Biomedical subjects

Paolo Carloni

Publications and source records attributed to Paolo Carloni.

46 records · Page 3Linked to original sources

PubMed2002

Potassium permeation through the KcsA channel: a density functional study.

We present a theoretical study on structural and electronic aspects of K+ permeation through the binding sites of the KcsA channel's selectivity filter. Density functional calculations are carried out on models taken from selected snapshots of a molecular dynamics simulation recently reported [FEBS Lett. 477 (2000) 37]. During the translocation process from one binding site to the other, the coordination number of the permeating K+ ion turns out to decrease and K+ ion polarizes significantly its ligands, backbone carbonyl groups and a water molecule. K+-induced polarization increases significantly at the transition state (TS) between the two binding sites. These findings suggest that polarization effects play a significant role in the microscopic mechanisms regulating potassium permeation.

Bacterial Proteins↗

PubMed2002

Role of conformational fluctuations in the enzymatic reaction of HIV-1 protease.

The emergence of compensatory drug-resistant mutations in HIV-1 protease challenges the common view of the reaction mechanism of this enzyme. Here, we address this issue by performing classical and ab initio molecular dynamics simulations (MD) on a complex between the enzyme and a peptide substrate. The classical MD calculation reveals large-scale protein motions involving the flaps and the cantilever. These motions modulate the conformational properties of the substrate at the cleavage site. The ab initio calculations show in turn that substrate motion modulates the activation free energy barrier of the enzymatic reaction dramatically. Thus, the catalytic power of the enzyme does not arise from the presence of a pre-organized active site but from the protein mechanical fluctuations. The implications of this finding for the emergence of drug-resistance are discussed.

Binding Sites↗

PubMed2002

Amino acid modification in the HIV-1 Tat basic domain: insights from molecular dynamics and in vivo functional studies.

Tat is an essential protein of the human immunodeficiency virus type 1 (HIV-1). It activates transcription by specifically binding a stem-loop element in the viral long terminal repeat through its highly basic arginine-rich domain. Conserved lysine residues at positions 50 and 51 inside this domain have been recently reported to be the targets of post-translational modification by acetylation, and mutation of these residues has pointed out its relevance to protein function. In an attempt to shed light on the molecular basis of the functional differences found for Tat mutants we have performed a series of molecular dynamics simulations on wt Tat, Lys50/51 --> Arg50/51, Lys50/51 --> Ala50/51 and acetylated Lys50 from HIV-1 strain Z2. Theoretical results are compared with a homogeneous set of in vivo transactivation assays on the corresponding Tat mutants from the strain B2, which exhibits high structural similarity with Tat from HIV-1 strain Z2. Remarkable correlation is found between the degree of structure conservation and the transactivation capabilities of Tat mutants.

Acetylation↗

PubMed2002

Enzymatic GTP hydrolysis: insights from an ab initio molecular dynamics study.

Ab initio methods were used to shed light on fundamental aspects of the enzymatic mechanism of guanosine triphosphate hydrolysis in the Cdc42/Cdc42GAP complex. The calculations focused on the nucleophilic addition of the catalytic water molecule to the gamma-phosphate phosphorus atom. A large model system was required to correctly reproduce the electrostatic properties on the active site. The model turned out to reproduce most of the electrostatic field of the biological complex at the reactants. Our calculations established the H-bond pattern of the catalytic water (WAT), which turned out to interact with Q61 and T35, in the most stable conformation. This ruled out the possibility that the catalytic water transferred its proton directly to the gamma-phosphate. Furthermore, the calculations suggested that the electronic structure of WAT was very different from that in the bulk. Finally, this study showed that during the reaction, WAT transferred a proton to Gln61, consistent with the available X-ray data on a transition-state analogue/enzyme complex(19) and with the decrease of activity in the Q61E mutant.

Binding Sites↗

PubMed2002

NADH interactions with WT- and S94A-acyl carrier protein reductase from Mycobacterium tuberculosis: an ab initio study.

We present an ab initio molecular dynamics study of the complex between acyl carrier protein reductase InhA from M. tuberculosis and isonicotinic acid hydrazide-NADH. We focus on wild-type (WT) InhA and a mutant causing drug resistance (S94A) for which structural information is available (Rozwarski et al., 1998;279:98--102; Dessen et al., 1995;267:1638--1641). Our calculations suggest that the water-mediated H-bond interactions between Ser94 side chain and NADH, present in WT InhA X-ray structure, can be lost during the dynamics. This conformational change is accompanied by a structural rearrangement of Gly14. The calculated structure of WT is rather similar to the X-ray structure of the S94A mutant in terms of geometrical parameters and chemical bonding. Further evidence for the mobility of Ser94 is provided by a 1-ns-long classical molecular dynamics on the entire protein. The previously unrecognized high mobility of Ser94 can provide a rationale of the small change in free binding energies on passing from WT to S94A InhA.

Acyl Carrier Protein↗

PubMed2002

Structural determinants and hydrogen-bond network of the mononuclear zinc(II)-beta-lactamase active site.

Zinc(II)-beta-lactamases are among the latest generation of antibiotic-resistant enzymes developed by bacteria against beta-lactams. Here we have used density functional theory to provide the full structure of the catalytic site from Bacillus cereus mononuclear beta-lactamase II. Calculations are carried out on relative large models built on the X-ray structure of the free enzyme at the highest available resolution (1.7 A, PDB entry 3BC2). The most stable conformation emerging from our calculations consists of a Zn(II)-bound hydroxide, which acts as nucleophilic agent in the enzymatic reaction, highly stabilized by a complex hydrogen-bond network, in which the protonation state of Asp90 plays a major role. The pattern differs from that previously proposed on the basis of smaller models. Furthermore, the calculations confirm that Arg91 contributes to determine the orientation and the protonation state of Asp90, as recently suggested by mutagenesis experiments.

Amino Acids↗

PubMed2002

The role and perspective of ab initio molecular dynamics in the study of biological systems.

Ab initio molecular dynamics (MD) allows realistic simulations to be performed without adjustable parameters. In recent years, the technique has been used on an increasing number of applications to biochemical systems. Here we describe the principles on which ab initio MD is based. We focus on the most popular implementation, based on density functional theory and plane wave basis set. By a survey of recent applications, we show that despite the current limitations of size and time scale, ab initio MD (and hybrid ab initio MD/MM approaches) can play an important role for the modeling of biological systems. Finally, we provide a perspective for the advancement of methodological approaches which may further expand the scope of ab initio MD in biomolecular modeling.

Animals↗

PubMed2002

Drug resistance in HIV-1 protease: Flexibility-assisted mechanism of compensatory mutations.

The emergence of drug-resistant variants is a serious side effect associated with acquired immune deficiency syndrome therapies based on inhibition of human immunodeficiency virus type 1 protease (HIV-1 PR). In these variants, compensatory mutations, usually located far from the active site, are able to affect the enzymatic activity via molecular mechanisms that have been related to differences in the conformational flexibility, although the detailed mechanistic aspects have not been clarified so far. Here, we perform multinanosecond molecular dynamics simulations on L63P HIV-1 PR, corresponding to the wild type, and one of its most frequently occurring compensatory mutations, M46I, complexed with the substrate and an enzymatic intermediate. The quality of the calculations is established by comparison with the available nuclear magnetic resonance data. Our calculations indicate that the dynamical fluctuations of the mutated enzyme differ from those in the wild type. These differences in the dynamic properties of the adducts with the substrate and with the gem-diol intermediate might be directly related to variations in the enzymatic activity and therefore offer an explanation of the observed changes in catalytic rate between wild type and mutated enzyme. We anticipate that this "flexibility-assisted" mechanism might be effective in the vast majority of compensatory mutations, which do not change the electrostatic properties of the enzyme.

Drug Resistance, Viral↗

PubMed2002

A novel class of peptides with facilitating action on neuronal nicotinic receptors of rat chromaffin cells in vitro: functional and molecular dynamics studies.

Peptides related to the N-terminal region of calcitonin gene-related peptide (CGRP) were tested for their ability to modulate neuronal nicotinic acetylcholine receptors (nAChRs) of rat cultured chromaffin cells under whole cell patch-clamp conditions. Although CGRP(1-7) and CGRP(2-7) depressed responses mediated by nAChRs, CGRP(1-6), CGRP(1-5), or CGRP(1-4) rapidly and reversibly potentiated submaximal nicotine currents while sparing maximal currents. CGRP(1-3) was inactive. The threshold concentration for the enhancing effect of CGRP(1-6) was 0.1 microM. CGRP(1-5) or CGRP(1-4) were less effective than CGRP(1-6). Coapplication of CGRP(1-6) and of the allosteric potentiator physostigmine (0.5 microM) gave additive effects on nicotine currents. CGRP(1-6) did not enhance responses generated by muscle-type nicotinic receptors of cultured myoblasts or by gamma-aminobutyric acid(A) receptors expressed by human embryonic kidney cells. Molecular dynamics (MD) simulations suggested that CGRP(1-7) exhibited a relatively rigid ring structure imparted by the disulfide bridge between Cys(2) and Cys(7). The circular dichroism (CD) spectrum recorded from the same peptide was in agreement with this result. Shorter peptides, missing such a bridge, exhibited propensity for alpha-helix configuration. Replacing Cys(7) with Ala yielded CGRP(1-7A), a fragment with partial alpha-helix structure and ability to enhance nicotine currents. CD measurements on CGRP(1-6) were compatible with these MD structural findings. Short terminal fragments of CGRP represent a novel class of substances with selective, rapid, and reversible potentiation of nAChRs.

Allosteric Regulation↗

PubMedDate not supplied

Tetraethylammonium binding to the outer mouth of the KcsA potassium channel: implications for ion permeation.

Extracellular tetraethylammonium (TEA+) inhibits the current carried out by K+ ions in potassium channels. Structural models of wild-type (WT) and Y82C KcsA K+ channel/TEA+ complexes are here built using docking procedures, electrostatics calculations and molecular dynamics simulations. The calculations are based on the structure determined by Doyle et al. (11) Our calculations suggest that in WT, the TEA+ cation turns binds at the outer mouth of the selectivity filter, stabilized by electrostatic and hydrophobic interactions with the four Tyr82 side chains. Replacement of Tyr82 with Cys causes a decrease of the affinity of the cation for the channel, consistently with the available site-directed mutagenesis data (16). An MD simulation in which K+ replaces TEA+ provides evidence that TEA+ binding site can accommodate a potassium ion, in agreement with the high-resolution structure recently reported by Zhou et al. (20)

Bacterial Proteins↗