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.

Molecular basis of the anchoring and stabilization of human islet amyloid polypeptide in lipid hydroperoxidized bilayers.

The molecular structure of membrane lipids is formed by mono- or polyunsaturations on their aliphatic tails that make them susceptible to oxidation, facilitating the incorporation of hydroperoxide (R-OOH) functional groups. Such groups promote changes in both composition and complexity of the membrane significantly modifying its physicochemical properties. Human Langerhans islets amyloid polypeptide (hIAPP) is the main component of amyloid deposits found in the pancreas of patients with type-2 diabetes (T2D). hIAPP in the presence of membranes with oxidized lipid species accelerates the formation of amyloid fibrils or the formation of intermediate oligomeric structures. However, the molecular bases at the initial stage of the anchoring and stabilization of the hIAPP in a hydroperoxidized membrane are not yet well understood. To shed some light on this matter, in this contribution, three bilayer models were modeled: neutral (POPC), anionic (POPS), and oxidized (POPCOOH), and full atom Molecular Dynamics (MD) simulations were performed. Our results show that the POPCOOH bilayer increases the helicity in hIAPP when compared to POPC or POPS bilayer. The modification in the secondary structure covers the residues of the so-called amyloidogenic core of the hIAPP. Overall, the hydroperoxidation of the neutral lipids modifies both the anchoring and the stabilization of the peptide hIAPP by reducing the random conformations of the peptide and increasing of hydrogen bond population with the hydroperoxidized lipids.

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 Characterization of TssL from Acinetobacter baumannii: a Key Component of the Type VI Secretion System.

The type VI secretion system (T6SS) is a complex molecular nanomachine used by Gram-negative bacteria to deliver diverse effectors into adjacent cells. A membrane complex (MC) anchors this transport system to the bacterial cell wall. One of the proteins forming the MC is TssL, a cytoplasmic protein bound to the inner membrane through a single transmembrane helix. Here, we report the structure of the cytoplasmic N-terminal region of TssL from Acinetobacter baumannii, a bacterium encoding in a single locus a secretion system that is a special case among other T6SSs. The protein structure, consisting of two antiparallel alpha-helical bundles connected by a short loop, reveals several interesting particularities compared with homologous proteins from other organisms. In addition, we demonstrate the structural significance of residues Asp98 and Glu99, which are strongly conserved among T6SS-encoding Gram-negative bacteria. Mutations in these two residues strongly impact protein dynamics, expression, and functionality. Our results improve our understanding of the T6SS of A. baumannii, which remains largely understudied compared with that of other pathogens.IMPORTANCE Several Acinetobacter species carry one functional type VI secretion system (T6SS). The T6SS is encoded in a single locus containing 16 conserved genes, most of which code for proteins essential to T6SS activity. One of these key components is TssL, a cytoplasmic protein bound to the inner membrane. Despite its importance and its particular characteristics, the structure of T6SS in A. baumannii remains understudied. Here, we present structural, in silico, and in vivo studies of TssL, highlighting the importance of two well-conserved residues and improving our understanding of this secretion system in this bacterium.

The role of hydrophobicity in the cold denaturation of proteins under high pressure: A study on apomyoglobin.

An exciting debate arises when microscopic mechanisms involved in the denaturation of proteins at high pressures are explained. In particular, the issue emerges when the hydrophobic effect is invoked, given that hydrophobicity cannot elucidate by itself the volume changes measured during protein unfolding. In this work, we study by the use of molecular dynamics simulations and essential dynamics analysis the relation between the solvation dynamics, volume, and water structure when apomyoglobin is subjected to a hydrostatic pressure regime. Accordingly, the mechanism of cold denaturation of proteins under high-pressure can be related to the disruption of the hydrogen-bond network of water favoring the coexistence of two states, low-density and high-density water, which directly implies in the formation of a molten globule once the threshold of 200 MPa has been overcome.

Mechanisms associated with the effects of urea on the micellar structure of sodium dodecyl sulphate in aqueous solutions.

We used simulations by Molecular Dynamics to characterize the mechanism whereby the variations in the urea concentration modifies the micellar structure of sodium dodecyl sulfate monomers in water. From a self-assembled micellar system, we observed that increasing urea concentration leads to a decrease in aggregation number. Likewise, when increasing urea concentration, the micelles increase their nonpolar surface exposed to solvent, while the polar surface exposed to solvent decreases. This rearrangement process of SDS micelles in presence of urea is mainly due to the fact that the ions of Na+ that stabilize the micellar structure increase its interaction with urea. In this process, the SDS hydrophilic head and Na+ ions increases its solvation by urea, destabilizing micellar structure and exponing the hydrophobic core to the solvent.

Pressure effect on micellization of non-ionic surfactant Triton X-100.

Micellar aggregates can be arranged in new types of conformational assemblies when they are isotropically compressed. Thus, the pressure effects in the underlying fundamental interactions leading to self-assembly of micellar aggregates can be represented by changes in the phase boundaries with increasing pressure. In this paper, we have employed molecular dynamics simulations to study the self-assembly of micelles composed of the non-ionic surfactant Triton X-100 at the atomic scale, monitoring the changes in the solvation dynamics when the micelles are subjected to a wide range of hydrostatic pressures. The computational molecular model was capable of self-assembling and forming a non-ionic micelle, which subsequently was coupled to a high-pressure barostat producing a geometric transition of the micelle due to changes in the solvation dynamics. Accordingly, under a high pressure regime, the hydrogen bonds are redistributed, the water density is modified, and water acts as an unstructured liquid, capable of penetrating into the micelle.

Essential dynamics of the cold denaturation: pressure and temperature effects in yeast frataxin.

The cold denaturation of globular proteins is a process that can be caused by increasing pressure or decreasing the temperature. Currently, the action mechanism of this process has not been clearly understood, raising an interesting debate on the matter. We have studied the process of cold denaturation using molecular dynamics simulations of the frataxin system Yfh1, which has a dynamic experimental characterization of unfolding at low and high temperatures. The frataxin model here studied allows a comparative analysis using experimental data. Furthermore, we monitored the cold denaturation process of frataxin and also investigated the effect under the high-pressure regime. For a better understanding of the dynamics and structural properties of the cold denaturation, we also analyzed the MD trajectories using essentials dynamic. The results indicate that changes in the structure of water by the effect of pressure and low temperatures destabilize the hydrophobic interaction modifying the solvation and the system volume leading to protein denaturation. Proteins 2016; 85:125-136. © 2016 Wiley Periodicals, Inc.