Biology subjects

Bottegoni, G.

Publications and source records attributed to Bottegoni, G..

4 recordsLinked to original sources

bioRxiv2026

Redox Control of S-sulfocysteine Formation in Adenosine Phosphosulfate Reductase

Sulfur assimilation fuels bacterial growth by supplying the reduced sulfur required for the biosynthesis of sulfur-containing biomolecules. Adenosine 5'-phosphosulfate reductase (APSR) catalyzes the first reductive step of this pathway, converting adenosine 5'-phosphosulfate (APS) to adenosine monophosphate. This reaction proceeds through nucleophilic attack by catalytic C256, located in the flexible C-terminal tail, on the sulfur atom of APS, forming a thiosulfonate enzyme intermediate. Here, we investigate this reaction in APSR from Pseudomonas aeruginosa, an opportunistic pathogen associated with severe infections, particularly in patients with cystic fibrosis. APSR contains an iron-sulfur [4Fe-4S] cluster, which participates in redox steps of the reaction. Here, we show that the redox state of the iron-sulfur cluster also controls the catalytic step. Multiscale molecular simulations investigate how oxidized and reduced cluster states affect APS binding, active site organization, and the nucleophilic attack step. Molecular dynamics (MD) simulations show that the oxidized [4Fe-4S]2+; cluster stabilizes substrate interactions and the conformation of the C-terminus, facilitating a catalytically productive orientation of C256. The activation barrier of 17.7 {+/-} 1.7 kcal mol-1 from quantum mechanics/molecular mechanics (QM/MM) umbrella sampling MD simulations at the B3LYP-D3(BJ)/6-31G(d) level of theory is in good agreement with the experimental kinetics. The redox state of the iron-sulfur cluster shows its role in modulating the conformation of conserved K144, which is important for transition state stabilization in the nucleophilic attack. These findings illuminate the mechanism of this P. aeruginosa target and provide broader insight into the roles of iron-sulfur clusters in controlling enzyme reactivity.

biochemistry↗

bioRxiv2026

Conformational Remodeling Underlies Activity Loss in Disease-Linked Asparagine Synthetase Variant

Asparagine synthetase deficiency (ASNSD) is a devastating congenital disorder characterized by profound neurological impairment and early childhood mortality. It is associated with pathogenic mutations in the asparagine synthetase (ASNS) gene. Despite the critical role of ASNS in the amino acid cycle, the molecular basis by which ASNSD-linked missense mutations impair enzyme function remains poorly understood. Here, we present a comprehensive characterization of a recurrent ASNSD-linked variant, R48Q. Steady-state kinetic assays reveal severe reductions in L-glutamine-dependent catalysis and disrupted product stoichiometry, implicating impaired interdomain communication. Cryogenic electron microscopy (cryo-EM) and 3D variable analysis of the EM map uncovers altered loop conformations at the N-terminal active site and subtle conformational changes at the C-terminal domain. Consistent with the structural data, molecular dynamics simulations support that the local disruption propagates across the protein, thereby decoupling coordinated domain motions essential for catalysis. Additionally, we demonstrate that the flanking arginine and the affected loop are evolutionarily conserved across Class II glutamine amidotransferases, highlighting their shared mechanistic importance. These findings provide the molecular basis of an ASNSD variant and establish a framework for understanding how point mutations disrupt complex enzyme dynamics, with broad implications for precision medicine. SignificanceUnderstanding how mutations affect multidomain enzymes is crucial for elucidating the molecular mechanisms underlying genetic disorders. Here, we examine the molecular consequences of the R48Q variant in human asparagine synthetase (ASNS), the sole enzyme responsible for de novo L-asparagine synthesis; mutations of this enzyme lead to a fatal neurometabolic disorder, asparagine synthetase deficiency (ASNSD). By combining biochemical, cryogenic electron microscopy, and molecular dynamics simulation, we show that a single N-terminal amino acid substitution disrupts both local and global coordination, impairing enzyme activity. Our work provides the first mechanistic blueprint of an ASNSD-linked variant. These findings not only deepen our understanding of ASNS but also offer a generalized framework for studying the dynamic regulation of multidomain enzymes in disease.

biochemistry↗

bioRxiv2024

Dual agonism of sodium iodide symporter function in vivo

New approaches are urgently needed to enhance the radioiodide (RAI) ablation of aggressive and metastatic thyroid cancer. We recently discovered that valosin-containing protein inhibitors (VCPi) such as clotrimazole and disulfiram transiently block sodium iodide symporter (NIS) proteasomal degradation, hence promoting RAI uptake. However, poor bioavailability diminishes their potential impact in vivo. Following 3D modelling and iterative drug design we appraised 26 novel analogues of clotrimazole, as well as albumin nano-encapsulated copper-diethyldithiocarbamate [Cu(DDC)2-alb] - a stabilised reformulation of a disulfiram metabolite. While several clotrimazole analogues specifically increased RAI uptake, the greatest impact was observed with Cu(DDC)2-alb in thyroid cancer cells as well as human primary thyrocytes from patients with thyroid hyperplasia. NanoBRET assays revealed that Cu(DDC)2 enhanced the plasma membrane accumulation of NIS in living cells. In BALB/c mice, both intraperitoneal and intravenous administration of Cu(DDC)2-alb significantly enhanced thyroidal 99mTc-uptake. RNA-Seq revealed the surprising observation that Cu(DDC)2-alb induced key thyroid transcription factors. Accordingly, expression of PAX8 and NKX2.1 was upregulated in thyroid glands from drug treated mice, with NIS levels correlating closely to 99mTc-uptake. As Cu(DDC)2 inhibits the VCP cofactor NPL4, with VCP being critical to the proteostatic processing of NIS protein, we identify a new dual agonist of RAI uptake in vivo, with the potential to directly impact RAI therapy for patients with aggressive thyroid cancer.

cancer biology↗

bioRxiv2023

Investigating the Unbinding of Muscarinic Antagonists from the Muscarinic 3 Receptor.

Patient symptom relief is often heavily influenced by the residence time of the inhibitor-target complex. For the human muscarinic receptor 3 (hMR3), tiotropium is a long-acting bronchodylator used in conditions such as asthma or chronic obstructive pulmonary disease (COPD). The mechanistic insights of this inhibitor remain unclear, specifically, elucidation of the main factors determining the unbinding rates could help develop the next generation of antimuscarinic agents. Using our novel unbinding algorithm, we were able to investigate ligand dissociation from hMR3. The unbinding paths of tiotropium and two of its analogues, N-methylscopolamin and homatropine methylbromide show a consistent qualitative mechanism and allowed us to identify the structural bottleneck of the process. Furthermore, our machine learning-based analysis identified key roles of the ECL2/TM5 junction involved at the transition state. Additionally, our results point at relevant changes at the intracellular end of the TM6 helix leading to the ICL3 kinase domain, highlighting the closest residue L482. This residue is located right between two main protein binding sites involved in signal transduction for hMR3s activation and regulation. We also highlight key pharmacophores of tiotropium that play determining roles in the unbinding kinetics and could aid towards drug design and lead optimization. Description O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/522558v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@2598b2org.highwire.dtl.DTLVardef@4bf425org.highwire.dtl.DTLVardef@89dfa8org.highwire.dtl.DTLVardef@1ba9f48_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstract of the work, showing the unbinding for ligands 1 (tiotropium, TTP), 2 (N-methylscopolamin, NMS) and 3 (homatropine methylbromide, CPD2). Using TTPs downhill simulations from its unbinding transition state, different protein-ligand and proteinprotein interactions were analyzed with MLTSA to find relevant CVs driving the different outcomes.

biophysics↗