Cardiolipin Models for Molecular Simulations of Bacterial and Mitochondrial Membranes

Present in bacterial and mitochondrial membranes, cardiolipins have a unique dimeric structure, which carries up to two charges (i.e., one per phosphate group) and, under physiological conditions, can be unprotonated or singly protonated. Exhaustive models and characterization of cardiolipins are to date scarce; therefore we propose an <i>ab initio</i> parametrization of cardiolipin species for molecular simulation consistent with commonly used force fields. Molecular dynamics simulations using these models indicate a protonation dependent lipid packing. A peculiar interaction with solvating mono- and divalent cations is also observed. The proposed models will contribute to the study of the assembly of more realistic bacterial and mitochondrial membranes and the investigation of the role of cardiolipins for the biophysical and biochemical properties of membranes and membrane-embedded proteins

Cardiolipin Models for Molecular Simulations of Bacterial and Mitochondrial Membranes

Present in bacterial and mitochondrial membranes, cardiolipins have a unique dimeric structure, which carries up to two charges (i.e., one per phosphate group) and, under physiological conditions, can be unprotonated or singly protonated. Exhaustive models and characterization of cardiolipins are to date scarce; therefore we propose an <i>ab initio</i> parametrization of cardiolipin species for molecular simulation consistent with commonly used force fields. Molecular dynamics simulations using these models indicate a protonation dependent lipid packing. A peculiar interaction with solvating mono- and divalent cations is also observed. The proposed models will contribute to the study of the assembly of more realistic bacterial and mitochondrial membranes and the investigation of the role of cardiolipins for the biophysical and biochemical properties of membranes and membrane-embedded proteins

Reaction Mechanism and Catalytic Fingerprint of Allantoin Racemase

The stereospecific oxidative decomposition of urate into allantoin is the core of purine catabolism in many organisms. The spontaneous decomposition of upstream intermediates and the nonenzymatic racemization of allantoin lead to an accumulation of (<i>R</i>)-allantoin, because the enzymes converting allantoin into allantoate are specific for the (<i>S</i>) isomer. The enzyme allantoin racemase catalyzes the reversible conversion between the two allantoin enantiomers, thus ensuring the overall efficiency of the catabolic pathway and preventing allantoin accumulation. On the basis of recent crystallographic and biochemical evidence, allantoin racemase has been assigned to the family of cofactor-independent racemases, together with other amino acid racemases. A detailed computational investigation of allantoin racemase has been carried out to complement the available experimental data and to provide atomistic insight into the enzymatic action. Allantoin, the natural substrate of the enzyme, has been investigated at the quantum mechanical level, in order to rationalize its conformational and tautomeric equilibria, playing a key role in protein–ligand recognition and in the following catalytic steps. The reaction mechanism of the enzyme has been elucidated through quantum mechanics/molecular mechanics (QM/MM) calculations. The potential energy surface investigation, carried out at the QM/MM level, revealed a stepwise reaction mechanism. A pair of cysteine residues promotes the stereoinversion of a carbon atom of the ligand without the assistance of cofactors. Electrostatic fingerprint calculations are used to discuss the role of the active site residues in lowering the p<i>K</i>_a of the substrate. The planar unprotonated intermediate is compared with the enolic allantoin tautomer observed in the active site of the crystallized enzyme. Finally, the enzymatic catalysis featured by allantoin racemase (AllR) is compared with that of other enzymes belonging to the same family

Reaction Mechanism and Catalytic Fingerprint of Allantoin Racemase

The stereospecific oxidative decomposition of urate into allantoin is the core of purine catabolism in many organisms. The spontaneous decomposition of upstream intermediates and the nonenzymatic racemization of allantoin lead to an accumulation of (<i>R</i>)-allantoin, because the enzymes converting allantoin into allantoate are specific for the (<i>S</i>) isomer. The enzyme allantoin racemase catalyzes the reversible conversion between the two allantoin enantiomers, thus ensuring the overall efficiency of the catabolic pathway and preventing allantoin accumulation. On the basis of recent crystallographic and biochemical evidence, allantoin racemase has been assigned to the family of cofactor-independent racemases, together with other amino acid racemases. A detailed computational investigation of allantoin racemase has been carried out to complement the available experimental data and to provide atomistic insight into the enzymatic action. Allantoin, the natural substrate of the enzyme, has been investigated at the quantum mechanical level, in order to rationalize its conformational and tautomeric equilibria, playing a key role in protein–ligand recognition and in the following catalytic steps. The reaction mechanism of the enzyme has been elucidated through quantum mechanics/molecular mechanics (QM/MM) calculations. The potential energy surface investigation, carried out at the QM/MM level, revealed a stepwise reaction mechanism. A pair of cysteine residues promotes the stereoinversion of a carbon atom of the ligand without the assistance of cofactors. Electrostatic fingerprint calculations are used to discuss the role of the active site residues in lowering the p<i>K</i>_a of the substrate. The planar unprotonated intermediate is compared with the enolic allantoin tautomer observed in the active site of the crystallized enzyme. Finally, the enzymatic catalysis featured by allantoin racemase (AllR) is compared with that of other enzymes belonging to the same family

TM connectivity and implications for signaling mechanism.

<p>The panels (A) and (B) indicate the projection for the F₀ and F₁ states, respectively, on the periplasmic (top) and cytoplasmic (bottom) surface of the membrane of the helical termini of the TM model. The contour lines represent the position of TM1 and TM2 during MD, and the dots represent the position of SD and HAMP available structures at the same section surface. (Central panel) At high mM concentration of Mg²⁺ and Ca²⁺ cations, metal bridges are formed between the SD acidic cluster and the negatively charged membrane. This conformation can be associated to the F₀ state of our model and to a kinase-dominant state (K⁺). When the concentration decreases, the metal ion bridges are disrupted, leading to repulsion between the membrane and SD active site. This triggers a conformational change of the TM domain (F₁ state associated to a phosphatase-dominant conformation, P⁺), and results in a rotation of TM2 at the cytoplasmic interface, that is then transmitted to the linked HAMP domain.</p

Free energy landscape for the TM conformational change.

<p>The free energy landscape defined by sampling inter-helical distances between TM1 C-termini and TM2 N-termini is reported. The conformational change observed in the unbiased MD simulations (orange points) occurs along a free energy valley, that connects a main equilibrium state (<i>F₀</i>) and a high-energy conformation, and can be associated with relevant states during the signaling process (<i>F₁</i>, ∼5 kcal/mol higher in free energy).</p

Effects of Asn202 mutation on the solvation of the TM domain.

<p>The kernel density estimation of water molecules for MD simulations of the wild-type TM bundle, and three relevant Asn202 mutants: N202A, N202H, and H202R. Residue 202 is localized in the middle of the membrane (at 0 Å). Conservative mutations preserve the hydration of the TM core, while substitution with alanine prevents water to enter the bundle. Distribution is calculated along the axis orthogonal to the membrane bilayer, and the transmembrane portion is schematically indicated by the grey area defined by the MD-averaged distance between bilayer polar heads (namely, phosphorus atoms).</p

Solvation-dependent dynamic features of the TM domain.

<p>(A) The rotation of the Cα atoms around the TM2 helix main axis is computed based on a principal component analysis, and the angle distribution is characterized by three major modes, that can be fitted using three Gaussians, (B) MD time series of the TM2 residue state corresponding to the angle distribution. The system is initially in a metastable state (black), before switching to a solvated state where the TM1-TM1 interface is tighter (grey). After ∼30 ns, the system, passing through to a metastable state, shifts to a stable state characterized by a larger TM1-TM1 distance (light grey). (C) The rotation per residue related to the switch between the 2 most relevant states in MD (B) is calculated. TM2 Pro208 acts like a hinge, and transforms the large movement of the N-terminus into a mild rotation (∼20 degrees) of the TM2 residue at the cytoplasmic interface.</p

TM structural validation using disulfide cross-linking scanning.

<p>MD-averaged contact maps for (A) TM1 and (B) TM2 interfaces within the assembled TM domain. A direct comparison with cross-linking efficiency of (A) TM1 and (B) TM2 is reported in the inset, and shows a strong correlation between the cross-linking (1-efficiency) (in black) and the MD-averaged Cα distance measured for the TM model structure (in red). The cross-linking efficiency for the whole TM1 and TM2 regions is reported in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002878#pcbi.1002878.s001" target="_blank">Figure S1</a>.</p

Structural Polymorphism of Alzheimer’s β‑Amyloid Fibrils as Controlled by an E22 Switch: A Solid-State NMR Study

The amyloid-β (Aβ) peptide of Alzheimer’s disease (AD) forms polymorphic fibrils on the micrometer and molecular scales. Various fibril growth conditions have been identified to cause polymorphism, but the intrinsic amino acid sequence basis for this polymorphism has been unclear. Several single-site mutations in the center of the Aβ sequence cause different disease phenotypes and fibrillization properties. The E22G (Arctic) mutant is found in familial AD and forms protofibrils more rapidly than wild-type Aβ. Here, we use solid-state NMR spectroscopy to investigate the structure, dynamics, hydration and morphology of Arctic E22G Aβ40 fibrils. ¹³C, ¹⁵N-labeled synthetic E22G Aβ40 peptides are studied and compared with wild-type and Osaka E22Δ Aβ40 fibrils. Under the same fibrillization conditions, Arctic Aβ40 exhibits a high degree of polymorphism, showing at least four sets of NMR chemical shifts for various residues, while the Osaka and wild-type Aβ40 fibrils show a single or a predominant set of chemical shifts. Thus, structural polymorphism is intrinsic to the Arctic E22G Aβ40 sequence. Chemical shifts and inter-residue contacts obtained from 2D correlation spectra indicate that one of the major Arctic conformers has surprisingly high structural similarity with wild-type Aβ42. ¹³C–¹H dipolar order parameters, ¹H rotating-frame spin–lattice relaxation times and water-to-protein spin diffusion experiments reveal substantial differences in the dynamics and hydration of Arctic, Osaka and wild-type Aβ40 fibrils. Together, these results strongly suggest that electrostatic interactions in the center of the Aβ peptide sequence play a crucial role in the three-dimensional fold of the fibrils, and by inference, fibril-induced neuronal toxicity and AD pathogenesis