Sergio Pantano

A Coarse Grained Model for Atomic-Detailed DNA Simulations with Explicit Electrostatics

Coarse-grain (CG) techniques allow considerable extension of the accessible size and time scales in simulations of biological systems. Although many CG representations are available for the most common biomacromolecules, very few have been reported for nucleic acids. Here, we present a CG model for molecular dynamics simulations of DNA on the multi-microsecond time scale. Our model maps the complexity of each nucleotide onto six effective superatoms keeping the chemical sense of specific Watson-Crick recognition. Molecular interactions are evaluated using a classical Hamiltonian with explicit electrostatics calculated under the framework of the generalized Born approach. This CG representation is able to accurately reproduce experimental structures, breathing dynamics, and conformational transitions from the A to the B form in double helical fragments. The model achieves a good qualitative reproduction of temperature-driven melting and its dependence on size, ionic strength, and sequence specificity. Reconstruction of atomistic models from CG trajectories give remarkable agreement with structural, dynamic, and energetic features obtained from fully atomistic simulation, opening the possibility to acquire nearly atomic detail data from CG trajectories.

The blockade of the neurotransmitter release apparatus by botulinum neurotoxins

The high toxicity of the seven serotypes of botulinum neurotoxins (BoNT/A to G), together with their specificity and reversibility, includes them in the list A of potential bioterrorism weapons and, at the same time, among the therapeutics of choice for a variety of human syndromes. They invade nerve terminals and cleave specifically the three proteins which form the heterotrimeric SNAP REceptors (SNARE) complex that mediates neurotransmitter release. The BoNT-induced cleavage of the SNARE proteins explains by itself the paralysing activity of the BoNTs because the truncated proteins cannot form the SNARE complex. However, in the case of BoNT/A, the most widely used toxin in therapy, additional factors come into play as it only removes a few residues from the synaptosomal associate protein of 25xa0kDa C-terminus and this results in a long duration of action. To explain these facts and other experimental data, we present here a model for the assembly of the neuroexocytosis apparatus in which Synaptotagmin and Complexin first assist the zippering of the SNARE complex, and then stabilize and clamp an octameric radial assembly of the SNARE complexes.

Unitary permeability of gap junction channels to second messengers measured by FRET microscopy.

Gap junction channels assembled from connexin protein subunits mediate intercellular transfer of ions and metabolites. Impaired channel function is implicated in several hereditary human diseases. In particular, defective permeation of cAMP or inositol-1,4,5-trisphosphate (InsP3) through connexin channels is associated with peripheral neuropathies and deafness, respectively. Here we present a method to estimate the permeability of single gap junction channels to second messengers. Using HeLa cells that overexpressed wild-type human connexin 26 (HCx26wt) as a model system, we combined measurements of junctional conductance and fluorescence resonance energy transfer (FRET) emission ratio of biosensors selective for cAMP and InsP3. The unitary permeabilities to cAMP (47 × 10−3 ± 15 × 10−3 μm3/s) and InsP3 (60 × 10−3 ± 12 × 10−3 μm3/s) were similar, but substantially larger than the unitary permeability to lucifer yellow (LY; 7 ± 3 × 10−3 μm3/s), an exogenous tracer. This method permits quantification of defects of metabolic coupling and can be used to investigate interdependence of intercellular diffusion and cross-talk between diverse signaling pathways.

Cloning, characterization, and subcellular localization of a Trypanosoma cruzi argonaute protein defining a new subfamily distinctive of trypanosomatids

Over the last years an expanding family of small non-coding RNAs (sRNA) has been identified in eukaryotic genomes which behave as sequence-specific triggers for mRNA degradation, translation repression, heterochromatin formation and genome stability. To achieve their effectors functions, sRNAs associate with members of the Argonaute protein family. Argonaute proteins are segregated into three paralogous groups: the AGO-like subfamily, the PIWI-like subfamily, and the WAGO subfamily (for Worm specific AGO). Detailed phylogenetic analysis of the small RNA-related machinery components revealed that they can be traced back to the common ancestor of eukaryotes. However, this machinery seems to be lost or excessively simplified in some unicellular organisms such as Saccharomyces cerevisiae, Trypanosoma cruzi, Leishmania major and Plasmodium falciparum which are unable to utilize dsRNA to trigger degradation of target RNAs. We reported here a unique ORF encoding for an AGO/PIWI protein in T. cruzi which was expressed in all stages of its life cycle at the transcript as well as the protein level. Database search for remote homologues, revealed the presence of a divergent PAZ domain adjacent to the well supported PIWI domain. Our results strongly suggested that this unique AGO/PIWI protein from T. cruzi is a canonical Argonaute in terms of its domain architecture. We propose to reclassify all Argonaute members from trypanosomatids as a distinctive phylogenetic group representing a new subfamily of Argonaute proteins and propose the generic designation of AGO/PIWI-tryp to identify them. Inside the Trypanosomatid-specific node, AGO/PIWI-tryps were clearly segregated into two paralog groups designated as AGO-tryp and PIWI-tryp according to the presence or absence of a functional link with RNAi-related phenomena, respectively.

The role of phosphorylation on the structure and dynamics of phospholamban: a model from molecular simulations.

Phospholamban (PLB) is a small membrane protein that regulates the activity of the calcium ATP‐ase in the cardiac, slow‐twitch, and smooth muscle sarcoplasmic reticulum through the reversible phosphorylation of Ser16. We present here a comparative molecular dynamics study of unmodified and phosphorylated PLB immersed in a phospholipid membrane. The study has been performed under different ionic strength conditions, using the NMR structures of two PLB variants determined in mixed organic solvent and dodecylphosphocholine micelles. The simulations indicate that all PLB forms studied display a highly dynamic behavior of the N‐terminal cytoplasmic moiety, with a decrease of its helical content in the phosphorylated forms. The cytoplasmic domain undergoes large collective motions sampling conformations parallel as well as perpendicular to the membrane surface in all the simulations. The transmembrane domain retains a tightly folded helical conformation with a small tilt with respect to the membrane plane probably induced by the presence of Asn30 and Asn34 within the hydrophobic environment. Furthermore, the phosphoric group on Ser16 establishes transient electrostatic interactions with the phospholipid heads. We propose a model in which phosphorylation diminishes the probability of interactions of PLB with residues near Lys400 in the SERCA pump, thus relieving itsinhibition. Proteins 2007

Transferable mixing of atomistic and coarse-grained water models.

Dual-resolution approaches for molecular simulations combine the best of two worlds, providing atomic details in regions of interest and coarser but much faster descriptions of less-relevant parts of molecular systems. Given the abundance of water in biomolecular systems, reducing the computational cost of simulating bulk water without perturbing the solutes properties is a very attractive strategy. Here we show that the coarse-grained model for water called WatFour (WT4) can be combined with any of the three most used water models for atomistic simulations (SPC, TIP3P, and SPC/E) without modifying the characteristics of the atomistic solvent and solutes. The equivalence of fully atomistic and hybrid solvation approaches is assessed by comparative simulations of pure water, electrolyte solutions, and the β1 domain of streptococcal protein G, for which comparisons between experimental and calculated chemical shifts at (13)Cα are equivalent.

SIRAH: A Structurally Unbiased Coarse-Grained Force Field for Proteins with Aqueous Solvation and Long-Range Electrostatics

Modeling of macromolecular structures and interactions represents an important challenge for computational biology, involving different time and length scales. However, this task can be facilitated through the use of coarse-grained (CG) models, which reduce the number of degrees of freedom and allow efficient exploration of complex conformational spaces. This article presents a new CG protein model named SIRAH, developed to work with explicit solvent and to capture sequence, temperature, and ionic strength effects in a topologically unbiased manner. SIRAH is implemented in GROMACS, and interactions are calculated using a standard pairwise Hamiltonian for classical molecular dynamics simulations. We present a set of simulations that test the capability of SIRAH to produce a qualitatively correct solvation on different amino acids, hydrophilic/hydrophobic interactions, and long-range electrostatic recognition leading to spontaneous association of unstructured peptides and stable structures of single polypeptides and protein-protein complexes.

Mixing Atomistic and Coarse Grain Solvation Models for MD Simulations: Let WT4 Handle the Bulk.

Accurate simulation of biomolecular systems requires the consideration of solvation effects. The arrangement and dynamics of water close to a solute are strongly influenced by the solute itself. However, as the solute-solvent distance increases, the water properties tend to those of the bulk liquid. This suggests that bulk regions can be treated at a coarse grained (CG) level, while keeping the atomistic details around the solute. Since water represents about 80% of any biological system, this approach may offer a significant reduction in the computational cost of simulations without compromising atomistic details. We show here that mixing the popular SPC water model with a CG model for solvation (called WatFour) can effectively mimic the hydration, structure, and dynamics of molecular systems composed of pure water, simple electrolyte solutions, and solvated macromolecules. As a nontrivial example, we present simulations of the SNARE membrane fusion complex, a trimeric protein-protein complex embedded in a double phospholipid bilayer. Comparison with a fully atomistic reference simulation illustrates the equivalence between both approaches.

Evidence for a radial SNARE super-complex mediating neurotransmitter release at the Drosophila neuromuscular junction

Summary The SNARE proteins VAMP/synaptobrevin, SNAP-25 and syntaxin are core components of the apparatus that mediates neurotransmitter release. They form a heterotrimeric complex, and an undetermined number of SNARE complexes assemble to form a super-complex. Here, we present a radial model of this nanomachine. Experiments performed with botulinum neurotoxins led to the identification of one arginine residue in SNAP-25 and one aspartate residue in syntaxin (R206 and D253 in Drosophila melanogaster). These residues are highly conserved and predicted to play a major role in the protein–protein interactions between SNARE complexes by forming an ionic couple. Accordingly, we generated transgenic Drosophila lines expressing SNAREs mutated in these residues and performed an electrophysiological analysis of their neuromuscular junctions. Our results indicate that SNAP-25-R206 and syntaxin-D253 play a major role in neuroexocytosis and support a radial assembly of several SNARE complexes interacting via the ionic couple formed by these two residues.

Coarse-grained models of water

Coarse‐grained (CG) models for macromolecules have become a standard in the study of biological systems, overcoming limitations in size and time scales encountered by atomistic molecular dynamics simulations. Just as in any biomolecular ensemble, water in CG models plays a key role in mediating intermolecular and intramolecular interactions. However, owing to the highly nontrivial properties of water, important simplifications have been commonly used to treat solvation effects. Recent developments of CG models for water are overviewed, comparing some characteristic features and limitations.