Role of <i>cis-trans</i> proline isomerization in the function of pathogenic enterobacterial Periplasmic Binding Proteins

<div><p>Periplasmic Binding Proteins (PBPs) trap nutrients for their internalization into bacteria by ABC transporters. Ligand binding triggers PBP closure by bringing its two domains together like a Venus flytrap. The atomic determinants that control PBP opening and closure for nutrient capture and release are not known, although it is proposed that opening and ligand release occur while in contact with the ABC transporter for concurrent substrate translocation. In this paper we evaluated the effect of the isomerization of a conserved proline, located near the binding site, on the propensity of PBPs to open and close. ArgT/LAO from <i>Salmonella typhimurium</i> and HisJ from <i>Escherichia coli</i> were studied through molecular mechanics at two different temperatures: 300 and 323 K. Eight microseconds were simulated per protein to analyze protein opening and closure in the absence of the ABC transporter. We show that when the studied proline is in <i>trans</i>, closed empty LAO and HisJ can open. In contrast, with the proline in <i>cis</i>, opening transitions were much less frequent and characterized by smaller changes. The proline in <i>trans</i> also renders the open trap prone to close over a ligand. Our data suggest that the isomerization of this conserved proline modulates the PBP mechanism: the proline in <i>trans</i> allows the exploration of conformational space to produce trap opening and closure, while in <i>cis</i> it restricts PBP movement and could limit ligand release until in productive contact with the ABC transporter. This is the first time that a proline isomerization has been related to the control of a large conformational change like the PBP flytrap mechanism.</p></div

HisJ domain separation during simulations at 323 K, monitored through distances, angles and dihedral between domains as a function of Pro16 isomerization state.

<p>Data were obtained, processed and presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188935#pone.0188935.g003" target="_blank">Fig 3</a>.</p

Changes in seven metrics during simulations of the closed/empty LAO with <i>cis</i> or <i>trans</i> Pro16 at 300 K.

<p>Ten different trajectories were concatenated and changes in distance, angle, RMSD, Rg, SAS, Q(NC) and q(similarity) were calculated. Each trajectory is separated by a vertical line. Distances and angles were measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188935#pone.0188935.g002" target="_blank">Fig 2</a>. Q(NC) and q(similarity) were ploted using the closed PDB ID 1LAF (black line) or the open 2LAO (red line), as reference. Simultaneous changes in the metrics that coincide with crossovers in q(similarity) are indicated by black bars at the bottom of the figure. LAO with <i>cis</i> Pro16 (A) displayed two concurrent changes in the metrics whereas with <i>trans</i> (B), seven concurrent peaks were detected.</p

HisJ domain separation during simulations at 300 K, monitored through distances, angles and dihedrals between domains as a function of Pro16 isomerization state.

<p>Data were obtained, processed and presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188935#pone.0188935.g002" target="_blank">Fig 2</a>. Dotted and solid lines across the graphs correspond to the reference values calculated from open (PDB ID 2M8C) or closed (PDB ID 1HSL) structures.</p

Changes in seven metrics during simulations of the open/with ligand LAO with <i>cis</i> or <i>trans</i> Pro16 at 323 K.

<p>Ten different trajectories were concatenated and metrics were calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188935#pone.0188935.g006" target="_blank">Fig 6</a>. Independent trajectories are divided by vertical lines. LAO with <i>cis</i> Pro16 (A) displayed one crossover in q(similarity) values, whereas five were detected with <i>trans</i> Pro16, concurrent with dips in distance and angle (indicated with black bars at the bottom of the figure).</p

β-Lactoglobulin's Conformational Requirements for Ligand Binding at the Calyx and the Dimer Interphase: a Flexible Docking Study

<div><p>β-lactoglobulin (BLG) is an abundant milk protein relevant for industry and biotechnology, due significantly to its ability to bind a wide range of polar and apolar ligands. While hydrophobic ligand sites are known, sites for hydrophilic ligands such as the prevalent milk sugar, lactose, remain undetermined. Through the use of molecular docking we first, analyzed the known fatty acid binding sites in order to dissect their atomistic determinants and second, predicted the interaction sites for lactose with monomeric and dimeric BLG. We validated our approach against BLG structures co-crystallized with ligands and report a computational setup with a reduced number of flexible residues that is able to reproduce experimental results with high precision. Blind dockings with and without flexible side chains on BLG showed that: i) 13 experimentally-determined ligands fit the calyx requiring minimal movement of up to 7 residues out of the 23 that constitute this binding site. ii) Lactose does not bind the calyx despite conformational flexibility, but binds the dimer interface and an alternate Site C. iii) Results point to a probable lactolation site in the BLG dimer interface, at K141, consistent with previous biochemical findings. In contrast, no accessible lysines are found near Site C. iv) lactose forms hydrogen bonds with residues from both monomers stabilizing the dimer through a claw-like structure. Overall, these results improve our understanding of BLG's binding sites, importantly narrowing down the calyx residues that control ligand binding. Moreover, our results emphasize the importance of the dimer interface as an insufficiently explored, biologically relevant binding site of particular importance for hydrophilic ligands. Furthermore our analyses suggest that BLG is a robust scaffold for multiple ligand-binding, suitable for protein design, and advance our molecular understanding of its ligand sites to a point that allows manipulation to control binding.</p> </div

β-lactoglobulin and its calyx binding site.

<p>The main BLG binding site (calyx or Site A) is shown empty from two perspectives: (<b>A</b>) top-down view and (<b>B</b>) bottom-up view. The seven flexible residues required for ligand binding are labeled. The secondary structure is colored from N-terminus in blue, to C-terminus in red. (<b>C</b>) Plot of binding energy calculated from docking vs ligand using rigid, monomeric, empty BLG structures with open (2BLG, black circles), or semi closed (2Q39, black squares) EF loops, and compared to experimentally determined data (open circles). Fatty acids are sorted by increasing size, or in the case of stearic, oleic and linoleic, by decreasing saturation. No experimental affinity has been reported for stearic or retinoic acids. (<b>D</b>) Weblogo of the sequence alignment of BLG from 7 mammals. Asterisks indicate the 5 residues made flexible for docking.</p

Effect of residue flexibility on fatty acid binding.

<p>(<b>A</b>) Docking to a rigid 2BLG allows only the three smallest lipids into the calyx (shown superposed in white, light blue and light purple), while excluding longer fatty acids to Site C (black square, fatty acids in different colors). The seven binding residues in the calyx are shown in light yellow. When five of these residues were allowed flexibility all fatty acids bind the calyx. Stearic acid (purple) is shown bound in (<b>B</b>) and (<b>C</b>) in a full BLG top down view and a side view magnification of the calyx, respectively. In (<b>C</b>) the five flexible residues (blue) are shown aligned to their XRD counterpart (light yellow) to highlight movements that enable docking. (<b>D</b>) Plot of binding energy from docking vs. ligand using the monomeric empty, 2BLG, either rigid (black circles) or with 5 (black squares) or 7 (black triangles) flexible residues. Experimentally determined energies are shown for comparison (open circles). Fatty acids are sorted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079530#pone-0079530-g001" target="_blank">Figure 1C</a>.</p

Lactose docking to the 2BLG dimer.

<p>(<b>A</b>) Lactose docking to a rigid BLG dimer. Notice both K138 and K141 pointing away from lactose. In (<b>B</b>), a side view of the rigid interface docking (lactose, K138 and K141 in yellow) is compared to the “fully flexible” results (green) where K141 shifts towards lactose. In (<b>C</b>) a top view of the best “fully flexible” result. Residues involved in lactose binding are highlighted by chain: chain B in purple and chain A in cyan. (<b>D</b>) close-up of the interfacial binding site showing chains from both BLG monomers and their respective hydrogen bonds to lactose.</p

New Insights on the Mechanism of the K⁺-Independent Activity of Crenarchaeota Pyruvate Kinases

<div><p>Eukarya pyruvate kinases have glutamate at position 117 (numbered according to the rabbit muscle enzyme), whereas in Bacteria have either glutamate or lysine and in Archaea have other residues. Glutamate at this position makes pyruvate kinases K⁺-dependent, whereas lysine confers K⁺-independence because the positively charged residue substitutes for the monovalent cation charge. Interestingly, pyruvate kinases from two characterized Crenarchaeota exhibit K⁺-independent activity, despite having serine at the equivalent position. To better understand pyruvate kinase catalytic activity in the absence of K⁺ or an internal positive charge, the <i>Thermofilum pendens</i> pyruvate kinase (valine at the equivalent position) was characterized. The enzyme activity was K⁺-independent. The kinetic mechanism was random order with a rapid equilibrium, which is equal to the mechanism of the rabbit muscle enzyme in the presence of K⁺ or the mutant E117K in the absence of K⁺. Thus, the substrate binding order of the <i>T</i>. <i>pendens</i> enzyme was independent despite lacking an internal positive charge. Thermal stability studies of this enzyme showed two calorimetric transitions, one attributable to the A and C domains (<i>T_m</i> of 99.2°C), and the other (<i>T_m</i> of 105.2°C) associated with the B domain. In contrast, the rabbit muscle enzyme exhibits a single calorimetric transition (<i>T_m</i> of 65.2°C). The calorimetric and kinetic data indicate that the B domain of this hyperthermophilic enzyme is more stable than the rest of the protein with a conformation that induces the catalytic readiness of the enzyme. B domain interactions of pyruvate kinases that have been determined in <i>Pyrobaculum aerophilum</i> and modeled in <i>T</i>. <i>pendens</i> were compared with those of the rabbit muscle enzyme. The results show that intra- and interdomain interactions of the Crenarchaeota enzymes may account for their higher B domain stability. Thus the structural arrangement of the <i>T</i>. <i>pendens</i> pyruvate kinase could allow charge-independent catalysis.</p></div