Juxta‐terminal Helix Unwinding as a Stabilizing Factor to Modulate the Dynamics of Transmembrane Helices

Transmembrane helices of integral membrane proteins often are flanked by interfacial aromatic residues that can serve as anchors to aid the stabilization of a tilted transmembrane orientation. Yet, physical factors that govern the orientation or dynamic averaging of individual transmembrane helices are not well understood and have not been adequately explained. Using solid‐state ²H NMR spectroscopy to examine lipid bilayer‐incorporated model peptides of the GWALP23 (acetyl‐GGALW(LA)₆LWLAGA‐amide) family, we observed substantial unwinding at the terminals of several tilted helices spanning the membranes of DLPC, DMPC, or DOPC lipid bilayers. The fraying of helix ends might be vital for defining the dynamics and orientations of transmembrane helices in lipid bilayer membranes.     

The Molecular Bases of the Interaction between a Saponin from the Roots of Gypsophila paniculata L. and Model Lipid Membranes

In view of the possible medical applications of saponins, the molecular structure of a GOTCAB saponin from the roots of Gypsophila paniculata L. was determined by NMR. The biological activity of saponins may depend on the interaction with cell membranes. To obtain more insight in the mechanism of membrane-related saponin function, an experimental and theoretical study was conducted. Ternary lipid systems composed of sphingomyelin, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and cholesterol were used as models of mammalian cell membranes. The membrane–saponin interaction was studied experimentally by monitoring surface pressure in the monomolecular films formed at the air–aqueous subphase interface. The behavior of GOTCAB saponin in a water box and model monolayer systems was characterized by molecular dynamics simulations. The results obtained showed that, in the systems used, cholesterol had a decisive effect on the interaction between GOTCAB and phosphocholine or sphingomyelin as well as on its location within the lipid film.     

The role of extra-membranous inter-helical loops in helix-helix interactions

The effect of a short loop connecting two transmembrane alpha-helices was studied using molecular dynamics simulations. Helices F and G from bacteriorhodopsin and two corresponding polyalanine helices were embedded in octane and POPC membranes in a transmembrane configuration both with and without the inter-helical loop. The results indicate that the membrane environment and the sequence of the loop are more influential on the dynamics and structure of the motif than the presence of a loop as such, at least for the time-scales investigated. The four residues in the FG loop are stabilized by four hydrogen bonds. These hydrogen bonds are not present in the polyalanine loop, causing it to be more flexible than the FG loop. This effect was observed independently of the protein environment, stressing the importance of the sequence. The structural analysis indicates that the loop has weak stabilizing properties in all environments. The stabilization due to the presence of the loop was strongest in a simulation of the FG fragment in a membrane-mimetic octane slab. In the simulations of the helix-loop-helix motif embedded in an explicit lipid bilayer model, the lipid bilayer interface compensates to a large extent for the absence of the loop.     

Structural characterization by computer experiments of the lipid-free LDL-receptor-binding domain of apolipoprotein E

The structure and dynamics of the lipid-free LDL-receptor-bindingdomain of apolipoprotein E (apoE-RBD) has been investigatedby Molecular Dynamics Simulations. ApoE-RBD in its monomericlipid-free form is a singular four-helix bundle made up of fourelongated amphipathic helices. Analysis of one 1.5 ns moleculardynamics trajectory of apoE-RBD performed in water indicatesthat the lipid-free domain adopts a structure that exhibitscharacteristics found in native proteins: it has very stablehelices and presents a compact structure. Yet its interior exhibitsa larger number of transient atomic-size cavities relative tothat found in other proteins of similar size and its apolarside chains are more mobile. The latter features distinguishthe elongated four-helix bundle as a slightly disordered structure,which shows a structural likeness with some de novo designedfour-helix bundle proteins and shares with the latter a leucine-richresidue composition. We anticipate that these unique propertiescompared with other native helix bundles may be related to thepostulated ability of apoE-RBD to undergo an opening of itsbundle upon interaction with phospholipids. The distributionof empty cavities computed along the trajectory in the interfaceregions between the different pairs of helices reveals thatthe tertiary contacts in one of the interfaces are weaker suggestingthat this particular interface could be more easily rupturedupon lipid association.     

Sequence divergence analysis for the prediction of seven-helix membrane protein structures: I. Comparison with bacteriorhodopsin

A method using protein sequence divergence to predict the three-dimensionalstructure of the transmembrane domain of seven-helix membraneproteins is described. The key component in the multistep procedureis the calculation of a hydrophilic and lipophilic variabilityindex for each amino acid in an alignment of a family of homologousproteins. The variability profile, a plot of the calculatedvariability index versus alignment position, can be used topredict a tertiary model of the backbone conformation of thetransmembrane domain. This method was applied to bacteriorhodopsin(BR) and the model obtained was compared with the known structureof this protein. Using an alignment of the amino acid sequencesof BR and closely related (20% identity) proteins, the boundariesof the transmembrane regions, their secondary structures andorientations inside the membrane bilayer were predicted basedon the variability profile. Additional information about theshape of the helix bundle was also obtained from the averagevariability of each transmembrane helix with the assumptionthat the helices are packed sequentially and form a closed helixbundle. Correct features of the known structure of BR were foundin the model structure, suggesting that a similar strategy canbe used to predict transmembrane helices and the packing shapeof other membrane proteins with seven transmembrane helices,such as the opsins and other G-protein coupled receptors.     

Stability of water/toluene interfaces saturated with adsorbed naphthenic acids—A molecular dynamics study

Stability of water/toluene interfaces saturated with adsorbed naphthenic acids with various sizes at room temperature was studied by molecular dynamics simulation. In direct comparison with our recent results on similar systems involving n-heptane as the oil phase, it has been found that toluene significantly enhances the stability of the water/oil interface, as suggested by the considerably reduced diffusion coefficients of the interface-bound naphthenic acids. The oil phase dependence may be rationalized in terms of the oil-naphthenic acid interactions, both electronically and sterically, and the intermolecular interactions between the molecules in the oil phase. Consistent with our previous studies, mobility of naphthenic acid has been found to be dependent on its size, with naphthenic acid featuring a longer carbon chain in between the carboxylic and cycloaliphatic groups showing lower mobility, leading to a more stable interface. However, such size dependence is much more pronounced for the water/toluene than the water/n-heptane interface. This may be understood in terms of the structural influence of toluene on the compactness of the liquid crystalline mesophase formed by naphthenic acids adsorbed at the interface.     

Missing Pieces in Structure Puzzles: How Hyperpolarized NMR Spectroscopy Can Complement Structural Biology and Biochemistry

Structure determination lies at the heart of many biochemical research programs. However, the “giants”: X-ray diffraction, electron microscopy, molecular dynamics simulations, and nuclear magnetic resonance, among others, leave quite a few dark spots on the structural pictures drawn of proteins, nucleic acids, membranes, and other biomacromolecules. For example, structural models under physiological conditions or of short-lived intermediates often remain out of reach of the established experimental methods. This account frames the possibility of including hyperpolarized, that is, dramatically signal-enhanced NMR in existing workflows to fill these spots with detailed depictions. We highlight how integrating methods based on dissolution dynamic nuclear polarization can provide valuable complementary information about formerly inaccessible conformational spaces for many systems. A particular focus will be on hyperpolarized buffers to facilitate the NMR structure determination of challenging systems.     

Archaeal Lipids Regulating the Trimeric Structure Dynamics of Bacteriorhodopsin for Efficient Proton Release and Uptake

S-TGA-1 and PGP-Me are native archaeal lipids associated with the bacteriorhodopsin (bR) trimer and contribute to protein stabilization and native dynamics for proton transfer. However, little is known about the underlying molecular mechanism of how these lipids regulate bR trimerization and efficient photocycling. Here, we explored the specific binding of S-TGA-1 and PGP-Me with the bR trimer and elucidated how specific interactions modulate the bR trimeric structure and proton release and uptake using long-term atomistic molecular dynamic simulations. Our results showed that S-TGA-1 and PGP-Me are essential for stabilizing the bR trimer and maintaining the coherent conformational dynamics necessary for proton transfer. The specific binding of S-TGA-1 with W80 and K129 regulates proton release on the extracellular surface by forming a “Glu-shared” model. The interaction of PGP-Me with K40 ensures proton uptake by accommodating the conformation of the helices to recruit enough water molecules on the cytoplasmic side. The present study results could fill in the theoretical gaps of studies on the functional role of archaeal lipids and could provide a reference for other membrane proteins containing similar archaeal lipids.     

Hinge-bending motions in annexins: molecular dynamics and essential dynamics of apo-annexin V and of calcium bound annexin V and I

Annexins are homologous proteins that bind to membranes in a calcium dependent manner, but for which precise physiological roles have yet to be defined. Most annexins are composed of a planar array of four homologous repeats, each containing five alpha-helices and associated into two modules. Annexin V forms a voltage-gated calcium channel in phospholipid bilayers. It has been proposed that the hydrophilic pore in the centre of the molecule may represent the ion conduction pathway and that a hinge movement in annexin V causes a variation of the inter- module angle and opens the calcium ion path. Here we present the results of molecular dynamics simulations of apo-annexin V and of calcium-bound annexin V and annexin I. The three simulations show significant differences in conformation and dynamics. The essential dynamics method was used to study the essential subspace of annexin V and showed that one of the essential motions corresponds to the postulated hinge motion. The hinge residues were located between repeats but belong to helices rather than to the links between helices. Calcium binding to annexin V led to a limitation of this hinge motion with more open conformations being favoured.      

Effects of Cardiolipin on the Conformational Dynamics of Membrane-Anchored Bcl-xL

The anti-apoptotic protein Bcl-xL regulates apoptosis by preventing the permeation of the mitochondrial outer membrane by pro-apoptotic pore-forming proteins, which release apoptotic factors into the cytosol that ultimately lead to cell death. Two different membrane-integrated Bcl-xL constructs have been identified: a membrane-anchored and a membrane-inserted conformation. Here, we use molecular dynamics simulations to study the effect of the mitochondrial specific lipid cardiolipin and the protein protonation state on the conformational dynamics of membrane-anchored Bcl-xL. The analysis reveals that the protonation state of the protein and cardiolipin content of the membrane modulate the orientation of the soluble head region (helices α1 through α7) and hence the exposure of its BH3-binding groove, which is required for its interaction with pro-apoptotic proteins.     

Insight into Conformational Change for 14-3-3σ Protein by Molecular Dynamics Simulation

14-3-3σ is a member of a highly conserved family of 14-3-3 proteins that has a double-edged sword role in human cancers. Former reports have indicated that the 14-3-3 protein may be in an open or closed state. In this work, we found that the apo-14-3-3σ is in an open state compared with the phosphopeptide bound 14-3-3σ complex which is in a more closed state based on our 80 ns molecular dynamics (MD) simulations. The interaction between the two monomers of 14-3-3σ in the open state is the same as that in the closed state. In both open and closed states, helices A to D, which are involved in dimerization, are stable. However, large differences are found in helices E and F. The hydrophobic contacts and hydrogen bonds between helices E and G in apo-14-3-3σ are different from those in the bound 14-3-3σ complex. The restrained and the mutated (Arg56 or Arg129 to alanine) MD simulations indicate that the conformation of four residues (Lys49, Arg56, Arg129 and Tyr130) may play an important role to keep the 14-3-3σ protein in an open or closed state. These results would be useful to evaluate the 14-3-3σ protein structure-function relationship.     

Structural and Functional Insights into the Transmembrane Domain Association of Eph Receptors

Eph receptors are the largest family of receptor tyrosine kinases and by interactions with ephrin ligands mediate a myriad of processes from embryonic development to adult tissue homeostasis. The interaction of Eph receptors, especially at their transmembrane (TM) domains is key to understanding their mechanism of signal transduction across cellular membranes. We review the structural and functional aspects of EphA1/A2 association and the techniques used to investigate their TM domains: NMR, molecular modelling/dynamics simulations and fluorescence. We also introduce transmembrane peptides, which can be used to alter Eph receptor signaling and we provide a perspective for future studies.     

Structural and Dynamical Insight into PPARγ Antagonism: In Silico Study of the Ligand-Receptor Interactions of Non-Covalent Antagonists

The structural and dynamical properties of the peroxisome proliferator-activated receptor γ (PPARγ) nuclear receptor have been broadly studied in its agonist state but little is known about the key features required for the receptor antagonistic activity. Here we report a series of molecular dynamics (MD) simulations in combination with free energy estimation of the recently discovered class of non-covalent PPARγ antagonists. Their binding modes and dynamical behavior are described in details. Two key interactions have been detected within the cavity between helices H3, H11 and the activation helix H12, as well as with H12. The strength of the ligand-amino acid residues interactions has been analyzed in relation to the specificity of the ligand dynamical and antagonistic features. According to our results, the PPARγ activation helix does not undergo dramatic conformational changes, as seen in other nuclear receptors, but rather perturbations that occur through a significant ligand-induced reshaping of the ligand-receptor and the receptor-coactivator binding pockets. The H12 residue Tyr473 and the charge clamp residue Glu471 play a central role for the receptor transformations. Our results also demonstrate that MD can be a helpful tool for the compound phenotype characterization (full agonists, partial agonists or antagonists) when insufficient experimental data are available.     

Alpha-Bulges in G Protein-Coupled Receptors

Agonist binding is related to a series of motions in G protein-coupled receptors (GPCRs) that result in the separation of transmembrane helices III and VI at their cytosolic ends and subsequent G protein binding. A large number of smaller motions also seem to be associated with activation. Most helices in GPCRs are highly irregular and often contain kinks, with extensive literature already available about the role of prolines in kink formation and the precise function of these kinks. GPCR transmembrane helices also contain many α-bulges. In this article we aim to draw attention to the role of these α-bulges in ligand and G-protein binding, as well as their role in several aspects of the mobility associated with GPCR activation. This mobility includes regularization and translation of helix III in the extracellular direction, a rotation of the entire helix VI, an inward movement of the helices near the extracellular side, and a concerted motion of the cytosolic ends of the helices that makes their orientation appear more circular and that opens up space for the G protein to bind. In several cases, α-bulges either appear or disappear as part of the activation process.     

Deciphering the Binding Interactions between Acinetobacter baumannii ACP and β-ketoacyl ACP Synthase III to Improve Antibiotic Targeting Using NMR Spectroscopy

Fatty acid synthesis is essential for bacterial viability. Thus, fatty acid synthases (FASs) represent effective targets for antibiotics. Nevertheless, multidrug-resistant bacteria, including the human opportunistic bacteria, Acinetobacter baumannii, are emerging threats. Meanwhile, the FAS pathway of A. baumannii is relatively unexplored. Considering that acyl carrier protein (ACP) has an important role in the delivery of fatty acyl intermediates to other FAS enzymes, we elucidated the solution structure of A. baumannii ACP (AbACP) and, using NMR spectroscopy, investigated its interactions with β-ketoacyl ACP synthase III (AbKAS III), which initiates fatty acid elongation. The results show that AbACP comprises four helices, while Ca²⁺ reduces the electrostatic repulsion between acid residues, and the unconserved F47 plays a key role in thermal stability. Moreover, AbACP exhibits flexibility near the hydrophobic cavity entrance from D59 to T65, as well as in the α₁α₂ loop region. Further, F29 and A69 participate in slow exchanges, which may be related to shuttling of the growing acyl chain. Additionally, electrostatic interactions occur between the α₂ and α₃-helix of ACP and AbKAS III, while the hydrophobic interactions through the ACP α₂-helix are seemingly important. Our study provides insights for development of potent antibiotics capable of inhibiting A. baumannii FAS protein–protein interactions.     

The activation of fibroblast growth factors by heparin: synthesis, structure, and biological activity of heparin-like oligosaccharides

An effective strategy has been designed for the synthesis of oligosaccharides of different sizes structurally related to the regular region of heparin; this is illustrated by the preparation of hexasaccharide 1 and octasaccharide 2. This synthetic strategy provides the oligosaccharide sequence containing a D-glucosamine unit at the nonreducing end that is not available either by enzymatic or chemical degradation of heparin. It may permit, after slight modifications, the preparation of oligosaccharide fragments with different charge distribution as well. NMR spectroscopy and molecular dynamics simulations have shown that the overall structure of 1 in solution is a stable right-hand helix with four residues per turn. Hexasaccharide 1 and, most likely, octasaccharide 2 are, therefore, chemically well-defined structural models of naturally occurring heparin-like oligosaccharides for use in binding and biological activity studies. Both compounds 1 and 2 induce the mitogenic activity of acid fibroblast growth factor (FGF1), with the half-maximum activating concentration of 2 being equivalent to that of heparin. Sedimentation equilibrium analysis with compound 2 suggests that heparin-induced FGF1 dimerization is not an absolute requirement for biological activity.     

The Folding of de Novo Designed Protein DS119 via Molecular Dynamics Simulations

As they are not subjected to natural selection process, de novo designed proteins usually fold in a manner different from natural proteins. Recently, a de novo designed mini-protein DS119, with a βαβ motif and 36 amino acids, has folded unusually slowly in experiments, and transient dimers have been detected in the folding process. Here, by means of all-atom replica exchange molecular dynamics (REMD) simulations, several comparably stable intermediate states were observed on the folding free-energy landscape of DS119. Conventional molecular dynamics (CMD) simulations showed that when two unfolded DS119 proteins bound together, most binding sites of dimeric aggregates were located at the N-terminal segment, especially residues 5–10, which were supposed to form β-sheet with its own C-terminal segment. Furthermore, a large percentage of individual proteins in the dimeric aggregates adopted conformations similar to those in the intermediate states observed in REMD simulations. These results indicate that, during the folding process, DS119 can easily become trapped in intermediate states. Then, with diffusion, a transient dimer would be formed and stabilized with the binding interface located at N-terminals. This means that it could not quickly fold to the native structure. The complicated folding manner of DS119 implies the important influence of natural selection on protein-folding kinetics, and more improvement should be achieved in rational protein design.     

静电作用力算法对分子动力学模拟结果的影响分析

蛋白质分子和界面之间的作用在药物输送以及生物分离等领域至关重要。利用分子动力学模拟考察蛋白质分子在界面附近的行为是最近10年研究的热点。在早期的工作中,Wang等发现同电荷离子交换介质可用于辅助蛋白质复性,但其机理不甚明确。在利用分子动力学模拟研究其分子机理时发现,不同静电作用力参数对模拟结果有直接的影响。因此,通过全原子分子动力学模拟考察不同静电参数条件对模拟结果的影响,展示此过程的构象和能量变化,分析了造成结果差异的原因。研究结果揭示了不同静电参数对模拟结果的影响,为进一步研究蛋白质在界面表面的行为奠定了一定的理论基础。     

Transmembrane helix prediction: a comparative evaluation and analysis

The prediction of transmembrane (TM) helices plays an important role in the study of membrane proteins, given the relatively small number (approximately 0.5% of the PDB) of high-resolution structures for such proteins. We used two datasets (one redundant and one non-redundant) of high-resolution structures of membrane proteins to evaluate and analyse TM helix prediction. The redundant (non-redundant) dataset contains structure of 434 (268) TM helices, from 112 (73) polypeptide chains. Of the 434 helices in the dataset, 20 may be classified as 'half-TM' as they are too short to span a lipid bilayer. We compared 13 TM helix prediction methods, evaluating each method using per segment, per residue and termini scores. Four methods consistently performed well: SPLIT4, TMHMM2, HMMTOP2 and TMAP. However, even the best methods were in error by, on average, about two turns of helix at the TM helix termini. The best and worst case predictions for individual proteins were analysed. In particular, the performance of the various methods and of a consensus prediction method, were compared for a number of proteins (e.g. SecY, ClC, KvAP) containing half-TM helices. The difficulties of predicting half-TM helices suggests that current prediction methods successfully embody the two-state model of membrane protein folding, but do not accommodate a third stage in which, e.g., short helices and re-entrant loops fold within a bundle of stable TM helices.     

Crystallization of glycine in water/saturated fatty acid emulsions

It was found that carbon chain length of fatty acids strongly affects polymorphic selection in the cooling crystallization of glycine from water/saturated fatty acid emulsions. Two-dimensional packing density of saturated fatty acid head groups, which is inversely proportional to the number of carbon atoms, was shown to be responsible for polymorphic selection of glycine: γ-glycine was obtained from the emulsions of hexanoic acid and octanoic acid, whereas α-glycine was found to crystallize from the emulsions of dodecanoic acid, tetradecanoic acid, hexadecanoic acid and octadecanoic acid. Those results indicate that molecular structure of γ-glycine is only well matched with molecular structure of head groups of hexanoic acid and octanoic acid at the interface of the emulsion, and thus such molecular interface provides the preferential site for the organization of γ-form crystal structure from the liquid-like cluster of glycine.