Accademia Lucchese di Science

Palazzo Canavese, Italy

Accademia Lucchese di Science

Palazzo Canavese, Italy
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Investigated here are interactions of C-terminal amidated peptides with the hASIC1a acid-sensing ion channel. The peptides comprise endogenous FMRFa, present in the western Atlantic clam Sunray Venus, and FIRFa, present in cephalopods, as well as non-endogenous ones for comparison. The interaction is investigated by automated docking. The resulting key hASIC1a-FMRFa complex, set in a lipidic POPC (=1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) membrane surrounded by H 2O and Na +-neutralized, was also investigated by molecular dynamics. It was observed that all investigated peptides become encapsulated into the ion channel, on one side by the thumb and finger of a subunit, and, on the opposite side, by the knuckle and β-ball of a second subunit. The third subunit is not involved. This is much the same binding site that was disclosed previously by both a similar computational approach, and electrophysiological and binding experiments for the hASIC1a ion channel-blocker tarantula toxin PCTX1. This paves the way to a better understanding of the role of these peptides in invertebrates. Copyright © 2011 Verlag Helvetica Chimica Acta AG, Zürich.


In this work, by applying a non-deterministic, randomly-oriented minimal force to the dissociated CO ligand of the MauG-CO system, the molecular-dynamics (MD) behavior of this system could be quickly unraveled. It turned out that CO has no marked directional egress from the high-spin c-heme iron distal pocket. Rather, CO is able to exploit all interstices created during the protein fluctuations. Nonetheless, no steady route toward the surrounding solvent was ever observed: CO jumped first into other binding pockets before being able to escape the protein. In a few cases, on hitting the surrounding H2O molecules, CO was observed to reverse direction, re-entering the protein. A contention that conformational inversion of the P107 ring provides a gate to the iron ion is not supported by the present simulations. Copyright © 2012 Verlag Helvetica Chimica Acta AG, Zürich.


In this work, two protein systems, Kij3D-FMN-AKM-O2 and Kij3DFMNO2, made of KijD3 N-oxygenase, flavin mononucleotide (FMN) cofactor, dTDP-3-amino-2,3,6-trideoxy-4-keto-3-methyl-D-glucose (AKM) substrate, and dioxygen (O2), have been assembled by adding a molecule of O2, and removing (or not) AKM, to crystal data for the Kij3D-FMN-AKM complex. Egress of AKM and O2 from these systems was then investigated by applying a tiny external random force, in turn, to their center of mass in the course of molecular dynamics in explicit H2O. It turned out that the wide AKM channel, even when emptied, does not constitute the main route for O2 egress. Other routes appear to be also viable, while various binding pockets (BPs) outside the active center are prone to trap O2. By reversing the reasoning, these can also be considered as routes for uptake of O2 by the protein, before or after AKM uptake, while BPs may serve as reservoirs of O2. This shows that the small molecule O2 is capable of permeating the protein by exploiting all nearby interstices that are created on thermal fluctuations of the protein, rather than having necessarily to look for farther, permanent channels. Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.


Pietra F.,Accademia Lucchese di Science
Chemistry and Biodiversity | Year: 2011

The pathways of escape of carbon monoxide (CO) from sperm whale myoglobin were investigated by means of a biased form of all-atoms molecular dynamics (RAMD), whereby a weak, randomly oriented force is applied to the center of mass of CO. The force only persists if the direction taken by CO continues for a given period of time, otherwise a new direction is randomly chosen. A statistically significant number of RAMD runs gave distinct responses according to the level of approximations used for the model. Thus, with rigid bonds to all H-atoms, several portals for CO egress toward the solvent, besides the main H64 gate, were identified, like in recently published unbiased massive MD, six orders of magnitude acceleration of CO escape in RAMD notwithstanding. In contrast, by removing the approximation of rigid bonds in the model, only one of these extra portals was identified, besides the H64 portal. Sticking to this all-free-bonds model, Perutz's early suggestion that the H64 imidazole must rotate 'out' toward the solvent in order that CO can cross the H64 gate was directly implemented. RAMD Simulations with this model led to CO egress from the H64 gate only, reconciling theory with experiments. Copyright © 2011 Verlag Helvetica Chimica Acta AG, Zürich.


Central inhibition of the acid-sensing hASIC1a channel, acting upstream of the opiate system, might serve to treat any type of pain, avoiding the unwanted addiction problems of the opioid drugs. To this end, inhibition of hASIC1a channel by PcTx1, a peptide from the Trinidad chevron tarantula, is under development. New inhibitors of the hASIC1a channel are also being sought, in the hope of further modulating the activity, from which antiplasmodial amidine and guanidine phenyl drugs have emerged as promising candidates. However, how such current inhibition takes place remains obscure from the molecular point of view, hindering any further progress in developing drugs. Therefore, the nature of the binding sites, and how they are reached by the amidine-guanidine drugs, was investigated here via automated docking and molecular dynamics with hASIC1a homology models. This study has revealed that this ion channel is rich in binding sites, and that flexible drugs, such as nafamostat, may penetrate it in a snake-like elongated conformation. Then, crawling like a snake through temporary holes in the protein, nafamostat either simply flips, or changes to a high-energy folded conformation to become adapted to the shape of the binding site. © 2012 Verlag Helvetica Chimica Acta AG, Zürich.


H-NOX (Heme Nitric Oxide/Oxygen) domain has widespread occurrence, either standalone or associated with functional proteins, sending signals for functions that span from modulating vasodilation and neurotransmission with humans to competition and symbiosis with bacteria. Understanding how H-NOX works, and possibly intervening on degeneration for health purposes, needs first clarifying how diatomic gases are relocated through this protein in relation to the deeply buried heme. To this end, a biased form of molecular dynamics, i.e., Random Accelaration Molecular Dynamics (RAMD), is used by applying a randomly oriented tiny force to heme-dissociated CO of Nostoc sp. H-NOX, while changing randomly the direction of the force, if CO travels less than specified for the evaluated block. The result is that a large area of the protein, comprising amino acids from serine 44 to leucine 67 along two adjacent helices, offers a broad portal to CO from the surrounding medium to the deeply buried heme. Most traffic is concentrated through a channel lined by tyrosine 49, valine 52, and leucine 67. This modifies the picture drawn from mapping Xe cavities on pressurizing Nostoc sp. H-NOX with Xe gas. What is the main pathway with Xe-cavity mapping becomes a minor pathway with RAMD, and vice versa. The reason is that the fluctuating protein under MD creates clefts for CO slipping through, as it is expected to occur in nature. Copyright © 2012 Verlag Helvetica Chimica Acta AG, Zürich.


Pietra F.,Accademia Lucchese di Science
Chemistry and Biodiversity | Year: 2012

Extensive random-acceleration molecular-dynamics (RAMD) simulations of the egress of dioxygen (O 2) from a model of rabbit 12/15-lipoxygenase- arachidonic acid complex disclosed several exit portals in addition to those previously described from implicit ligand sampling calculations and limited MD simulations. © 2012 Verlag Helvetica Chimica Acta AG.


Pietra F.,Accademia Lucchese di Science
Chemistry and Biodiversity | Year: 2013

This work deals with two neuroglobins from phylogenetically distant organisms. Deriving from the acoelomorph Symsagittifera roscoffensis, SrNgb is functionally pentacoordinated, and is assumed to function as a reserve of dioxygen (O2). Obtained from mice, mNgb is functionally hexacoordinated, and presumably triggers signals from sensing O2. Here, it is investigated how these two globins are permeated by diatomic gases, SrNgb by O2 and mNgb by CO. With protein atomic coordinates available from high-resolution X-ray diffraction analysis, O2 and CO pathways were traced from molecular-dynamics simulations in H2O solution, which makes no difference between the two gases, accelerated by applying an external randomly-oriented minimal force to the center of mass of the diatomic gas molecule. This allowed us to explore a statistically significant large number of trajectories. It emerged that CO leaves mNgb from preferentially peripheral gates located on the side of the heme propionate chains, whereas O2 leaves SrNgb from the opposite side. This shows no analogy with either the functionally pentacoordinated, O2-transporting, myoglobin (Mgb), or the hexacoordinated, O2-sensing, cytoglobin, despite the same three-over-three typical α-helical globin folding. The sole analogy that could be observed was a preference for the shortest diatomic gas pathways with both SrNgb and Mgb. It is tempting to speculate that this fulfills the need of being quick in delivering O2 to depleted organs. Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.


Pietra F.,Accademia Lucchese di Science
Chemistry and Biodiversity | Year: 2013

This work discloses two bona fide gates through which the CO ligand can leave the distal cavity of carboxy human cytoglobin, reaching the solvent. The investigation was based on molecular dynamics, aided by a minimal randomly-oriented force applied to the ligand. The shortest pathway progresses toward the main gate, H81-R84, in the open state, with the H81 imidazole moiety turned toward the solvent. A longer pathway develops toward the diametrically opposed W31-W151 gate. In between, CO may be entrapped into binding cavities, either along the path toward the gates, or in a cul-de-sac, from which CO may even be incapable to escape. This behavior contrasts with carboxy myoglobin, where the corresponding H64 gate, when opened, is the sole used by CO to get to the solvent. These observations, which could hold also for other small ligands of biological interest, such as O2, NO, and NO3, provide an answer to a neglected aspect of the mysterious six-coordinated globins. Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.


This work deals with dioxygen (O2) binding sites and pathways through inducible human heme oxygenase (HO-1). The experimentally known distal binding site 1, and sites 2-3 above it, could be reproduced by means of non-deterministic random-acceleration molecular-dynamics (RAMD) simulations. In addition, RAMD revealed the proximal binding site 5, a deeply-seated binding site 4, which lies behind heme, as well as a few gates communicating with the external medium. In getting from site 1 to the main gate, which lies on the protein front opposed to site 4, O2 follows chiefly the shortest direct pathway. Less frequently, O2 visits intermediate sites 2, 4, or 5 along longer pathways. A similarity between HO-1, myoglobin, and cytoglobin in using, for diatomic gas delivery, the direct shortest pathway from the heme center to the surrounding medium, is emphasized. Otherwise, comparing other proteins and diatomic gases, each system reveals its peculiarities as to sites, gates, and pathways. Thus, relating these properties to the physiological functions of the proteins remains in general a challenge for future studies. Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.

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