Structure and Behavior of Human α-Thrombin upon Ligand Recognition: Thermodynamic and Molecular Dynamics Studies

Thrombin is a serine proteinase that plays a fundamental role in coagulation. In this study, we address the effects of ligand site recognition by alpha-thrombin on conformation and energetics in solution. Active site occupation induces large changes in secondary structure content in thrombin as shown by circular dichroism. Thrombin-D-Phe-Pro-Arg-chloromethyl ketone (PPACK) exhibits enhanced equilibrium and kinetic stability compared to free thrombin, whose difference is rooted in the unfolding step. Small-angle X-ray scattering (SAXS) measurements in solution reveal an overall similarity in the molecular envelope of thrombin and thrombin-PPACK, which differs from the crystal structure of thrombin. Molecular dynamics simulations performed with thrombin lead to different conformations than the one observed in the crystal structure. These data shed light on the diversity of thrombin conformers not previously observed in crystal structures with distinguished catalytic and conformational behaviors, which might have direct implications on novel strategies to design direct thrombin inhibitors

Secondary structure effects of αTh active site occupation.

<p>(<b>A</b>) CD spectra of free (continuous line) and PPACK-bound (dashed line) αTh. Spectra were collected at 30 µM for both proteins in 0.10 mm pathlength quartz cells. (<b>B</b>) Thermal unfolding of free (closed symbols) and PPACK-bound (open symbols) αTh monitored by changes in ellipticity at 222 nm throughout the process. Solid lines are linear regression. Details in the <i>Experimental Methods</i> section.</p

Overall properties assessed by MD simulations.

<p>RMSD values for free enzyme (black curve), αTh-PPACK (red curve), secondary structure deviation in free αTh (green curve) and in complexed αTh (blue curve), for light chain (“L”, panel <b>A</b>) and heavy chain (“H”, panel <b>B</b>); (<b>C</b>) H-bonds formed between coil-surrounded PPACK site in free αTh (black curve) and complexed form (red curve); (<b>D</b>) Total intramolecular H-bonds formed between residues for free αTh (black curve) and complexed αTh (red curve). Details in the <i>Experimental Methods</i> section.</p

Stability effects of αTh active site occupation.

<p>Evaluation of PPACK binding to αTh was probed by equilibrium denaturation induced by GdmCl and monitored by intrinsic fluorescence spectroscopy. (<b>A</b>) Fluorescence spectra of free and PPACK-bound αTh in the presence and absence of 4 M GdmCl. (<b>B</b>) Denaturation curve of free (closed circles) and PPACK-bound (open circles) αTh was monitored by changes in the spectral center of mass. Solid lines are non-linear regression fitting with Eq [12]. (<b>C</b>) Changes in intrinsic fluorescence emission of free (closed circles) and PPACK-bound (open circles) αTh as a function of GdmCl concentration. Note the increase in fluorescence emission in a pre-transition region for αTh-PPACK, corresponding to the transition between the initial ‘native’ and the intermediate states. Excitation was set at 280 nm and emission scanned from 300 to 420 nm; both the spectral center of mass and spectral area were calculated. Details in the <i>Experimental Methods</i> section.</p

Structural and thermodynamic parameters of αTh and αTh-PPACK.

1<p>) from SAXS measurements;</p>2<p>) Resolution is calculated as 2π/q<i>_max</i>;</p>3<p>) from equilibrium GdmCl induced denaturation;</p>4<p>) from heat induced denaturation, with equivalent results for both 1 and 2°C/min heating rate;</p>5<p>) from kinetic measurements.</p

Superposition of unbounded αTh (salmon) and αTh-PPACK complex (blue) after 50 ns MD simulation.

<p>The loops surrounding the PPACK binding site in the catalytic cleft are shown in detail. Both structures deviate from the original crystallographic model used in the MD simulations (PDB ID 1PPB). Details in the <i>Experimental Methods</i> section.</p

Interaction energies assessed by MD for both thrombin forms.

<p>Interaction energies assessed by MD for both thrombin forms.</p

Kinetic measurements of GdmCl induced folding and unfolding transitions of αTh and αTh -PPACK.

<p>Kinetic traces (A and C) and residuals (B and D) of unfolding (A; B) and refolding (C;D) transitions of αTh (closed circles) and αTh-PPACK (open circles). The curves were fit with a single exponential decay, and the rate constant was calculated. Fitting residuals are shown for αTh (closed circles) and αTh-PPACK (open circles); (E) Chevron plot – GdmCl dependence of the apparent rate constant of folding and unfolding of αTh and αTh-PPACK. The lack of linearity in the Chevron plot indicates a kinetic mechanism more complex than a simple two-state model, and thus limits a precise quantitative analysis of the kinetic process. (F) The dependence of the refolding lag phase as a function of GdmCl. (G) Temperature dependence on the kinetics of GdmCl-induced unfolding of αTh and αTh-PPACK by jumping the GdmCl concentration from 0 to 4 M and monitoring by the changes in fluorescence intensity, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024735#pone-0024735-g004" target="_blank">Fig. 4A</a>. Lines represent the best fit with Eq 22. Details in the <i>Experimental Methods</i> section.</p

Small-angle X-ray scattering analysis of αTh and αT-PPACK.

<p>SAXS measurements of free (closed circles) and active site-bound (open symbols) αTh (<b>A</b>) Experimental scattering curves of αTh and αTh-PPACK; solid lines correspond to fits to data with the crystal structure of αTh-PPACK (1PPB) using Crysol. <i>Inset</i>: linear <i>q</i> scale. (<b>B</b>) Guinier plot of the scattering function; solid lines correspond to first-order linear regression of the data. The linearity of the Guinier plot indicated that both samples are monodispersed and constitute a unique species; (<b>C</b>) Kratky plot from raw data and fitting from panel (A); (<b>D</b>) Distance distribution functions. Details in the <i>Experimental Methods</i> section.</p