Dynamic Consequences of Mutation of Tryptophan 215 in Thrombin

Abstract

Thrombin normally cleaves fibrinogen to promote coagulation; however, binding of thrombomodulin to thrombin switches the specificity of thrombin toward protein C, triggering the anticoagulation pathway. The W215A thrombin mutant was reported to have decreased activity toward fibrinogen without significant loss of activity toward protein C. To understand how mutation of Trp215 may alter thrombin specificity, hydrogen-deuterium exchange experiments (HDXMS), accelerated molecular dynamics (AMD) simulations, and activity assays were carried out to compare the dynamics of Trp215 mutants with those of wild type (WT) thrombin. Variation in NaCl concentration had no detectable effect on the sodium-binding (220s_CT) loop, but appeared to affect other surface loops. Trp215 mutants showed significant increases in amide exchange in the 170s_CT loop consistent with a loss of H-bonding in this loop identified by the AMD simulations. The W215A thrombin showed increased amide exchange in the 220s_CT loop and in the N-terminus of the heavy chain. The AMD simulations showed that a transient conformation of the W215A thrombin has a distorted catalytic triad. HDXMS experiments revealed that mutation of Phe227, which engages in a π-stacking interaction with Trp215, also caused significantly increased amide exchange in the 170s_CT loop. Activity assays showed that only the F227V mutant had wild type catalytic activity, whereas all other mutants showed markedly lower activity. Taken together, the results explain the reduced pro-coagulant activity of the W215A mutant and demonstrate the allosteric connection between Trp215, the sodium-binding loop, and the active site.

Figure 1

A) Deuterium incorporation into residues 212-227_CT (residues 260-275; MH+ 1673.760 and 1788.801 for W215A and WT respectively) over 5 min is shown for WT thrombin at 100 mM NaCl (grey) and 300 mM NaCl (black), and for the W215A mutant at 100 mM NaCl (cyan) and 300 mM NaCl (blue). The mutant residue, is underlined in the peptide sequence shown. B) Structure of WT thrombin (PDB 1PPB) highlighting residues 212-227_CT (residues 260-275; brown). The sidechains of Trp215_CT (blue) and the catalytic triad (black) are shown as sticks.

Figure 2

Structure of WT thrombin (PDB 1PPB) highlighting residues 81-85_CT (residues 113-117; red), residues 86-102_CT (residues 118-135; orange), and residues 129A-130_CT (residues 163-166; green). Colored residues specify regions affected by the concentration of NaCl after subtraction of deuteron uptake of overlapping peptides 66-80_CT (residues 97-112; MH+ 2014.105) from 66-85_CT (residues 97-117; MH+ 2586.441) and 117-129_CT (residues 150-162; MH+ 1513.747) from 117-130_CT (residues 150-166; MH+ 1897.984) as well as residues 85-102_CT (residues 117-135; MH+ 2530.295). The sidechains of Trp215_CT (blue) and the catalytic triad (black) are shown as sticks. Deuteron incorporation into these residues over 5 min is shown for WT thrombin at 100 mM NaCl (grey) and 300 mM NaCl (black), and for the W215A mutant at 100 mM NaCl (cyan) and 300 mM NaCl (blue).

Figure 3

Structure of WT thrombin (PDB 1PPB) highlighting 156-181_CT (residues 197-222; green). The sidechains of Trp215CT (blue) and the catalytic triad (black) are shown as sticks. Deuteron incorporation over 5 min into residues 156-180_CT (residues 197-221; MH+ 2896.550), 161-170_CT (residues 202-211; MH+ 1155.619), and 161-181_CT (residues 202-222; MH+ 2490.296) is shown for WT thrombin at 100 mM NaCl (grey) and 300 mM NaCl (black), and for the W215A mutant at 100 mM NaCl (cyan) and 300 mM NaCl (blue).

Figure 4

A) Deuterium incorporation over 5 min into residues 16-23_CT (residues 36-44; MH+ 819.373) for WT thrombin at 100 mM NaCl (grey) and 300 mM NaCl (black), and for the W215A mutant at 100 mM NaCl (cyan) and 300 mM NaCl (blue). B) Structure of WT thrombin (wheat; PDB 1PPB) highlighting residues 16-23_CT (pink). The sidechains of Trp215_CT (blue), Phe227_CT (cyan), and the catalytic triad (black) are shown as sticks. Accelerated MD simulations identified 5 H-bonds (red-dotted lines) within WT thrombin (left) that broke during simulations of W215A. The transient structure observed during the W215A simulation (brown) overlaying the structure of WT thrombin (right). The side chains of His57_CT, Asp102_CT, Thr172_CT, Arg173_CT, Arg 187_CT, Asp 189_CT, Asp194_CT, Ser195_CT, Trp215_CT (blue), Glu217_CT, and Phe227_CT (cyan). The 140s_CT loop is hidden for clarity. The backbone of Ile16_CT (pink) is also shown as sticks. The corresponding side chains in the W215A structure, including Ala215_CT, are colored green and are shown as sticks.

Figure 5

Structure of WT thrombin (PDB 1PPB) highlighting residues 156-181_CT (residues 197-222; green), residues 208-215_CT (residues 256-263; red), and residues 216-228_CT (residues 264-276; brown). The sidechains of Trp215_CT (blue), Phe227_CT (cyan), and the catalytic triad (black) are shown as sticks. A) Uptake plots corresponding to residues 156-180_CT (residues 197-221; MH+ 2896.550), 161-170_CT (residues 202-211; MH+ 1155.619), and 161-181_CT (residues 202-222; MH+ 2490.296). B) Uptake plots corresponding to residues 208-227_CT (residues 256-275; MH+ 2152.943, MH+ 2191.954, and MH+ 2267.985 for W215A, F227A, and WT respectively), residues 212-227_CT (residues 260-275; MH+ 1673.760, MH+ 1715.806, and MH+ 1788.801 for W215A, W215I, and WT respectively), and residues 212-228_CT (residues 260-276; MH+ 1903.864 for F227V). The mutant residue, if present, is underlined in the peptide sequence shown. C) Uptake plots corresponding to residues 208-214_CT (residues 256-262; MH+ 797.386) and 208-215_CT (residues 256-263; MH+ 983.466). Deuterium incorporation over 5 min into the multiple peptides that cover these regions are shown for WT thrombin (grey) as well as the F227A (orange), F227V (red), W215A (cyan), and W215I (purple) mutants under experimental conditions of 100 mM NaCl.