Biomedical subjects

Paolo Carloni

Publications and source records attributed to Paolo Carloni.

At least 37 records · Page 2Linked to original sources

PubMed2005

Molecular basis of the allosteric mechanism of cAMP in the regulatory PKA subunit.

The second messenger cyclic Adenosine MonoPosphate (cAMP) mediates many biological process by interacting with structurally conserved nucleotide binding domains (cNBD's). Here, we use molecular dynamics simulations on RIIbeta-PKA, one of the best characterized members of the cNBD family, in presence and absence of cAMP. The results of our calculations are fully consistent with the available experimental data and suggest that the key factor of the cAMP allosteric mechanism in cNBDS's is the increased flexibility of the protein upon ligand release along with a mechanical coupling between helical segments. In addition, our calculations provide a rationale for the experimentally observed cAMP selective binding to PKA.

Allosteric Regulation↗

PubMed2005

Structural basis of gating of CNG channels.

Cyclic nucleotide-gated (CNG) ion channels, underlying sensory transduction in vertebrate photoreceptors and olfactory sensory neurons, require cyclic nucleotides to open. Here, we present structural models of the tetrameric CNG channel pore from bovine rod in both open and closed states, as obtained by combining homology modeling-based techniques, experimentally derived spatial constraints and structural patterns present in the PDB database. Gating is initiated by an anticlockwise rotation of the N-terminal region of the C-linker, which is then, transmitted through the S6 transmembrane helices to the P-helix, and in turn from this to the pore lumen, which opens up from 2 to 5A thus allowing for ion permeation. The approach, here presented, is expected to provide a general methodology for model ion channels and their gating when structural templates are available and an extensive electrophysiological analysis has been performed.

Amino Acid Sequence↗

PubMed2005

Evolutionarily conserved functional mechanics across pepsin-like and retroviral aspartic proteases.

The biological function of the aspartic protease from HIV-1 has recently been related to the conformational flexibility of its structural scaffold. Here, we use a multistep strategy to investigate whether the same mechanism affects the functionality in the pepsin-like fold. (i) We identify the set of conserved residues by using sequence-alignment techniques. These residues cluster in three distinct regions: near the cleavage-site cavity, in the four beta-sheets cross-linking the two lobes, and in a solvent-exposed region below the long beta-hairpin in the N-terminal lobe. (ii) We elucidate the role played by the conserved residues for the enzymatic functionality of one representative member of the fold family, the human beta-secretase, by means of classical molecular dynamics (MD). The conserved regions exhibit little overall mobility and yet are involved into the most important modes of structural fluctuations. These modes influence the substrate-catalytic aspartates distance through a relative rotation of the N- and C-terminal lobes. (iii) We investigate the effects of this modulation by estimating the reaction free energy at different representative substrate/enzyme conformations. The activation free energy is strongly affected by large-scale protein motions, similarly to what has been observed in the HIV-1 enzyme. (iv) We extend our findings to all other members of the two eukaryotic and retroviral fold families by recurring to a simple, topology-based, energy functional. This analysis reveals a sophisticated mechanism of enzymatic activity modulation common to all aspartic proteases. We suggest that aspartic proteases have been evolutionarily selected to possess similar functional motions despite the observed fold variations.

Amino Acid Sequence↗

PubMed2005

Comparative analysis of HIV-1 Tat variants.

HIV-1 Tat protein is a crucial element for viral replication; therefore, its inhibition might be exploited against the AIDS infection. To gain insights on the natural variability of this protein, we present a comparative investigation on the relationship between the primary sequences and the experimentally available three-dimensional structures from the HIV-1 Tat variants Z2, BRU, and MAL. Our computational tools include sequence conservation algorithms, structural analysis, electrostatic modeling, and molecular dynamics (MD) simulations. We find that two regions located between residues 10-18 and 41-52 display the highest primary sequence conservation, while the most conserved region among the available structures corresponds approximately to the segment between positions approximately 44 and 50. Furthermore, in spite of their large structural divergence, Tat variants share a common mode for long-range intramolecular interactions. Finally, the flexibility of the Z2, BRU, and MAL variants, as emerging from multinanosecond MD simulations, is rather similar. Based on this work, we conclude that the turnlike region between amino acids 44 and 50 is structurally most conserved, emerging as an important motif for pharmaceutical targeting aimed toward inhibiting Tat action.

Algorithms↗

PubMed2004

A proficient enzyme: insights on the mechanism of orotidine monophosphate decarboxylase from computer simulations.

Decarboxylation of orotidine 5'-monophosphate (Omp) to uridine 5'-monophosphate by orotidine 5'-monophosphate decarboxylase (ODCase) is currently the object of vivid debate. Here, we clarify its enzymatic activity with long time scale classical molecular dynamics and hybrid ab initio Car-Parrinello/molecular mechanics simulations. The lack of structural (experimental) information on the ground state of ODCase/Omp complex is overcome by a careful construction of the model and the analysis of three different strains of the enzyme. We find that the ODCase/substrate complex is characterized by a very stable charged network Omp-Lys-Asp-Lys-Asp, which is incompatible with the previously proposed direct decarboxylation driven by a ground-state destabilization. A direct decarboxylation induced by a transition-state electrostatic stabilization is consistent with our findings. The calculated activation free energy for the direct decarboxylation with the formation of a C6 carboanionic intermediate yields an overall rate enhancement by the enzyme (k(cat)/k(wat) = 3.5 x 10(16)) in agreement with experiments (k(cat)/k(wat) = 1.7 x 10(17)). The decarboxylation is accompanied by the movement of a fully conserved lysine residue toward the developing negative charge at the C6 position.

Computer Simulation↗

PubMed2004

Water-assisted reaction mechanism of monozinc beta-lactamases.

Hybrid Car-Parrinello QM/MM calculations are used to investigate the reaction mechanism of hydrolysis of a common beta-lactam substrate (cefotaxime) by the monozinc beta-lactamase from Bacillus cereus (BcII). The calculations suggest a fundamental role for an active site water in the catalytic mechanism. This water molecule binds the zinc ion in the first step of the reaction, expanding the zinc coordination number and providing a proton donor adequately oriented for the second step. The free energy barriers of the two reaction steps are similar and consistent with the available experimental data. The conserved hydrogen bond network in the active site, defined by Asp120, Cys221, and His263, not only contributes to orient the nucleophile (as already proposed), but it also guides the second catalytic water molecule to the zinc ion after the substrate is bound. The hydrolysis reaction in water has a relatively high free energy barrier, which is consistent with the stability of cefotaxime in water solution. The modeled Michaelis complexes for other substrates are also characterized by the presence of an ordered water molecule in the same position, suggesting that this mechanism might be general for the hydrolysis of different beta-lactam substrates.

Bacillus cereus↗

PubMed2004

Dynamics and energetics of water permeation through the aquaporin channel.

Structural properties of water inside bovine aquaporin-1 are investigated by molecular simulation. The calculations, which are based on the recently determined X-ray structure at 2.2 A resolution (Sui et al., Nature 2001;414:872-878), are carried out on one monomeric subunit immersed in a water-n-octane-water bilayer. Molecular dynamics (MD) simulations suggest that His182, a fully conserved residue in the channel pore, is protonated in the delta position. Furthermore, they reveal a highly ordered water structure in the channel, induced by the electrostatic properties of the protein. Multiple-steering MD simulations are used to calculate the free-energy of water diffusion. To the best of our knowledge, this represents the first free-energy calculation based on the new, high-resolution structure of the pore. The calculated barrier is 2.5 kcal/mol, and it is associated to water permeation through the Asn-Pro-Ala (NPA) region of the pore, where water molecules are only hydrogen-bonded with themselves. These findings are fully consistent with those based on the previous MD studies on the human protein (de Groot and Grubmüller, Science 2001;294:2353-2357).

Animals↗

PubMed2004

Accurate and efficient description of protein vibrational dynamics: comparing molecular dynamics and Gaussian models.

Current all-atom potential based molecular dynamics (MD) allows the identification of a protein's functional motions on a wide-range of timescales, up to few tens of nanoseconds. However, functional, large-scale motions of proteins may occur on a timescale currently not accessible by all-atom potential based MD. To avoid the massive computational effort required by this approach, several simplified schemes have been introduced. One of the most satisfactory is the Gaussian network approach based on the energy expansion in terms of the deviation of the protein backbone from its native configuration. Here, we consider an extension of this model that captures in a more realistic way the distribution of native interactions due to the introduction of effective side-chain centroids. Since their location is entirely determined by the protein backbone, the model is amenable to the same exact and computationally efficient treatment as previous simpler models. The ability of the model to describe the correlated motion of protein residues in thermodynamic equilibrium is established through a series of successful comparisons with an extensive (14 ns) MD simulation based on the AMBER potential of HIV-1 protease in complex with a peptide substrate. Thus, the model presented here emerges as a powerful tool to provide preliminary, fast yet accurate characterizations of protein near-native motion.

Computer Simulation↗

PubMed2004

Substrate binding to mononuclear metallo-beta-lactamase from Bacillus cereus.

Structure and dynamics of substrate binding (cefotaxime) to the catalytic pocket of the mononuclear zinc-beta-lactamase from Bacillus cereus are investigated by molecular dynamics simulations. The calculations, which are based on the hydrogen-bond pattern recently proposed by Dal Peraro et al. (J Biol Inorg Chem 2002; 7:704-712), are carried out for both the free and the complexed enzyme. In the resting state, active site pattern and temperature B-factors are in agreement with crystallographic data. In the complexed form, cefotaxime is accommodated into a stable orientation in the catalytic pocket within the nanosecond timescale, interacting with the enzyme zinc-bound hydroxide and the surrounding loops. The beta-lactam ring remains stable and very close to the hydroxide nucleophile agent, giving a stable representation of the productive enzyme-substrate complex.

Bacillus cereus↗

PubMed2004

Molecular dynamics study of the KcsA channel at 2.0-A resolution: stability and concerted motions within the pore.

The stability of the KcsA channel accommodating more than one ion in the pore has been studied with molecular dynamics. We have used the very last X-ray structure of the KcsA channel at 2.0-A resolution determined by Zhou et al. [Nature 414 (2001) 43]. In this channel, six of the seven experimentally evidenced sites have been considered. We show that the protein remains very stable in the presence of four K+ ions (three in the selectivity filter and one in the cavity). The locations and the respective distances of the different K+ ions and water molecules (W), calculated within our KWKWKK sequence, also fits well with the experimental observations. The analysis of the K+ ions and water molecules displacements shows concerted file motions on the simulated time scale (approximately 1 ns), which could act as precursor to the diffusion of K+ ions inside the channel. A simple one-dimensional dynamical model is used to interpret the concerted motions of the ions and water molecules in the pore leading ultimately to ion transfer.

Computer Simulation↗

PubMed2003

Molecular dynamics simulations on HIV-1 Tat.

Molecular dynamics simulations are used to investigate dynamics and intramolecular interactions of the HIV-1 transactivator (Tat) in aqueous solution. The calculations are based on the AMBER force field with particle mesh Ewald treatment for long-range electrostatics. The Tat structure exhibits a large flexibility, consistent with its absence of secondary structure elements. From an analysis of the correlation matrix and of electrostatic interactions we suggest that segments expressed by the two exons (amino acids 1-72 and 73-86, respectively) exhibit rather separated dynamic and energetic properties. We also identify intramolecular interactions of importance for structure stabilization. In particular, significant electrostatic interactions are recognized between the N-terminus and the basic domain of the protein, consistent with site-directed mutagenesis performed in this work.

Amino Acid Sequence↗

PubMed2003

A structural model of agonist binding to the alpha3beta4 neuronal nicotinic receptor.

(alpha3)2(beta4)3 is the most abundant type of neuronal nicotinic ACh receptor (nAChR) mediating cholinergic actions on the autonomic nervous system. Studies to refine or devise drugs selectively acting on (alpha3)2(beta4)3 receptors would benefit from a detailed description of the hitherto unclear agonist-binding domain. The present study reports a three-dimensional model for the ligand-binding domain (LBD) of this receptor either in its unoccupied or agonist-bound conformation. The receptor model was based on the structure of the acetylcholine-binding protein (AChBP), and was obtained using molecular modelling techniques. ACh, nicotine and cytisine (full agonists), muscarine (a selective agonist of muscarinic ACh receptors) and the allosteric modulator eserine were docked into the binding pockets of the receptor model. Simulated agonist-receptor complexes were compared with the agonist-binding complex of the AChBP, as well as of the (alpha4)2(beta2)3 type of nAChR, which is the commonest in the brain. Agonist docking identified discrete amino-acid residues of the beta subunits important for pharmacological selectivity of nAChRs. The predicted affinity of muscarine for the nAChR was low, suggesting the present model to be suitable for effective discrimination of nicotinic agonist binding versus nonselective cholinergic binding. Furthermore, the current model outlined a potential binding site for the allosteric modulator eserine, the site of action of which has remained elusive. The present LBD model of the receptor in its free state provides a novel structural framework to interpret experimental observations and a useful template for future investigations to develop (alpha3)2(beta4)3-selective ligands.

Amino Acid Sequence↗

PubMed2003

Molecular modeling studies on CNG channel from bovine retinal rod: a structural model of the cyclic nucleotide-binding domain.

A dimeric model of the cyclic nucleotide-binding domain of the all-alpha homomeric cyclic nucleotide-gated channel from bovine retinal rod is constructed. The model, based on the structure of the fairly homologous catabolite gene activator protein (Weber and Steitz, J Mol Biol 1987;198:311-326), is obtained by use of comparative modeling and molecular dynamics simulations. Our model provides a structural basis for the experimentally measured difference in activity between cAMP and cGMP, as well as the different solvent accessibilities of GLY597 in the complex with cGMP, with cAMP and in the protein in free state. In addition, it provides support for the rearrangement of the domain C helix on ligand binding and releasing proposed by Matulef et al. (Neuron 1999;24:443-452).

Animals↗

PubMed2003

Protonation state of Asp120 in the binuclear active site of the metallo-beta-lactamase from Bacteroides fragilis.

The determination of the protonation state of enzyme active sites may be crucial for the investigation of their mechanism of action. In the bizinc beta-lactamase family of enzymes, no consensus has been reached on the protonation state of a fully conserved amino acid present in the active site, Asp120. To address this issue, we carry out here density functional theory (DFT) calculations on large models (based on Bacteroides fragilis X-ray structure) which include the metal coordination polyhedron and groups interacting with it. Our calculations suggest that Asp120 is ionized. The relevance of this finding for site-directed mutagenesis experiments on the 120 position and on the mechanism of action is discussed.

Aspartic Acid↗

PubMed2003

Calcium binding to the transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase: insights from molecular modeling.

Sarcoplasmic reticulum Ca(2+)- ATPase pumps Ca(2+) ions from muscle cells to the sarcoplasmic reticulum. Here we use molecular dynamics and electrostatic modeling to investigate structural and dynamical features of key intermediates in the Ca(2+) binding process of the protein. Structural models of the protein (containing either two, one, or no calcium ions in the transmembrane domain) are constructed based on the X-ray structure by Toyoshima et al. (Nature 2000;405:647-655). The protein is embedded in a water/octane bilayer, which mimics the water/membrane environment. Our calculations provide information on the hydration of the two Ca(2+) ions, not emerging from the X-ray structure. Furthermore, they indicate that uptake of the metal ions causes large structural rearrangements of the metal binding sites. In addition, they suggest that the two ions reach their binding sites via two specific pathways. Finally, they allow identification of residues in the outer mouth of the protein that might interact with the Ca(2+) ions during the binding process.

Binding Sites↗

PubMed2003

Molecular dynamics simulations of the NGF-TrkA domain 5 complex and comparison with biological data.

The nerve growth factor (NGF) is an important pharmacological target for Alzheimer's and other neurodegenerative diseases. Its action derives partly from its binding to the tyrosine kinase A receptor (TrkA). Here we study energetics and dynamics of the NGF-TrkA complex by carrying out multinanosecond molecular dynamics simulations, accompanied by electrostatic calculations based on the Poisson-Boltzmann equation. Our calculations, which are based on the x-ray structure of the complex, suggest that some of the mutations affecting dramatically the affinity of the complex involve residues that form highly favorable, direct or water-mediated hydrogen bond interactions at the ligand-receptor interface and, in some cases, that also critically participate to the large-scale motions of the complex. Furthermore, our calculations offer a rationale for the small effect on binding affinity observed upon specific mutations involving large changes in electrostatics (i.e., the charged-to-neutral mutations). Finally, these calculations, used along with the mutagenesis data, provide a basis for designing new peptides that mimic NGF in TrkA binding function.

Binding Sites↗

PubMed2003

Molecular modeling of ion channels: structural predictions.

Recent advances in membrane protein crystallography have greatly increased structural information of channels permeating metal ions. Structural bioinformatics techniques and molecular dynamics calculations are providing structural models of ion channels for which the three-dimensional structure is not known. Most of the reported structure prediction studies focus on K(+) channels and are based on the KcsA K(+) channel structure.

Computational Biology↗

PubMed2002

Electronic structure of wet DNA.

The electronic properties of a Z-DNA crystal synthesized in the laboratory are investigated by means of density-functional theory Car-Parrinello calculations. The electronic structure has a gap of only 1.28 eV. This separates a manifold of 12 occupied states which came from the pi guanine orbitals from the lowest empty states in which the electron is transferred to the Na+ from PO-4 groups and water molecules. We have evaluated the anisotropic optical conductivity. At low frequency the conductivity is dominated by the pi-->Na+ transitions. Our calculation demonstrates that the cost of introducing electron holes in wet DNA strands could be lower than previously anticipated.

Crystallization↗