Theoretical Study of the Remarkably Diverse Linkages in Lignin

Lignin in plant cell walls is a potential renewable source of biofuels, chemicals, and value-added products. It consists of various aryl ethers, irregularly connected by a variety of linkages creating a complex structural network; hence, it is difficult to identify selective bond breaking events. In this study, we predict dissociation tendencies of a diverse set of lignin linkages encompassing 65 lignin model compounds using the density functional theoretical (DFT) approach. The chosen 65 lignin model compounds represent the most prevalent carbon–oxygen (ether) and carbon–carbon (C–C) bond linkages. Results from our systematic study identify the weakest and strongest linkages connecting arene rings in different classes of lignin model compounds. Also, the dissociating linkages can have different adjacent substituents, such as the methoxy group on the arene ring and hydrocarbon, methyl, and hydroxyl group substitutions on aliphatic carbon atoms. These substituents affect the ease of dissociation of lignin linkages and can be used to develop predictive models for delignification

A Mechanistic Understanding of Allosteric Immune Escape Pathways in the HIV-1 Envelope Glycoprotein

<div><p>The HIV-1 envelope (Env) spike, which consists of a compact, heterodimeric trimer of the glycoproteins gp120 and gp41, is the target of neutralizing antibodies. However, the high mutation rate of HIV-1 and plasticity of Env facilitates viral evasion from neutralizing antibodies through various mechanisms. Mutations that are distant from the antibody binding site can lead to escape, probably by changing the conformation or dynamics of Env; however, these changes are difficult to identify and define mechanistically. Here we describe a network analysis-based approach to identify potential allosteric immune evasion mechanisms using three known HIV-1 Env gp120 protein structures from two different clades, B and C. First, correlation and principal component analyses of molecular dynamics (MD) simulations identified a high degree of long-distance coupled motions that exist between functionally distant regions within the intrinsic dynamics of the gp120 core, supporting the presence of long-distance communication in the protein. Then, by integrating MD simulations with network theory, we identified the optimal and suboptimal communication pathways and modules within the gp120 core. The results unveil both strain-dependent and -independent characteristics of the communication pathways in gp120. We show that within the context of three structurally homologous gp120 cores, the optimal pathway for communication is sequence sensitive, i.e. a suboptimal pathway in one strain becomes the optimal pathway in another strain. Yet the identification of conserved elements within these communication pathways, termed inter-modular hotspots, could present a new opportunity for immunogen design, as this could be an additional mechanism that HIV-1 uses to shield vulnerable antibody targets in Env that induce neutralizing antibody breadth.</p> </div

Mycolactone acts as a linactant.

<p><b>A)</b> Spontaneous Lo (green lipids)-Ld (red lipids) domain formation in a ternary lipid system (diC16-PC, diC18:2-PC and cholesterol) at 4:3:3 lipid ratio. <b>B)</b> The addition of 5% mycolactone, however, decreases the line tension between the domains (see main text). <b>C)</b> Electron density profile along the interface of both domains. Clearly the higher peak corresponding to mycolactone (yellow line) is localized in the interface of both domains. Green and red lines highlight the electron-density profiles for the ordered and disordered lipids (diC16-PC and diC18:2-PC respectively). The white line corresponds to the electron-density of cholesterol within the Lo and Ld domains. <b>D)</b> Radial distribution function of mycolactone with the center of mass of ordered lipids-saturated tails (green line) and disordered lipids-unsaturated tails (red line) clearly show a slight preference for the disordered region, which is expressed by the free energy difference between the red and green (black dashed line). The radial distributions were also split by considering the head <b>(E)</b> and tail <b>(F)</b> regions of mycolactone, suggesting that both regions prefer the Ld region.</p

Membrane perturbing properties of toxin mycolactone from <i>Mycobacterium ulcerans</i>

<div><p>Mycolactone is the exotoxin produced by <i>Mycobacterium ulcerans</i> and is the virulence factor behind the neglected tropical disease Buruli ulcer. The toxin has a broad spectrum of biological effects within the host organism, stemming from its interaction with at least two molecular targets and the inhibition of protein uptake into the endoplasmic reticulum. Although it has been shown that the toxin can passively permeate into host cells, it is clearly lipophilic. Association with lipid carriers would have substantial implications for the toxin’s distribution within a host organism, delivery to cellular targets, diagnostic susceptibility, and mechanisms of pathogenicity. Yet the toxin’s interactions with, and distribution in, lipids are unknown. Herein we have used coarse-grained molecular dynamics simulations, guided by all-atom simulations, to study the interaction of mycolactone with pure and mixed lipid membranes. Using established techniques, we calculated the toxin’s preferential localization, membrane translocation, and impact on membrane physical and dynamical properties. The computed water-octanol partition coefficient indicates that mycolactone prefers to be in an organic phase rather than in an aqueous environment. Our results show that in a solvated membrane environment the exotoxin mainly localizes in the water-membrane interface, with a preference for the glycerol moiety of lipids, consistent with the reported studies that found it in lipid extracts of the cell. The calculated association constant to the model membrane is similar to the reported association constant for Wiskott-Aldrich syndrome protein. Mycolactone is shown to modify the physical properties of membranes, lowering the transition temperature, compressibility modulus, and critical line tension at which pores can be stabilized. It also shows a tendency to behave as a linactant, a molecule that localizes at the boundary between different fluid lipid domains in membranes and promotes inter-mixing of domains. This property has implications for the toxin’s cellular access, T-cell immunosuppression, and therapeutic potential.</p></div

Mycolactone modifies different physical properties of a lipid membrane.

<p><b>A)</b> gel-liquid transition temperature for a pure diC16-PC lipid bilayer (black line) and when combined with 5% mycolactone (red line). The transition is given as the change in area per lipid. As previously referenced[<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005972#pcbi.1005972.ref040" target="_blank">40</a>], in the pure diC16-PC membrane, our data shows that gel-liquid transition occurs at ~295 K and with a mean area per lipid of 0.6 nm². However, the addition of mycolactone drops this temperature by ~ 5 K. <b>B)</b> Pure diC16-PC lipid bilayer under 60 mN/m surface tension. The membrane thins, but no pore formation is observed. <b>C)</b> The addition of 5% mycolactone reduces its resistance to stretching, leading to the formation of a pore with the concomitant rupture of the bilayer at 55 mN/m surface tenstion. Once formed pores were observed to be stabilized at ~ 20 mN/m tension. Pore formation was observed in several independent simulations.</p

Effect of Glycosylation on an Immunodominant Region in the V1V2 Variable Domain of the HIV-1 Envelope gp120 Protein

<div><p>Heavy glycosylation of the envelope (Env) surface subunit, gp120, is a key adaptation of HIV-1; however, the precise effects of glycosylation on the folding, conformation and dynamics of this protein are poorly understood. Here we explore the patterns of HIV-1 Env gp120 glycosylation, and particularly the enrichment in glycosylation sites proximal to the disulfide linkages at the base of the surface-exposed variable domains. To dissect the influence of glycans on the conformation these regions, we focused on an antigenic peptide fragment from a disulfide bridge-bounded region spanning the V1 and V2 hyper-variable domains of HIV-1 gp120. We used replica exchange molecular dynamics (MD) simulations to investigate how glycosylation influences its conformation and stability. Simulations were performed with and without N-linked glycosylation at two sites that are highly conserved across HIV-1 isolates (N156 and N160); both are contacts for recognition by V1V2-targeted broadly neutralizing antibodies against HIV-1. Glycosylation stabilized the pre-existing conformations of this peptide construct, reduced its propensity to adopt other secondary structures, and provided resistance against thermal unfolding. Simulations performed in the context of the Env trimer also indicated that glycosylation reduces flexibility of the V1V2 region, and provided insight into glycan-glycan interactions in this region. These stabilizing effects were influenced by a combination of factors, including the presence of a disulfide bond between the Cysteines at 131 and 157, which increased the formation of beta-strands. Together, these results provide a mechanism for conservation of disulfide linkage proximal glycosylation adjacent to the variable domains of gp120 and begin to explain how this could be exploited to enhance the immunogenicity of those regions. These studies suggest that glycopeptide immunogens can be designed to stabilize the most relevant Env conformations to focus the immune response on key neutralizing epitopes.</p></div

Mycolactone in model lipid membrane.

<p><b>A)</b> All-atom and coarse-grained representations of diC16-PC (cyan and green) and mycolactone (cyan and yellow). The atomistic representation is characterized by an 8-undecenolide region (blue box), the C12-C20 northern fragment (red box), and the pentanoic acid ester southern fragment (yellow box). The CG representation can be described in terms of the head and tail regions. Different bead types (N1-N13) capture the general topology of the CG resolution, according to the definition of MARTINI force field (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005972#sec007" target="_blank">Methods</a>). <b>B)</b> The CG set-up of pure diC16-PC bilayer system in combination with 5% mycolactone. Notice that the simulation box is enclosed by the gray square. <b>C)</b> Cross section snapshot of the equilibrated membrane simulation. For clarity, diC16-PC lipid has been depicted by its head group (orange), the glycerol moiety (red) and the aliphatic tails (dark gray). <b>D)</b> Histograms correspond to probability distribution of two common configurations of mycolactone in lipid bilayer; either at the surface (black) or spanning the bilayer (red). The distributions are calculated as function of the distance between CG beads N1 and N13 for the bilayer system with 5% mycolactone as shown in the set-up of panel B. The dashed lines show the accumulation of population of these two configurations. Insets in both C and D (enclosed in circles) show representative configurations from all atom MD simulations.</p

Fraction of configurations as a function of number of hydrogen bonds between different components of the system.

<p>(A) Total hydrogen bonding within the peptide, between peptide and solvent, and between glycan and peptide. (B) Hydrogen bonding between the glycans and charged residues. Pep stands for peptide, Sol for solvent water, Gly for glycan, and Charged for the charged residues of the peptide.</p

Systems studied by MD simulations.

<p>Systems studied by MD simulations.</p

Effects of glycosylation on V1V2 peptide region in the context of the BG505 Env trimeric spike.

<p><b>(A)</b> Root mean square fluctuation of the backbone atoms corresponding to residues 129–134 and 152–184 (HBX2 numbering) and computed for either the glycosylated (black line) and non-glycosylated (red line) protein. Error bars were estimated from calculation in each of the independent protomers. <b>(B)</b> Cumulative configurational entropy for the backbone atoms corresponding to the same residues as in panel A. Values were estimated by considering the total entropy from the three promoters. <b>(C)</b> Total interaction energy from the representative sequence as in B. The energy corresponds to the total value calculated among the three protomers and during 1us trajectory simulation. <b>(D)</b> Secondary structural percentage as computed from 1us MD simulations of the full Env spike. Four stretches were considered for the analysis, each featuring disulfide bonds and glycosylation sites. Computed secondary structure percentage for amino acid stretches that contain glycans adjacent to Cysteins (HXB2 numbering): 131–157 (analogous to the V1V2 peptide), 385–418 and 296–331. It further demonstrates, in the context of Env trimer, that glycosylation decreases the amount of alpha-helix, beta strands, bridge and turns in these regions.</p