accuracy and provides novel quality control criteria for XL-MS experiments. We anticipate that X-SPAN and our short-range cross-link database will serve as a valuable resource for exploring local secondary structure rearrangements and their potential roles in protein function and allosteric regulation.

molecular biology↗

bioRxiv2024

Twisting Urea- to Imide-Based Mass Spectrometry-Cleavable Cross-Linkers Enables Affinity Tagging

Disuccinimidyl dibutyric urea (DSBU) is a mass spectrometry (MS)-cleavable cross-linker that has multiple applications in structural biology, ranging from isolated protein complexes to comprehensive system-wide interactomics. DSBU facilitates a rapid and reliable identification of cross-links through the dissociation of its urea group in the gas-phase. In this study, we further advance the structural capabilities of DSBU by twisting the urea group into an imide, thus introducing a novel class of cross-linkers. This modification preserves the MS-cleavability of the amide bond, granted by the two acyl groups of the imide function. The central nitrogen atom enables the introduction of affinity purification tags. Here, we introduce disuccinimidyl disuccinic imide (DSSI) as prototype of this class of cross-linkers. It features a phosphonate handle for immobilized metal ion affinity chromatography (IMAC) enrichment. We detail DSSI synthesis and describe its behavior in solution and in the gas-phase while cross-linking isolated proteins and human cell lysates. DSSI and DSBU cross-links are compared at the same enrichment depths to bridge these two cross-linker classes. We validate DSSI cross-links by mapping them in high-resolution structures of large protein assemblies. The cross-links observed yield insights into the morphology of intrinsically disordered proteins (IDPs) and their complexes. The DSSI linker might spearhead a novel class of MS-cleavable and enrichable cross-linkers.

biochemistry↗

bioRxiv2024

A Workflow for Improved Analysis of Cross-linking Mass Spectrometry Data Integrating Parallel Accumulation-Serial Fragmentation with MeroX and Skyline

Cross-linking mass spectrometry (XL-MS) has evolved into a pivotal technique for probing protein interactions. This study describes the implementation of Parallel Accumulation-Serial Fragmentation (PASEF) on timsTOF instruments, enhancing the detection and analysis of protein interactions by XL-MS. Addressing the challenges in XL-MS, such as the interpretation of complex spectra, low abundant cross-linked peptides, and a data acquisition bias, our current study integrates a peptide-centric approach for the analysis of XL-MS data and presents the foundation for integrating data-independent acquisition (DIA) in XL-MS with a vendor-neutral and open-source platform. A novel workflow is described for processing data-dependent analysis (DDA) of PASEF-derived information. For this, software by Bruker Daltonics is used, enabling the conversion of these data into a format that is compatible with MeroX and Skyline software tools. Our approach significantly improves the identification of cross-linked products from complex mixtures, allowing the XL-MS community to overcome current analytical limitations.

bioinformatics↗

bioRxiv2022

Structural Assessment of the Full-Length Wild-Type Tumor Suppressor Protein p53 by Mass Spectrometry-Guided Computational Modeling

The tetrameric tumor suppressor p53 represents a great challenge for 3D-structural analysis due to its high degree of intrinsic disorder (ca. 40%). We aim to shed light on the structural and functional roles of p53s C-terminal region in full-length, wild-type human p53 tetramer and their importance for DNA binding. For this, we employed complementary techniques of structural mass spectrometry (MS) in an integrated approach with AI-based computational modeling. Our results show no major conformational differences in p53 between DNA-bound and DNA-free states, but reveal a substantial compaction of p53s C-terminal region. This supports the proposed mechanism of unspecific DNA binding to the C-terminal region of p53 prior to transcription initiation by specific DNA binding to the core domain of p53. The synergies between complementary structural MS techniques and computational modeling as pursued in our integrative approach is envisioned to serve as general strategy for studying intrinsically disordered proteins (IDPs) and intrinsically disordered region (IDRs).

biochemistry↗

bioRxiv2022

First direct observation of 'elongated' conformational states in α-synuclein upon liquid-liquid phase separation

-Synuclein (-syn) is an intrinsically disordered protein (IDP) that undergoes liquid-liquid phase separation (LLPS), fibrillation, and forms insoluble intracellular Lewys bodies in neurons, which are the hallmark of Parkinsons Disease (PD). Neurotoxicity precedes the formation of aggregates and is probably related to LLPS of -syn in the cell. The molecular mechanisms underlying the early stages of LLPS are still elusive. To obtain structural insights into -syn upon LLPS, we take advantage of cross-linking/mass spectrometry (XL-MS) and introduce an innovative approach, termed COMPASS (COMPetitive PAiring StatisticS). COMPASS unravels transient interactions between -syn molecules in liquid droplets. In this work, we show that the conformational ensemble of -syn shifts from a hairpin-like structure towards more elongated conformational states upon LLPS. We obtain insights into the critical initial stages of PD and establish a novel mass spectrometry-based approach that will aid to solve open questions in LLPS structural biology.

biochemistry↗

bioRxiv2020

Structural Analysis of 70S Ribosomes by Cross-Linking/Mass Spectrometry Reveals Conformational Plasticity

The ribosome is not only a highly complex molecular machine that executes translation according to the central dogma of molecular biology, but also an exceptional specimen for testing and optimizing cross-linking/mass spectrometry (XL-MS) workflows. Due to its high abundance, ribosomal proteins are frequently identified in proteome-wide XL-MS studies of cells or cell extracts. Here, we performed in-depth cross-linking of the E. coli ribosome using the amine-reactive cross-linker diacetyl dibutyric urea (DSAU). We analyzed 143 E. coli ribosomal structures, mapping a total of 10,771 intramolecular distances for 126 cross-link-pairs and 3,405 intermolecular distances for 97 protein pairs. Remarkably, 44% of intermolecular cross-links covered regions that have not been resolved in any high-resolution E. coli ribosome structure and point to a plasticity of cross-linked regions. We systematically characterized all cross-links and discovered flexible regions, conformational changes, and stoichiometric variations in bound ribosomal proteins, and ultimately remodeled 2,057 residues (15,794 atoms) in total. Our working model explains more than 95% of all cross-links, resulting in an optimized E. coli ribosome structure based on the cross-linking data obtained. Our study might serve as benchmark for conducting biochemical experiments on newly modeled protein regions, guided by XL-MS. Data are available via ProteomeXchange with identifier PXD018935.

biochemistry↗

bioRxiv2019

Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin receptor assemblies

Cullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5 and their ubiquitylation targets, AUX/IAAs, sense auxin concentrations in the nucleus. TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, we resolved TIR1{middle dot}auxin{middle dot}IAA7 and TIR1{middle dot}auxin{middle dot}IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degrons vicinity, cooperatively position AUX/IAAs on TIR1. The AUX/IAA PB1 interaction domain also assists in non-native contacts, affecting AUX/IAA dynamic interaction states. Our results establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation, and might provide conformational flexibility for adopting a multiplicity of functional states. We postulate IDRs in distinct members of the AUX/IAA family to be an adaptive signature for protein interaction and initiation region for proteasome recruitment.

plant biology↗

bioRxiv2019

A Simple Cross-Linking/Mass Spectrometry Workflow to Study System-Wide Protein Interactions

We present a cross-linking/mass spectrometry (XLMS) workflow for performing proteome-wide cross-linking analyses within one week. The workflow is based on the commercially available MS-cleavable cross-linker disuccinimidyl dibutyric urea (DSBU) and can be employed by every lab having access to a mass spectrometer with tandem MS capabilities. We provide an updated version 2.0 of the freeware software tool MeroX, available at www.StavroX.com, that allows conducting fully automated and reliable studies delivering insights into protein-protein interaction networks and protein conformations at the proteome level. We exemplify our optimized workflow for mapping protein-protein interaction networks in Drosophila melanogaster embryos on a system-wide level. From cross-linked Drosophila embryo extracts, we detected 18,037 cross-link spectrum matches corresponding to 5,129 unique cross-linked residues in biological triplicate experiments at 5% FDR (3,098 at 1% FDR). Among these, 1,237 interprotein cross-linking sites were identified that contain valuable information on protein-protein interactions. The remaining 3,892 intra-protein cross-links yield information on conformational changes of proteins in their cellular environment.

molecular biology↗

bioRxiv2018

Cross-linking/Mass Spectrometry: A Community-Wide, Comparative Study Towards Establishing Best Practice Guidelines

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has largely increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats that are generally accepted in the field and that have shown to lead to high-quality results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. From the results obtained, common protocols will be established. Our study serves as basis for establishing best practice guidelines in the field for conducting cross-linking experiments, performing data analysis, and reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

biochemistry↗