Biology subjects

Sicoli, G.

Publications and source records attributed to Sicoli, G..

5 recordsLinked to original sources

bioRxiv2025

Domain-Specific EPR Spectroscopy to Monitor Facilitated Dissociation of a DNA-Transcription Factor Complex

Transcription factors (TFs) regulate gene expression by interacting with specific DNA sequences in various manners, yet the mechanisms by which they dissociate from high-affinity DNA sites under physiological conditions remain not completely understood. Here, we employ continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy in native solution state to resolve, with domain-level precision, the dissociation dynamics of the MYC-associated factor X (MAX) from its cognate EBOX DNA motif. Site-directed spin labeling reveals that in the absence of DNA, MAX undergoes a stepwise dissociation process--beginning with melting of the N-terminal disordered region (NTD), followed by the helix-loop-helix (HLH) domain, and culminating in leucine zipper (LZ) dimer dissociation. DNA binding reorganizes this process into a cooperative all-or-none transition, wherein destabilization of the LZ triggers concerted collapse of the entire trimeric MAX:MAX-DNA complex. Strikingly, the addition of a disordered BRCA1 fragment (residues 219-504) disrupts this cooperativity by selectively destabilizing DNA contacts in the NTD and HLH regions, without perturbing the LZ. This results in facilitated dissociation via competitive DNA binding, observed here directly at domain-level resolution. Our findings establish EPR spectroscopy as a uniquely sensitive tool for dissecting TF-DNA dynamics under physiological conditions, offering mechanistic insight into regulated unbinding processes inaccessible to ensemble methods.

biochemistry↗

bioRxiv2024

A widespread family of ribosomal peptide metallophores involved in bacterial adaptation to copper stress

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a structurally diverse group of natural products that bacteria employ in their survival strategies. Herein, we characterized the structure, the biosynthetic pathway and the mode of action of a new RiPP family called bufferins. With thousands of homologous biosynthetic gene clusters throughout the eubacterial phylogenetic tree, bufferins form by far the largest family of RiPPs modified by multinuclear non-heme iron-dependent oxidases (MNIO, DUF692 family). Using Caulobacter vibrioides bufferins as a model, we showed that the conserved Cys residues of their precursors are transformed into 5-thiooxazoles, further expanding the reaction range of MNIO enzymes. This rare modification is installed in conjunction with a partner protein of the DUF2063 family. Bufferin precursors are the first examples of bacterial RiPPs found to feature an N-terminal Sec signal peptide and thus to be exported by the ubiquitous Sec pathway, a new paradigm in the RiPP field. Other original features of bufferins are their large size and protein-like fold, which blurs the line between modified peptides and proteins. We reveal that bufferins are involved in copper homeostasis, and their metal-binding propensity requires the thiooxazole heterocycles. Bufferins enhance bacterial growth under copper stress by sequestering excess metal ions in the periplasm. Our study thus describes a large family of RiPP metallophores and unveils a widespread but overlooked metal homeostasis mechanism in eubacteria likely to be relevant to One-Health strategies. Significance statementCopper is both essential and toxic in excess. Bacteria face copper in their environments, notably in phagocytes, hence they have developed several defense mechanisms. We discovered a widespread strategy of protection from copper, through the biosynthesis of natural products that we call bufferins. Bufferins are ribosomally synthesized post-translationally modified peptides (RiPPs), natural products with key roles in bacterial physiology and ecology. Bufferins enhance bacterial growth under copper stress by complexing with the metal using thiooxazole heterocycles that result from enzymatic modification of cysteine residues. With thousands of homologs throughout the eubacterial phylogenetic tree, bufferins represent a highly prevalent strategy of adaptation to metal stress. They are larger in size than most RiPPs, expanding the concept of RiPPs to modified proteins.

microbiology↗

bioRxiv2023

Cleaving DNA with DNA: cooperative tuning of structure and reactivity driven by copper ions

Abstract/SummaryA copper-dependent self-cleaving DNA (DNAzyme or dexoxyribozyme) previously isolated by in vitro selection has been analyzed by a combination of Molecular Dynamics simulations and advanced EPR/ESR spectroscopy, providing insights on the structural and mechanistic features of the cleavage reaction at unprecedented resolution. The minimized 46-nucleotide deoxyribozyme forms duplex and triplex substructures that flank a highly conserved catalytic core. The self-cleaving construct forms a bimolecular complex that has a distinct substrate and enzyme domains. Cleavage of the substrate is directed at one of two adjacent nucleotides and proceeds via an oxidative cleavage mechanism that is unique to the position cleaved. The use of isotopologues of nucleotides allowed us to provide atomic resolution for the copper-substrate complex. The spectroscopic analysis overcomes the major drawbacks related to the metal-soup scenario, also known as super-stoichiometric ratios of cofactors versus substrate, conventionally required for the cleavage reaction within those nucleic acids-based enzymes. Our results pave the way for analysis on mixtures where metals/lanthanides are used as cofactors, having demonstrated that our approach may reach resolution of single nucleotide and beyond. Furthermore, the insertion of cleavage reaction within more complex architectures is now a realistic option towards the applicability of spectroscopic studies, both in vitro and in vivo matrices.

biophysics↗

bioRxiv2021

Large-scale conformational changes of FhaC provide insights into the two-partner secretion mechanism

The Two-Partner secretion pathway mediates protein transport across the outer membrane of Gram-negative bacteria. TpsB transporters belong to the Omp85 superfamily, whose members catalyze protein insertion into, or translocation across membranes without external energy sources. They are composed of a transmembrane {beta} barrel preceded by two periplasmic POTRA domains that bind the incoming protein substrate. Here we used an integrative approach combining in vivo assays, mass spectrometry, nuclear magnetic resonance and electron paramagnetic resonance techniques suitable to detect minor states in heterogeneous populations, to explore transient conformers of the TpsB transporter FhaC. This revealed substantial, spontaneous conformational changes with a portion of the POTRA2 domain coming close to the lipid bilayer and surface loops. Specifically, the amphipathic {beta} hairpin immediately preceding the first barrel strand can insert into the {beta} barrel. We propose that these motions enlarge the channel and hoist the substrate into it for secretion. An anchor region at the interface of the {beta} barrel and the POTRA2 domain stabilizes the transporter in the course of secretion. Our data propose a solution to the conundrum how these transporters mediate protein secretion without the need for cofactors, by utilizing intrinsic protein dynamics.

biophysics↗

bioRxiv2019

The 'hidden side' of spin labeled oligonucleotides: Molecular Dynamics study focusing on the EPR-silent components of base pairing

Nitroxide labels are combined with nucleic acid structures and studied using electron paramagnetic resonance experiments (EPR). As X-ray/NMR structures are unavailable with the nitroxide labels, detailed residue level information, down to atomic resolution, about the effect of these nitroxide labels on local RNA structures is currently lacking. This information is critical to evaluate the choice of spin label. In this study, we compare and contrast the effect of TEMPO-based (NT) and rigid spin (C) labels (in both 2-O methylated and not-methylated forms) on RNA duplexes. We also investigate sequence-dependent effects of NT label on RNA duplex along with the more complex G-quadruplex RNA. Distances measured from molecular dynamics simulations between the two spin labels are in agreement with the EPR experimental data. To understand the effect of labeled oligonucleotides on the structure, we studied the local base pair geometries and global structure in comparison with the unlabeled structures. Based on the structural analysis, we can conclude that TEMPO-based and C labels do not significantly perturb the base pair arrangements of the native oligonucleotide. When experimental structures for the spin labelled DNA/RNA molecules are not available, general framework offered by the current study can be used to provide information critical to the choice of spin labels to facilitate future EPR studies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/537324v3_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1b37634org.highwire.dtl.DTLVardef@11556f3org.highwire.dtl.DTLVardef@1ff6c13org.highwire.dtl.DTLVardef@1063ce_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗