Lipid exchange in crystal-confined fatty acid binding proteins: X-ray evidence and molecular dynamics explanation.

Fatty acid binding proteins (FABPs) are responsible for the long-chain fatty acids (FAs) transport inside the cell. However, despite the years, since their structure is known and the many studies published, there is no definitive answer about the stages of the lipid entry-exit mechanism. Their structure forms a ß -barrel of 10 anti-parallel strands with a cap in a helix-turn-helix motif, and there is some consensus on the role of the so-called portal region, involving the second α -helix from the cap ( α 2), ß C- ß D, and ß E- ß F turns in FAs exchange. To test the idea of a lid that opens, we performed a soaking experiment on an h-FABP crystal in which the cap is part of the packing contacts, and its movement is strongly restricted. Even in these conditions, we observed the replacement of palmitic acid by 2-Bromohexadecanoic acid (Br-palmitic acid). Our MD simulations reveal a two-step lipid entry process: (i) The travel of the lipid head through the cavity in the order of tens of nanoseconds, and (ii) The accommodation of its hydrophobic tail in hundreds to thousands of nanoseconds. We observed this even in the cases in which the FAs enter the cavity by their tail. During this process, the FAs do not follow a single trajectory, but multiple ones through which they get into the protein cavity. Thanks to the complementary views between experiment and simulation, we can give an approach to a mechanistic view of the exchange process.

Arginine-based surfactants alter the rheological and in-plane structural properties of stratum corneum model membranes.

HYPOTHESIS: Amino acid-based surfactants have been proposed as skin permeation enhancers. In this work, we investigated the potentiality of two arginine-based amphiphiles as permeation enhancers by studying their interaction with stratum corneum (SC) model lipid membranes. EXPERIMENTS: Nα-benzoyl arginine decyl- and dodecylamide were tested in comparison with the classical enhancer, oleic acid, and the non-enhancer, stearic acid. Two complementary approaches were used: lipid monolayers, taken as models of the unit film layer of SC, and atomistic molecular dynamics simulations. FINDINGS: The arginine-based amphiphiles studied were able to be incorporated into the SCM membrane and alter its rheological and structural properties by disordering the lipid chains, enhancing membrane elasticity, and thinning the overall membrane. They also affected the lateral structure of heterogeneous SC membranes at the nanoscale by relaxing and rounding the domain borders. Our work shows that the alteration observed of the overall rheological and structural properties of the SC membranes appears to be a shared ability for several amphiphilic permeation enhancers. Our results encourage future exploration of those amphiphiles as skin permeation enhancers.

Molecular dynamics simulation of the heart type fatty acid binding protein in a crystal environment.

Crystallographic data comes from a space-time average over all the unit cells within the crystal, so dynamic phenomena do not contribute significantly to the diffraction data. Many efforts have been made to reconstitute the movement of the macromolecules and explore the microstates that the confined proteins can adopt in the crystalline network. We explored different strategies to simulate a heart fatty acid binding protein (H-FABP) crystal by means of Molecular Dynamics (MD) simulations. We evaluate the effect of introducing restraints according to experimental isotropic B-factors and we analyzed the H-FABP motions in the crystal using Principal Component Analysis (PCA), isotropic and anisotropic B-factors. We compared the behavior of the protein simulated in the crystal confinement versus in solution, and we observed the effect of that confinement in the mobility of the protein residues. Restraining one-third of Cα atoms based on experimental B-factors produce lower B-factors than simulations without restraints, showing that the position restraint of the atoms with the lowest experimental B-factor is a good strategy to maintain the geometry of the crystal with an obvious decrease in the degrees of motion of the protein. PCA shows that, as position restraint reduces the conformational space explored by the system, the motion of the crystal is better recovered, for an essential subspace of the same size, in the simulations without restraints. Restraining only one Cα seems to be a good balance between giving flexibility to the system and preserving its structure. Communicated by Ramaswamy H. Sarma.

Structural Basis of Outstanding Multivalent Effects in Jack Bean α-Mannosidase Inhibition.

Multivalent design of glycosidase inhibitors is a promising strategy for the treatment of diseases involving enzymatic hydrolysis of glycosidic bonds in carbohydrates. An essential prerequisite for successful applications is the atomic-level understanding of how outstanding binding enhancement occurs with multivalent inhibitors. Herein we report the first high-resolution crystal structures of the Jack bean α-mannosidase (JBα-man) in apo and inhibited states. The three-dimensional structure of JBα-man in complex with the multimeric cyclopeptoid-based inhibitor displaying the largest binding enhancements reported so far provides decisive insight into the molecular mechanisms underlying multivalent effects in glycosidase inhibition.

Dynamic Regulation of Cell Volume and Extracellular ATP of Human Erythrocytes.

INTRODUCTION: The peptide mastoparan 7 (MST7) triggered in human erythrocytes (rbcs) the release of ATP and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe), interacting with P (purinergic) receptors, can affect cell volume (Vr), we explored the dynamic regulation between Vr and ATPe. METHODS AND TREATMENTS: We made a quantitative assessment of MST7-dependent kinetics of Vr and of [ATPe], both in the absence and presence of blockers of ATP efflux, swelling and P receptors. RESULTS: In rbcs 10 µM MST7 promoted acute, strongly correlated changes in [ATPe] and Vr. Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%. Pre-incubation of rbcs with 10 µM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40-50% and swelling by 40-60%, while in the presence of 80 U/mL apyrase, an ATPe scavenger, cell swelling was prevented. While exposure to 10 µM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced [ATPe] by 48%, and swelling by 80%, incubation of cells in sodium free medium reduced swelling by 92%. ANALYSIS AND DISCUSSION: Results were analyzed by means of a mathematical model where ATPe kinetics and Vr kinetics were mutually regulated. Model dependent fit to experimental data showed that, upon MST7 exposure, ATP efflux required a fast 1960-fold increase of ATP permeability, mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. Both experimental and theoretical results suggest that, following MST7 exposure, ATP is released via two conduits, one of which is mediated by pannexin 1. The accumulated ATPe activates P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activates ATP release. Thus swelling and P2X receptors constitute essential components of a positive feedback loop underlying ATP-induced ATP release of rbcs.

Molecular dynamics of water in the neighborhood of aquaporins.

Present knowledge obtained by molecular dynamics (MD) simulation studies regarding the dynamics of water, both in the vicinity of biological membranes and within the proteinaceous water channels, also known as aquaporins (AQPs), is reviewed. A brief general summary of the water models most extensively employed in MD simulations (SPC, SPC/E, TIP3P, TIP4P), indicating their most relevant pros and cons, is likewise provided. Structural considerations of water are also discussed, based on different order parameters, which can be extracted from MD simulations as well as from experiments. Secondly, the behaviour of water in the neighbourhood of membranes by means of molecular dynamics simulations is addressed. Consequently, the comparison with previous experimental evidence is pointed out. In living cells, water is transported across the plasma membrane through the lipid bilayer and the aforementioned AQPs, which motivates this review to focus mostly on MD simulation studies of water within AQPs. Relevant contributions explaining peculiar properties of these channels are discussed, such as selectivity and gating. Water models used in these studies are also summarised. Finally, based on the information presented here, further MD studies are encouraged.

A molecular dynamics approach to ligand-receptor interaction in the aspirin-human serum albumin complex.

In this work, we present a study of the interaction between human serum albumin (HSA) and acetylsalicylic acid (ASA, C(9)H(8)O(4)) by molecular dynamics simulations (MD). Starting from an experimentally resolved structure of the complex, we performed the extraction of the ligand by means of the application of an external force. After stabilization of the system, we quantified the force used to remove the ASA from its specific site of binding to HSA and calculated the mechanical nonequilibrium external work done during this process. We obtain a reasonable value for the upper boundary of the Gibbs free energy difference (an equilibrium thermodynamic potential) between the complexed and noncomplexed states. To achieve this goal, we used the finite sampling estimator of the average work, calculated from the Jarzynski Equality. To evaluate the effect of the solvent, we calculated the so-called "viscous work," that is, the work done to move the aspirin in the same trajectory through the solvent in absence of the protein, so as to assess the relevance of its contribution to the total work. The results are in good agreement with the available experimental data for the albumin affinity constant for aspirin, obtained through quenching fluorescence methods.