12 research outputs found
Development of CHARMM-Compatible Force-Field Parameters for Cobalamin and Related Cofactors from Quantum Mechanical Calculations
Corrinoid
cofactors such as cobalamin are used by many enzymes
and are essential for most living organisms. Therefore, there is broad
interest in investigating cobalamin–protein interactions with
molecular dynamics simulations. Previously developed parameters for
cobalamins are based mainly on crystal structure data. Here, we report
CHARMM-compatible force field parameters for several corrinoids developed
from quantum mechanical calculations. We provide parameters for corrinoids
in three oxidation states, Co<sup>3+</sup>, Co<sup>2+</sup>, and Co<sup>1+</sup>, and with various axial ligands. Lennard-Jones parameters
for the cobalt center in the CoÂ(II) and CoÂ(I) states were optimized
using a helium atom probe, and partial atomic charges were obtained
with a combination of natural population analysis (NPA) and restrained
electrostatic potential (RESP) fitting approaches. The Force Field
Toolkit was used to optimize all bonded terms. The resulting parameters,
determined solely from calculations of cobalamin alone or in water,
were then validated by assessing their agreement with density functional
theory geometries and by analyzing molecular dynamics simulation trajectories
of several corrinoid proteins for which X-ray crystal structures are
available. In each case, we obtained excellent agreement with the
reference data. In comparison to previous CHARMM-compatible parameters
for cobalamin, we observe a better agreement for the fold angle and
lower RMSD in the cobalamin binding site. The approach described here
is readily adaptable for developing CHARMM-compatible force-field
parameters for other corrinoids or large biomolecules
Thrombin generation parameters of FVIII variants.
<p>FVIII concentration for each variant was adjusted to 5 ng/ml in complete medium. Subsequently each variant was diluted 1:1 in FVIII deficient plasma and triggered with 4 μM of phospholipids. The mean is calculated from three independent experiments performed in duplicate. (WT: wild-type; ETP: Endogenous thrombin potential; ttpeak: time to peak; SD: Standard deviation of mean).</p
The cleavage site region interface.
<p>The panels A and B in this image illustrate a close up view of the Arg<sup>391</sup> (a1-A2 junction), Arg<sup>759</sup> (a2-B junction) cleavage site regions and their putative interactions in the crystal structure of FVIII. The cleavage site regions Arg<sup>391</sup> (a1-A2 junction) and Arg<sup>759</sup> (a2-B junction) have been remodeled. Both panels show the same view but are colored differently. In both panels the backbone is depicted in ribbon format. Panel A illustrates the region surrounding the Arg<sup>391</sup> (a1-A2 junction) as a red ribbon while the Arg<sup>759</sup> (a2-B junction) region is depicted in blue ribbon format. In Panel B both regions are colored based on their secondary structure (i.e. blue: helix, cyan:coil,yellow:short helix, red:beta sheet). The cleavage site P1 and P1´ residues are depicted in stick format. In Panel A they are colored green while in Panel B they are colored based on atom (i.e. white:hydrogen, blue: nitrogen and cyan:carbon). In both panels the hydrogen bonds are depicted as yellow colored dots extending from their donor to the acceptor atom. The interface region between the two cleavage site regions is marked with a blue shaded area.</p
Structural changes in the thrombin active site cleft upon cleavage site regions peptide with consensus cleavage sequence insertion.
<p>This figure illustrates the differences in structure of the thrombin active site cleft upon the insertion of each cleavage site peptide with the consensus sequence. The central figure is a multiple alignment of post insertion thrombin structures for all three cleavage sites with the apoenzyme; all of them depicted in ribbon format. Color coding: Green: Apoenzyme, Red: O-TCS 1 cleavage site inserted thrombin, Yellow: O-TCS 2 cleavage site inserted thrombin, Cyan: O-TCS 3 cleavage site inserted thrombin. The catalytic Ser<sup>195</sup> residue of thrombin is depicted in stick form. The panels surrounding the central inset figure are close up views of each cleavage site inserted thrombin active site cleft structure aligned against the apoenzyme (and therefore of the region specified in the shaded area in the central figure). Depiction code follows that of the central figure.</p
An <i>in silico</i> and <i>in vitro</i> approach to elucidate the impact of residues flanking the cleavage scissile bonds of FVIII
<div><p>Coagulation Factor VIII is activated by an ordered limited thrombin proteolysis with different catalytic efficiency at three P1 Arginine residues: Arg<sup>759</sup>> Arg<sup>1708</sup>>Arg<sup>391</sup>, indicating the flanking residues of the latter to be less optimal. This study aimed to investigate, <i>in silico</i> and <i>in vitro</i>, the impact of possessing hypothetically optimized residues at these three catalytic cleavage sites. The structural impact of the residues flanking Arginine cleavage sites was studied by <i>in silico</i> analysis through comparing the cleavage cleft of the native site with a hypothetically optimized sequence at each site. Moreover, recombinant FVIII proteins were prepared by replacing the sequences flanking native thrombin cleavage sites with the proposed cleavage-optimized sequence. FVIII specific activity was determined by assessing the FVIII activity levels in relation to FVIII antigen levels. We further investigated whether thrombin generation could reflect the haemostatic potential of the variants. Our <i>in silico</i> results show the impact of the residues directly in the cleavage bond, and their neighboring residues on the insertion efficiency of the loop into the thrombin cleavage cleft. Moreover, the <i>in vitro</i> analysis shows that the sequences flanking the Arg<sup>1708</sup> cleavage site seem to be the most close to optimal residues for achieving the maximal proteolytic activation and profactor activity of FVIII. The residues flanking the scissile bonds of FVIIII affect the cleavage rates and modulate the profactor activation. We were able to provide insights into the mechanisms of the specificity of thrombin for the P1 cleavage sites of FVIII. Thus, the P4-P2´ residues surrounding Arg<sup>1708</sup> of FVIII have the highest impact on rates of thrombin proteolysis which contributes to thrombin activation of the profactor and eventually to the thrombin generation potential.</p></div
Thrombin generation parameters of FVIII variants.
<p>FVIII concentration for each variant was adjusted to 5 ng/ml in complete medium. Subsequently each variant was diluted 1:1 in FVIII deficient plasma and triggered with 4 μM of phospholipids. The mean is calculated from three independent experiments performed in duplicate. (WT: wild-type; ETP: Endogenous thrombin potential; ttpeak: time to peak; SD: Standard deviation of mean).</p
Specific activity of FVIII variants possessing an optimization of sequences flanking the thrombin cleavage site based on FVIII activity levels measured by FVIII:C<sub>1st</sub> and FVIII:C<sub>chr</sub>.
<p>Mean values with standard deviation from three independent experiments in duplicates are shown (* = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001).</p
Thrombin generation potential of FVIII variants possessing an optimization of sequences flanking the thrombin cleavage site.
<p>FVIII-depleted plasma was reconstituted with FVIII variants and thrombin generation was measured in the absence of TF. All experiments were performed at least three times. Representative thrombin generation curves are shown.</p
Illustration of the three modeled holoenzyme complexes of the optimized cleavage sites.
<p>This figure illustrates a close up view of three modeled holoenzyme complexes (for consensus cleavage site peptides) along with the change in the consensus cleavage site peptide structures pre and post insertion into thrombin. The central figure is the close up view of each consensus cleavage site peptide inserted thrombin holoenzyme structure. The backbone depiction is in ribbon format with the thrombin consensus cleavage site Factor VIII peptide colored green and the thrombin colored cyan. The catalytic Ser<sup>195</sup> residue is also shown in blue stick format. Also the region on the consensus cleavage site peptide where the scissile bond exists is colored red. The left and the right images corresponding to the central image each represent the pre and post-insertion structures of three consensus cleavage site peptides. The left image i.e. the pre-insertion structures are depicted in ribbon format with coloring as per secondary structure i.e. coil: cyan, helix: blue, beta strand: red. The scissile bond region is colored yellow. The right image is a green colored ribbon format description of the post-insertion consensus cleavage site peptide structure. It also shows their molecular surface colored as per their coulombic charges i.e. blue represents positive and red negative charge.</p
Mean of relative FVIII activity levels (FVIII:C) and FVIII antigen (FVIII:Ag) levels after exchange of native cleavage sites into consensus sequence.
<p>The mean is calculated from three independent experiments performed in duplicate. (WT, wild-type; SD, Standard deviation of mean).</p