Light Chain Mutation Effects on the Dynamics of Thrombin

Abstract

Thrombin plays an important role in the process of hemostasis and blood coagulation. Studies in thrombin can help us find ways to treat cancer because thrombin is able to reduce the characteristic hypercoagulability of cancer. Thrombin is composed of two chains, the light chain and the heavy chain. The function of the heavy chain has been largely explored, while the function of the light chain was obscured until several disease-associated mutations in the light chain come to light. In this study, we want to explore the dynamic and conformation effects of mutations on the light chain further to determine possible associations between mutation, conformational changes, and disease. The study, which is a follow-up for our studies on apo thrombin and the mutant, ΔK9, mainly focuses on the mutants E8K and R4A. E8K is a disease-associated mutation, and R4A is used to study the role of Arg4, which is suggested experimentally to play a critical role for thrombin's catalytic activities. We performed five all-atom one microsecond-scale molecular dynamics (MD) simulations for both E8K and R4A, and quantified the changes in the conformational ensemble of the mutants. From the root-mean-square fluctuations (RMSF) for the α-carbons, we find that the atomic fluctuations change in the mutants in the 60s loop and γ loop. The correlation coefficients for the α-carbons indicate that the correlation relation for atom-pairs in the protein is also impacted. The clustering analysis and the principal component analysis (PCA) consistently tell us that the catalytic pocket and the regulatory loops are destabilized by the mutations. We also find that there are two binding modes for Na⁺ by clustering the vector difference between the Na⁺ ions and the 220s loop. After further analysis, we find that there is a relation between the Na⁺ binding and the rigidification of the γ loop, which may shed light on the mysterious role of the γ loop in thrombin.

Figure 1.

Thrombin-Na⁺ complex structure. The heavy chain is shown in transparent green. The light chain is shown in purple. The 60s (pink), 220s (tan), and γ (black) loops and exosite I (orange) and II (yellow) are highlighted as the known regulatory regions. Side chains of the catalytic triad are displayed in red. The bound Na⁺ is shown as the blue bead.

Figure 2.

(a) Root-mean-square fluctuations (RMSF) for the α-carbons of WT, ΔK9, E8K, and R4A. Residue number follows the sequential residue numbering scheme that all residues are numbered from 1 to 295 (MD notation). As LYS9 with residue ID 17 is deleted in ΔK9, we exclude the RMSF of LYS9 in our calculation. (b) Thrombin is colored based on the RMSF of the WT and the RMSF difference between the mutants (ΔK9, E8K, and R4A) and WT. The scale is on the left bottom.

Figure 3.

(a–d) Correlation matrices of WT, ΔK9, E8K, and R4A. (e–h) Visualizations of the correlation matrix for WT, ΔK9, E8K, and R4A, respectively. The representation method is the same as Figure 1 for comparison. In graph e, light-red lines indicate residue pairs (excluding adjacent residue pairs) with the absolute value of correlation coefficients larger than 0.7. In graphs f–h, both light-red lines and light-blue lines represent pairs of residues with positive and negative subtractions (mutants – WT) of correlation matrices whose absolute value is larger than 0.5.

Figure 4.

Amorim–Hennig clustering distribution of ΔK9 and WT thrombin. Panels a and d represent the clustering results of the heavy atoms of the catalytic pocket and regulatory loops. The percentage for each cluster is on top of each column. Panels b and c are the representative structures for cluster 0 and 1 of catalytic pocket, and panels e–g are the representative structures for cluster 0 to 2 of regulatory loops. They are shown in NewCartoon representation with transparent green. Side chains in the catalytic pocket or regulatory loops are displayed by the Licorice in red. Gray shadows represent the variances in the catalytic pocket or regulatory loops. Catalytic triad are shown in blue.

Figure 5.

(a) Comparisons of conformational free energy surfaces of the catalytic pocket of different forms of thrombin. (b) Comparisons of conformational free energy surfaces of the regulatory loops of different forms of thrombin. The basis set of the structural projections was obtained from all conformations in the WT, ΔK9, E8K, and R4A forms of thrombin.

Figure 6.

Comparisons of conformational free energy surfaces of the catalytic pocket for different forms of thrombin. The basis set of the structural projections was obtained from all conformations in the WT, ΔK9, E8K, and R4A forms of thrombin. Na⁺-binding/unbinding status are respectively discussed. The white number represent the minimum free energy in each well. The black number respresent the saddle point (i.e., energy barrier) between each pair of wells.

Figure 7.

Comparisons of conformational free energy surfaces of the regulatory loops for different forms of thrombin. The basis set of the structural projections was obtained from all conformations in the WT, ΔK9, E8K, and R4A forms of thrombin. Na⁺-binding/unbinding status are discussed. The white number represent the minimum free energy in each well. The black number respresent the saddle point (i.e., energy barrier) between each pair of wells.

Figure 8.

(a–h) Representative structures for catalytic pocket in wells 1–8. They are shown in NewCartoon representation with transparent green. Side chains in the catalytic pocket are displayed by the Licorice in red. Gray shadows represent the variances in the catalytic pocket. Catalytic triad are shown in blue.

Figure 9.

(a–j) Representative structures for regulatory loops in wells 1–10. They are shown in NewCartoon representation with transparent green. Side chains in the regulatory loops are displayed by the Licorice in red. Gray shadows represent the variances in the regulatory loops. Catalytic triad are shown in blue.

Figure 10.

Probability of the closest mean distance between Na⁺ ions and the 220s loop. The 220s loop is defined as residue 264 to 274. Distances between each atom in the 220s loop and each Na⁺ ion are calculated. We calculated the mean distance between each Na⁺ ion and the 220s loop and selected the smallest one.

Figure 11.

(a) Amorim–Hennig clustering distribution of WT, ΔK9, E8K, and R4A. Panels b–e use the NewCartion representation to display the representative structure and the shadow to display the variances for cluster 0 to 3. “Unbinding” represents the frames whose closest mean distance between the Na⁺ ions and the 220s loop is bigger than 12 Å. In this distance, Na⁺ binding is impossible.

Figure 12.

(a and b) Comparisons of conformational free energy surfaces for catalytic pocket and regulatory loops of different forms of thrombin. The basis set of the structural projections was obtained from all conformations in the WT, ΔK9, E8K, and R4A forms of thrombin. Na⁺ inner/outer binding status are discussed, respectively.