Molecular Dynamics Simulation of Ligand Dissociation from Liver Fatty Acid Binding Protein

The mechanisms of how ligands enter and leave the binding cavity of fatty acid binding proteins (FABPs) have been a puzzling question over decades. Liver fatty acid binding protein (LFABP) is a unique family member which accommodates two molecules of fatty acids in its cavity and exhibits the capability of interacting with a variety of ligands with different chemical structures and properties. Investigating the ligand dissociation processes of LFABP is thus a quite interesting topic, which however is rather difficult for both experimental approaches and ordinary simulation strategies. In the current study, random expulsion molecular dynamics simulation, which accelerates ligand motions for rapid dissociation, was used to explore the potential egress routes of ligands from LFABP. The results showed that the previously hypothesized “portal region” could be readily used for the dissociation of ligands at both the low affinity site and the high affinity site. Besides, one alternative portal was shown to be highly favorable for ligand egress from the high affinity site and be related to the unique structural feature of LFABP. This result lends strong support to the hypothesis from the previous NMR exchange studies, which in turn indicates an important role for this alternative portal. Another less favored potential portal located near the N-terminal end was also identified. Identification of the dissociation pathways will allow further mechanistic understanding of fatty acid uptake and release by computational and/or experimental techniques

Transcriptome analysis of stem development in the tumourous stem mustard Brassica juncea var. tumida Tsen et Lee by RNA sequencing

<p>Abstract</p> <p>Background</p> <p>Tumourous stem mustard (<it>Brassica juncea </it>var. <it>tumida </it>Tsen et Lee) is an economically and nutritionally important vegetable crop of the <it>Cruciferae </it>family that also provides the raw material for <it>Fuling </it>mustard. The genetics breeding, physiology, biochemistry and classification of mustards have been extensively studied, but little information is available on tumourous stem mustard at the molecular level. To gain greater insight into the molecular mechanisms underlying stem swelling in this vegetable and to provide additional information for molecular research and breeding, we sequenced the transcriptome of tumourous stem mustard at various stem developmental stages and compared it with that of a mutant variety lacking swollen stems.</p> <p>Results</p> <p>Using Illumina short-read technology with a tag-based digital gene expression (DGE) system, we performed <it>de novo </it>transcriptome assembly and gene expression analysis. In our analysis, we assembled genetic information for tumourous stem mustard at various stem developmental stages. In addition, we constructed five DGE libraries, which covered the strains <it>Yong'an </it>and <it>Dayejie </it>at various development stages. Illumina sequencing identified 146,265 unigenes, including 11,245 clusters and 135,020 singletons. The unigenes were subjected to a BLAST search and annotated using the GO and KO databases. We also compared the gene expression profiles of three swollen stem samples with those of two non-swollen stem samples. A total of 1,042 genes with significantly different expression levels occurring simultaneously in the six comparison groups were screened out. Finally, the altered expression levels of a number of randomly selected genes were confirmed by quantitative real-time PCR.</p> <p>Conclusions</p> <p>Our data provide comprehensive gene expression information at the transcriptional level and the first insight into the understanding of the molecular mechanisms and regulatory pathways of stem swelling and development in this plant, and will help define new mechanisms of stem development in non-model plant organisms.</p

Residues constituting the portal regions.

§<p>Coordinates of the backbone nitrogen atoms of these residues determined the reference plane.</p>#<p>Residues constituting each portal were determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006081#s2" target="_blank">Methods</a> section. Totally 11, 30, 4 and 36 trajectories were used for the analysis of portal I, portal II, portal III of LFABP and the helical portal of IFABP, respectively. For portals I and III of LFABP, only the residues which were recorded as potential portal residues in at least two different trajectories were finally reported; for portal II of LFABP and the helical portal of IFABP, only the residues which were recorded in at least four different trajectories were finally reported.</p

Dissociation of OLA129.

<p>(A–C) Snapshots showing three different dissociation processes of OLA129 by portal I, II and III, respectively; these three representative trajectories are also used for the calculation of RMSF (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006081#pone-0006081-g005" target="_blank">Figure 5</a>). The starting conformation (yellow) is aligned with the protein conformations in the moment of ligand expulsions (blue). The sidechains of residues which are surrounding OLA129 are shown in the ball-and-stick representation. (D) Structural alignment of rat-LFABP (blue) and rat-IFABP (green). βH and βG strands of IFABP are significantly longer than those of LFABP.</p

Snapshots of 1,8-ANS exiting the cavity.

<p>(A) ANS128 exiting from portal I. (B–D) ANS129 exiting from portal I, portal II and portal III, respectively. 1,8-ANS is displayed in a sphere representation (orange).</p

Front views of three portals.

<p>Three portal areas in the starting structure of REMD (time at zero) are shown in ball-and-stick (A–C) and surface (D–F) representations. Residues constituting portal I, II, and III are colored green, yellow, and red, respectively. Phe3 is colored pink (in C and F). Oleate molecules are not shown in the structure.</p

REMD simulation of ligand exiting from the cavity of IFABP and LFABP.

<p>The time interval (Δt) of REMD was kept constant at 0.25 ps for all trajectories.</p>#<p>Random force was applied on OLA129 only, while OLA128 was present in the low affinity binding site.</p>†<p>The number of trajectories in which the ligand successfully egressed using the individual portals.</p>‡<p>The number of trajectories in which the ligand failed to exit from the cavity in 200 ps.</p>**<p>For IFABP, portal I refers to the helical portal.</p>*<p>In this single trajectory, OLA129 partially protruded outside from portal II initially, then tried to slide over βE/βF loop, and eventually dissociated from the gap between the βE/βF loop and α-helix cap.</p>§<p>Trajectories were used for the analysis of residues constituting individual portals of LFABP (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006081#pone-0006081-t002" target="_blank">Table 2</a>).</p>§§<p>Trajectories of successful dissociations were used for the analysis of residues constituting the portal of IFABP (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006081#pone-0006081-t002" target="_blank">Table 2</a>).</p

Backbone residue-wise root mean square fluctuations (RMSF) of LFABP.

<p>The backbone RMSF of LFABP for the dissociation processes from portal I (black), portal II (green), and portal III (red) are plotted against the residue number; calculations are based on the last 10 ps, 13.5 ps (which is the whole trajectory) and last 15 ps of trajectories for portals I, II and III, respectively.</p

Dissociation of palmitate (purple) from the cavity of IFABP (dark green).

<p>Residues constituting the helical portal (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006081#pone-0006081-t002" target="_blank">Table 2</a>) are shown in yellow. In all the runs of successful dissociations, the palmitate molecule came out from essentially the same region.</p