15 research outputs found
Simulation systems.
<p>The mutation induces type 2A VWD. Tensile force was applied by pulling the C-terminus at constant velocity from the N-terminus. Three simulations were started for the wild-type and each mutant with and without applied tensile force; they are labeled with 1, 2 and 3, respectively. The pulling runs were started from snapshots sampled during the simulations with no tensile force, more specifically after 10 ns of run XXX_1 and after 10 and 20 ns of run XXX_2, where XXX is either WT or one of the mutations.</p
Analysis of mutations predicted to destabilize the A2 domain.
<p>(<b>A</b>) C root mean square fluctuations of mutants compared to the wild-type. The values reported are averages over three runs. The bars indicate the standard error of the mean and the labels are the correlation p-values from a single-tailed student t-test (mutant versus wild-type). Reported are only p-values which are not larger than 0.1. (<b>B</b>) Van der Waals interaction energy between a side chain and the rest of the simulated system (i.e., rest of the protein and solvent). Compared are the energy of the wild-type residue (black) and the mutated side chain (red). The values are averages over three runs using the last 30 ns of in total 40 ns long runs at 300 K under static conditions (i.e., no force was applied). Electrostatic interaction energies are not shown because they are negligible compared to vdW. (<b>C</b>) Tensile force during exposure of the C-terminal hydrophobic core (see “Materials and Methods” for details about its measurement) in constant velocity pulling simulations with the mutants compared to the wild-type. The values are averaged over three runs and the standard error of the mean is indicated by error bars. The correlation p-values from the single tailed student t-test (mutant versus wild-type) are reported.</p
Structural Basis of Type 2A von Willebrand Disease Investigated by Molecular Dynamics Simulations and Experiments
<div><p>The hemostatic function of von Willebrand factor is downregulated by the metalloprotease ADAMTS13, which cleaves at a unique site normally buried in the A2 domain. Exposure of the proteolytic site is induced in the wild-type by shear stress as von Willebrand factor circulates in blood. Mutations in the A2 domain, which increase its susceptibility to cleavage, cause type 2A von Willebrand disease. In this study, molecular dynamics simulations suggest that the A2 domain unfolds under tensile force progressively through a series of steps. The simulation results also indicated that three type 2A mutations in the C-terminal half of the A2 domain, L1657I, I1628T and E1638K, destabilize the native state fold of the protein. Furthermore, all three type 2A mutations lowered <em>in silico</em> the tensile force necessary to undock the C-terminal helix 6 from the rest of the A2 domain, the first event in the unfolding pathway. The mutations F1520A, I1651A and A1661G were also predicted by simulations to destabilize the A2 domain and facilitate exposure of the cleavage site. Recombinant A2 domain proteins were expressed and cleavage assays were performed with the wild-type and single-point mutants. All three type 2A and two of the three predicted mutations exhibited increased rate of cleavage by ADAMTS13. These results confirm that destabilization of the helix 6 in the A2 domain facilitates exposure of the cleavage site and increases the rate of cleavage by ADAMTS13.</p> </div
Tertiary and secondary structure of the A2 domain.
<p>(<b>A</b>) Stereoview of the A2 domain X-ray structure (PDB code 3GXB). The backbone of the proteolysis site is indicated in red and the rest of the protein is colored according to secondary structure elements with helices in purple, 3 helices in blue, strands in green and turns in cyan. The side chains of Tyr and Met, which are part of the cleavage site, and of the mutation sites Leu, Ile and Glu are drawn in the ball and stick representation. The three mutation sites are also indicated by blue circles. The labeling of helices and strands is the same as in reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045207#pone.0045207-Zhang1" target="_blank">[13]</a>. (<b>B</b>) Secondary structure sequence. The cleavage site and the location of the three type 2A mutations investigated here are indicated. This figure was created with VMD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045207#pone.0045207-Humphrey1" target="_blank">[49]</a>.</p
Cleavage rates of wild-type and mutant A2 fragments with ADAMTS13.
<p>Reported are the initial rates of cleavage expressed as the percent of A2 fragment cleaved per minute. The error bars indicate the standard deviation of three independent measurements.</p
Analysis of key properties during pulling with the wild-type versus mutants.
<p>(<b>A</b>) Time series of the applied force and SASA of the C-terminal hydrophobic core. The force peak was determined by identifying the time point when the SASA exceeds 50 Ă… and searching for the highest value of the force within a 400 ps time window. 20-ps running average is indicated in red for the force and in blue for the SASA, respectively. (<b>B</b>) Mean and standard error of the force peaks corresponding to disruption of the C-terminal hydrophobic core averaged over three simulations (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045207#pone.0045207.s015" target="_blank">Figures S15</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045207#pone.0045207.s016" target="_blank">S16</a>). Reported are the correlation values (p-values) calculated from a single tailed student t-test.</p
Effects of mutations onto the structural stability of the A2 domain.
<p>(<b>A</b>) C root mean square fluctuations of the C-terminal helix, 6, and strand 6. The values are calculated over the last 30 ns of in total 40-ns long room temperature simulations. Averages and standard error of the means are calculated over three independent runs for each mutant and the wild-type. The values reported next to the error bars are the correlation factors calculated from a single tailed student t-test. A difference is said to be statistically significant if the correlation factor is not larger than 0.05, whereas values between 0.05 and 0.10 are referred to as marginally statistically significant. Only correlation factors not larger than 0.10 are reported. (<b>B</b>) Interaction energy between a mutated side chain and (upper panel) the rest of the system (i.e., solvent and protein excluding the mutated side chain), or (lower panel) rest of the protein, subtracted from the interaction energy of the wild-type residue.</p
Inhibition and Reversal of Microbial Attachment by an Antibody with Parasteric Activity against the FimH Adhesin of Uropathogenic <i>E</i>. <i>coli</i>
<div><p>Attachment proteins from the surface of eukaryotic cells, bacteria and viruses are critical receptors in cell adhesion or signaling and are primary targets for the development of vaccines and therapeutic antibodies. It is proposed that the ligand-binding pocket in receptor proteins can shift between inactive and active conformations with weak and strong ligand-binding capability, respectively. Here, using monoclonal antibodies against a vaccine target protein - fimbrial adhesin FimH of uropathogenic <i>Escherichia coli</i>, we demonstrate that unusually strong receptor inhibition can be achieved by antibody that binds within the binding pocket and displaces the ligand in a non-competitive way. The non-competitive antibody binds to a loop that interacts with the ligand in the active conformation of the pocket but is shifted away from ligand in the inactive conformation. We refer to this as a parasteric inhibition, where the inhibitor binds adjacent to the ligand in the binding pocket. We showed that the receptor-blocking mechanism of parasteric antibody differs from that of orthosteric inhibition, where the inhibitor replaces the ligand or allosteric inhibition where the inhibitor binds at a site distant from the ligand, and is very potent in blocking bacterial adhesion, dissolving surface-adherent biofilms and protecting mice from urinary bladder infection.</p></div
The overlap between the FimH pocket residues and mAb926 and mAb475 epitopes.
<p>Distances between mannose ligand and hydrogen bond forming FimH amino acids in the active and inactive conformation of the binding pocket are also shown.</p><p><sup>1</sup> Distance between hydrogen bond forming atoms of α-D-mannose and FimH amino acid residues in the active- (1UWF) and the inactive (3JWN) conformers of lectin domain as measured by PyMole.</p><p>*AA involved in hydrophobic interactions with mannose.</p><p>The overlap between the FimH pocket residues and mAb926 and mAb475 epitopes.</p
Inhibitory potency of mAb926 and mAb475.
<p>Binding of FimH<sup>wt</sup>-expressing <i>E</i>. <i>coli</i> to surface-immobilized yeast mannan in the presence of different concentrations of anti-FimH antibodies. Data are mean ± SEM (n = 5 independent experiments). The IC<sub>50</sub> values were calculated from the fitted curves shown using Prism GraphPad 6 software. **, <i>P</i> ≤ 0.005 (t-test).</p