Arnau Cordomí

Membrane Protein Simulations Using AMBER Force Field and Berger Lipid Parameters.

AMBER force fields are among the most commonly used in molecular dynamics (MD) simulations of proteins. Unfortunately, they lack a specific set of lipid parameters, thus limiting its use in membrane protein simulations. In order to overcome this limitation we assessed whether the widely used united-atom lipid parameters described by Berger and co-workers could be used in conjunction with AMBER force fields in simulations of membrane proteins. Thus, free energies of solvation in water and in cyclohexane, and free energies of water to cyclohexane transfer, were computed by thermodynamic integration procedures for neutral amino acid side-chains employing AMBER99, AMBER03, and OPLS-AA amino acid force fields. In addition, MD simulations of three membrane proteins in a POPC lipid bilayer, the β2 adrenergic G protein-coupled receptor, Aquaporin-1, and the outer membrane protein Omp32, were performed with the aim of comparing the AMBER99SB/Berger combination of force fields with the OPLS-AA/Berger combination. We have shown that AMBER99SB and Berger force fields are compatible, they provide reliable free energy estimations relative to experimental values, and their combination properly describes both membrane and protein structural properties. We then suggest that the AMBER99SB/Berger combination is a reliable choice for the simulation of membrane proteins, which links the easiness of ligand parametrization and the ability to reproduce secondary structure of AMBER99SB force field with the largely validated Berger lipid parameters.

Effect of ions on a dipalmitoyl phosphatidylcholine bilayer. a molecular dynamics simulation study.

The effect of physiological concentrations of different chlorides on the structure of a dipalmitoyl phosphatidylcholine (DPPC) bilayer has been investigated through atomistic molecular dynamics simulations. These calculations provide support to the concept that Li+, Na+, Ca2+, Mg2+, Sr2+, Ba2+, and Ac3+, but not K+, bind to the lipid-head oxygens. Ion binding exhibits an influence on lipid order, area per lipid, orientation of the lipid head dipole, the charge distribution in the system, and therefore the electrostatic potential across the head-group region of the bilayer. These structural effects are sensitive to the specific characteristics of each cation, i.e., radius, charge, and coordination properties. These results provide evidence aimed at shedding some light into the apparent contradictions among different studies reported recently regarding the ordering effect of ions on zwitterionic phosphatidylcholine lipid bilayers.

Interactions between intracellular domains as key determinants of the quaternary structure and function of receptor heteromers.

G protein-coupled receptor (GPCR) heteromers are macromolecular complexes with unique functional properties different from those of its individual protomers. Little is known about what determines the quaternary structure of GPCR heteromers resulting in their unique functional properties. In this study, using resonance energy transfer techniques in experiments with mutated receptors, we provide for the first time clear evidence for a key role of intracellular domains in the determination of the quaternary structure of GPCR heteromers between adenosine A2A, cannabinoid CB1, and dopamine D2 receptors. In these interactions, arginine-rich epitopes form salt bridges with phosphorylated serine or threonine residues from CK1/2 consensus sites. Each receptor (A2A, CB1, and D2) was found to include two evolutionarily conserved intracellular domains to establish selective electrostatic interactions with intracellular domains of the other two receptors, indicating that these particular electrostatic interactions constitute a general mechanism for receptor heteromerization. Mutation experiments indicated that the interactions of the intracellular domains of the CB1 receptor with A2A and D2 receptors are fundamental for the correct formation of the quaternary structure needed for the function (MAPK signaling) of the A2A-CB1-D2 receptor heteromers. Analysis of MAPK signaling in striatal slices of CB1 receptor KO mice and wild-type littermates supported the existence of A1-CB1-D2 receptor heteromer in the brain. These findings allowed us to propose the first molecular model of the quaternary structure of a receptor heteromultimer.

Cognitive impairment induced by delta9-tetrahydrocannabinol occurs through heteromers between cannabinoid CB1 and serotonin 5-HT2A receptors

Activation of cannabinoid CB1 receptors (CB1R) by delta9-tetrahydrocannabinol (THC) produces a variety of negative effects with major consequences in cannabis users that constitute important drawbacks for the use of cannabinoids as therapeutic agents. For this reason, there is a tremendous medical interest in harnessing the beneficial effects of THC. Behavioral studies carried out in mice lacking 5-HT2A receptors (5-HT2AR) revealed a remarkable 5-HT2AR-dependent dissociation in the beneficial antinociceptive effects of THC and its detrimental amnesic properties. We found that specific effects of THC such as memory deficits, anxiolytic-like effects, and social interaction are under the control of 5-HT2AR, but its acute hypolocomotor, hypothermic, anxiogenic, and antinociceptive effects are not. In biochemical studies, we show that CB1R and 5-HT2AR form heteromers that are expressed and functionally active in specific brain regions involved in memory impairment. Remarkably, our functional data shows that costimulation of both receptors by agonists reduces cell signaling, antagonist binding to one receptor blocks signaling of the interacting receptor, and heteromer formation leads to a switch in G-protein coupling for 5-HT2AR from Gq to Gi proteins. Synthetic peptides with the sequence of transmembrane helices 5 and 6 of CB1R, fused to a cell-penetrating peptide, were able to disrupt receptor heteromerization in vivo, leading to a selective abrogation of memory impairments caused by exposure to THC. These data reveal a novel molecular mechanism for the functional interaction between CB1R and 5-HT2AR mediating cognitive impairment. CB1R-5-HT2AR heteromers are thus good targets to dissociate the cognitive deficits induced by THC from its beneficial antinociceptive properties.

Structural Rearrangements of Rhodopsin Subunits in a Dimer Complex: a Molecular Dynamics Simulation Study

Abstract The present work reports a 0.1 μs molecular dynamics simulation of a bovine rhodopsin dimer based on a recently reported semi-empirical model obtained by fitting two monomers from the crystal structure to atomic force microscopy maps (Fotiadis et al. Curr Opin Struct Biol 16, 252, 2006). The simulation shows that the quaternary arrangement is stable although subtle rearrangements in its tertiary elements are observed during the first 60 ns of the trajectory. The comparison with a parallel 0.1 μs simulation of a single monomer in the same conditions and using the same protocol allows the study of subunit-subunit interactions on the dimer interface together with the structural effects associated to the dimer formation. The present study describes the interface of a TM4/TM5-TM4/TM5 dimer at an atomistic level including an analysis of the energy contributions to the interaction of each part of the protein involved. We also compare the differences in the structure of the single monomer with those of the dimer subunits with the aim of understanding the changes required for the dimer formation.

Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with Gi and Gs

BackgroundG-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown.ResultsWe used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins.ConclusionsThe combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function.

Effect of Force Field Parameters on Sodium and Potassium Ion Binding to Dipalmitoyl Phosphatidylcholine Bilayers.

The behavior of electrolytes in molecular dynamics simulations of zwitterionic phospholipid bilayers is very sensitive to the force field parameters used. Here, several 200 ns molecular dynamics of simulations of dipalmitoyl phosphotidylcholine (PC) bilayers in 0.2 M sodium or potassium chloride using various common force field parameters for the cations are presented. All employed parameter sets give a larger number of Na(+) ions than K(+) ions that bind to the lipid heads, but depending on the parameter choice quite different results are seen. A wide range of coordination numbers for the Na(+) and K(+) ions is also observed. These findings have been analyzed and compared to published experimental data. Some simulations produce aggregates of potassium chloride, indicating (in accordance with published simulations) that these force fields do not reproduce the delicate balance between salt and solvated ions. The differences between the force fields can be characterized by one single parameter, the electrostatic radius of the ion, which is correlated to σMO (M represents Na(+)/K(+)), the Lennard-Jones radius. When this parameter exceeds a certain threshold, binding to the lipid heads is no longer observed. One would, however, need more accurate experimental data to judge or rank the different force fields precisely. Still, reasons for the poor performance of some of the parameter sets are clearly demonstrated, and a quality control procedure is provided.

Impact of Helix Irregularities on Sequence Alignment and Homology Modeling of G Protein‐Coupled Receptors

Comparison of the crystal structures of G protein‐coupled receptors (GPCRs) revealed backbone irregularities in the majority of the transmembrane (TM) helices. Among these, wide (π bulge) and tight (310) helical turns on TM2 and TM5 deserve special attention because of their proximity to the ligand binding site. These irregularities are related to residue insertion or deletion (reflected by inclusion of gaps in sequence alignments) accumulated during the evolution of these two helices. These findings have direct implications for the sequence alignments, phylogeny reconstruction, and homology modeling of class A GPCRs.

Structures for G-Protein-Coupled Receptor Tetramers in Complex with G Proteins

G-Protein-coupled receptors (GPCRs) were classically described as monomers. We now appreciate that they also function as homo- and hetero-oligomers, for which structural information is lacking. Here, we use available 3D structures and biochemical considerations to present and evaluate experimentally testable structural models for GPCR oligomers and associated G proteins.

The G-protein Coupled Receptor Family: Actors with Many Faces

G-protein coupled receptors (GPCRs) comprise the largest family of proteins in our body, which have many important physiological functions and are implicated in the pathophysiology of many serious diseases. GPCRs therefore are significant targets in pharmaceutical research. GPCRs share the common architecture of seven plasma membrane-spanning segments connected to each other with three extracellular and three intracellular loops. In addition, GPCRs contain an extracellular N-terminal region and an intracellular C-terminal tail. GPCRs could stimulate different intracellular G-proteins (internal stimuli) and signaling pathways after their interaction with different ligands (external stimuli). The exceptional functional plasticity of GPCRs could be attributed to their inherent dynamic nature to adopt different active conformations, which are stabilized differentially by different stimuli as well as by several mutations. This review describes the structural changes of GPCRs associated with their activation. Understanding the dynamic nature of GPCRs could potentially contribute in the development of future structure-based approaches to design new receptor-specific, signaling-selective ligands, which will enrich the pharmaceutical armamentarium against various diseases.