The Bacterial Fimbrial Tip Acts as a Mechanical Force Sensor

The subunits that constitute the bacterial adhesive complex located at the tip of the fimbria form a hook-chain that acts as a rapid force-sensitive anchor at high flow

Destabilization of the von Willebrand factor A2 domain under oxidizing conditions investigated by molecular dynamics simulations.

The protein von Willebrand factor (VWF) is key for the adhesion of blood platelets to sites of vascular injury. Recent studies have shown that the release of oxidative agents during inflammation increases the platelet-tethering activity of VWF contributing to a pro-thrombotic state. This has been linked to the oxidation of methionine residues in the A1, A2 and A3 domains of VWF. The A1 domain binds to platelet surface receptors glycoprotein Ib α (GpIbα). This interaction has been shown to be inhibited under static conditions by the neighboring A2 domain. Tensile force exerted by blood flow unfolds the A2 domain normally leading to its cleavage by the metalloprotease ADAMTS13 preventing pathological thrombus formation. However, oxidizing conditions inhibit proteolysis through ADAMTS13. Here, molecular dynamics simulations tested the hypothesis whether methionine oxidation induced by inflammatory conditions favors unfolding of the A2 domain contributing to the experimentally observed activation of VWF. The results indicate that oxidation of methionine residues located near the C-terminal helix of the A2 domain reduce the force necessary to initiate unfolding. Furthermore, oxidation of methionine residues shifts the thermodynamic equilibrium of the A2 domain fold towards the denatured state. This work suggests a mechanism whereby oxidation reduces the kinetic and thermodynamic stability of the A2 domain removing its inhibitory function on the binding of the A1 domain to GpIbα

Characterization and further stabilization of designed ankyrin repeat proteins by combining molecular dynamics simulations and experiments

Multiple molecular dynamics simulations with explicit solvent at room temperature and at 400 K were carried out to characterize designed ankyrin repeat (AR) proteins with full-consensus repeats. Using proteins with one to five repeats, the stability of the native structure was found to increase with the number of repeats. The C-terminal capping repeat, originating from the natural guanine-adenine-binding protein, was observed to denature first in almost all high-temperature simulations. Notably, a stable intermediate is found in experimental equilibrium unfolding studies of one of the simulated consensus proteins. On the basis of simulation results, this intermediate is interpreted to represent a conformation with a denatured C-terminal repeat. To validate this interpretation, constructs without C-terminal capping repeat were prepared and did not show this intermediate in equilibrium unfolding experiments. Conversely, the capping repeats were found to be essential for efficient folding in the cell and for avoiding aggregation, presumably because of their highly charged surface. To design a capping repeat conferring similar solubility properties yet even higher stability, eight point mutations adapting the C-cap to the consensus AR and adding a three-residue extension at the C-terminus were predicted in silico and validated experimentally. The in vitro full-consensus proteins were also compared with a previously published designed AR protein, E3_5, whose internal repeats show 80% identity in primary sequence. A detailed analysis of the simulations suggests that networks of salt bridges between beta-hairpins, as well as additional interrepeat hydrogen bonds, contribute to the extraordinary stability of the full consensus

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

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 Å 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

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

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 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

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