Biology subjects

Gianti, E.

Publications and source records attributed to Gianti, E..

4 recordsLinked to original sources

bioRxiv2026

Interfacial water in the PRDX1-sulfiredoxin repair intermediate: an all-atom molecular dynamics study

Peroxiredoxins protect cells from oxidative damage, and sulfiredoxin (Srx) restores their activity by repairing the overoxidized catalytic cysteine; how the two proteins recognize one another is central to redox signaling and to oxidative-stress-associated disease. We characterize the human peroxiredoxin-1 (PRDX1)-Srx repair intermediate (PDB 2RII) and four related catalytic-cysteine states by all-atom molecular dynamics (three replicas of 200 ns per system; 3.0 microsecond total), using geometric and kinetic observables only, with across-replica statistics. The disulfide-linked complex is stable (backbone RMSD 2.5- 3.2 Angstrom) and the peroxidatic Cys52 is buried in the interface (buried surface area 71-76 Angstrom^2). The interface is extensive, with 202 consensus residue-residue contacts, and predominantly water-mediated: 20 reproducible, hydrogen-bond-validated water bridges centered on the PRDX1 165-170 region, against three persistent salt bridges. Removing the engineered tether and modeling Cys52 as the sulfinate on which Srx acts leaves the interface intact (189 consensus contacts) and the water network larger (36 bridges), new bridges linking the sulfinate to the Srx catalytic pocket. At the free Cys52, first-shell water responds to charge state as electrostatics predicts, and at matched water counts the thiolate shows no additional clustering, separating generic hydration from the specific interfacial organization. An equalized, permutation-controlled comparison of the Srx-bound and free intra-PRDX1 contact networks leaves them statistically indistinguishable: of 14,127 residue pairs only one exceeds both floors, and it does not reproduce across independent trajectory sets. A covalent celastrol-Cys173 adduct retains its thioether bond while the tethered ligand reorients widely, so covalent attachment fixes the anchor rather than the pose.

pharmacology and toxicology↗

bioRxiv2026

Preorganized RdRp-Thumb Dynamics Drives SARS-CoV-2 Polymerase Function

The SARS-CoV-2 RNA-dependent RNA polymerase drives viral genome replication and is a major antiviral target. How intrinsic conformational dynamics organize functional states of the polymerase, however, remains incompletely understood. Here, molecular dynamics (MD) simulations combined with free-energy landscape analysis reveal that the apo polymerase samples preexisting conformational states defined by coordinated thumb-subdomain motions. Projection of experimental structures, representative of the polymerase nucleic acid cycle, onto the conformational landscape identified discrete basins spanning apo-like and elongation-like states and revealed a coherent structural axis coupling global polymerase compaction (radius of gyration, Rg) with thumb-interface separation (center-of-mass distance, COM). These motions connect catalytic motifs, RNA-binding regions, and distal regulatory elements across the polymerase ensemble. The observed conformational organization is not apparent from static structures alone and supports a model in which functional transitions arise from intrinsic collective dynamics of the apo enzyme. Intermediate conformational ensembles combine structural stability with retained inter-domain flexibility, identifying mechanically responsive states favorable for allosteric modulation. Together, these findings define structurally coupled regulatory regions within the coronavirus polymerase and support conformational trapping of thumb-subdomain dynamics as a potential strategy for antiviral design targeting RNA virus replication machinery.

biophysics↗

bioRxiv2022

Computational and functional characterization of the PI(4,5)P2 binding site of the TRPM3 ion channel

Transient Receptor Potential Melastatin 3 (TRPM3) is a heat-activated ion channel expressed in peripheral sensory neurons and the central nervous system. TRPM3 activity depends on the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], but the molecular mechanism of activation by PI(4,5)P2 is not known. As no experimental structure of TRPM3 is available, we built a homology model of the channel in complex with PI(4,5)P2 via molecular modeling. We identified putative contact residues for PI(4,5)P2 in the pre-S1 segment, the S4-S5 linker, and the proximal C-terminal TRP-domain. Mutating these residues increased sensitivity to inhibition of TRPM3 by decreasing PI(4,5)P2 levels by phosphatidylinositol 4-kinase inhibition. Changes in ligand-binding affinities via MM/GBSA showed reduced PI(4,5)P2 affinity for the mutants. Mutating PI(4,5)P2 interacting residues also reduced sensitivity for activation by the endogenous ligand pregnenolone sulfate (PregS), pointing to an allosteric interaction between PI(4,5)P2 and PregS. Mutating residues in the PI(4,5)P2 binding site in TRPM8 had similar effects, increased sensitivity to PI(4,5)P2 depletion, and reduced sensitivity to menthol. Mutation of most PI(4,5)P2 interacting residues in TRPM3 also increased sensitivity to inhibition by G{beta}{gamma}, indicating allosteric interaction between G{beta}{gamma} and PI(4,5)P2. Disease-associated gain of function TRPM3 mutations on the other hand, resulted in no change of PI(4,5)P2 sensitivity, indicating that mutations did not increase channel activity via increasing PI(4,5)P2 interactions. Our data provide insight into the mechanism of regulation of TRPM3 by PI(4,5)P2, its relationship to endogenous activators and inhibitors of TRPM3, as well as identify similarities and differences between PI(4,5)P2 regulation of TRPM3 and TRPM8.

biophysics↗

bioRxiv2019

Phosphoinositides regulate the TRPV1 channel via two functionally distinct binding sites

Regulation of the heat- and capsaicin-activated Transient Receptor Potential Vanilloid 1 (TRPV1) channel by phosphoinositides is controversial. In a recent cryoEM structure, an endogenous phosphoinositide was detected in the vanilloid binding site, and phosphoinositides were proposed to act as competitive vanilloid antagonists. This model is difficult to reconcile with phosphatidylinositol 4,5- bisphosphate [PtdIns(4,5)P2] being a well established positive regulator of TRPV1. To resolve this controversy, we propose that phosphoinositides regulate TRPV1 via two functionally distinct binding sites. Our molecular dynamics simulations show that phosphatidylinositol (PtdIns) is more stable in the vanilloid binding site, whereas a distinct site responsible for activation is preferentially occupied by PtdIns(4,5)P2. Consistently, we show that in the presence of PtdIns(4,5)P2 in excised patches PtdIns partially inhibited TRPV1 activity induced by low, but not high capsaicin concentrations. In the absence of PtdIns(4,5)P2 on the other hand, PtdIns partially stimulated TRPV1 activity presumably by binding to the activating site. Overall, our data resolve a major controversy in the regulation of TRPV1.

biophysics↗