Crystal Structure of Prothrombin Reveals Conformational Flexibility and Mechanism of Activation

Abstract

Background: The x-ray structure and mechanism of activation of prothrombin remain elusive. Results: X-ray and solution studies document conformation flexibility of prothrombin. The two sites of cleavage at Arg-271 and Arg-320 have distinct solvent accessibility. Conclusion: Burial of Arg-320 prevents prothrombin autoactivation and directs prothrombinase to cleave at Arg-271 first. Significance: A structure-based mechanism of prothrombin activation emerges.

Histone H4 Promotes Prothrombin Autoactivation

“…In the absence of histone H4, the slowest lifetime of 603 s refers to the decay of the donor-only control, the short lifetime of 28 Ϯ 3 s with 45% population has a distance between Ser-101 in kringle-1 and Ser-210 in kringle-2 of Յ34 Å, and the long lifetime of 272 Ϯ 8 s with 55% population has a distance of 54 Ϯ 2 Å. These results are practically identical to those reported recently (5). Under saturating concentrations of histone H4 the lifetime of the collapsed conformation remains unchanged, but a new lifetime of 170 Ϯ 5 s corresponding to a distance between the probes of 47 Ϯ 2 Å replaces the one pertaining to the fully extended conformation.…”

“…The recent crystal structure of prothrombin along with LRET measurements have revealed that kringle-1 moves similar to a dumbbell relative to the rest of the zymogen due to the extreme flexibility of the linker connecting the two kringles (5). As a result, prothrombin assumes two alternative conformations in solution: a fully extended conformation with a population of 55% where Ser-101 in kringle-1 and Ser-210 in kringle-2 are separated by a distance of 54 Ϯ 2 Å, and a collapsed conformation with a population of 45% where Ser-101 in kringle-1 and Ser-210 in kringle-2 are separated by a distance of Յ34 Å or are at least 20 Å closer than in the fully extended conformation (5).…”

“…Semilog plot of lifetime data for the LRET donor-acceptor pair conjugated to residues 101 in kringle-1 and 210 in kringle-2 of the full-length prothrombin mutant S101C/S210C (150 nM) in the absence (A) or presence (B) of 0.9 M histone H4. The donor quenching (red dots) and acceptor sensitization (gray dots) curves of prothrombin both fit to a triple-exponential decay (green line) and two populations, collapsed and fully extended (5). Decomposition of the triple exponential fit into its individual components (dotted lines) reveals a short relaxation with a lifetime 1 ϭ 28 Ϯ 3 s associated with the collapsed conformation and a longer relaxation with 2 ϭ 272 Ϯ 8 s associated with the fully extended conformation.…”

“…Histones are positively charged and may interact non-specifically with the surface of prothrombin that is mostly negatively charged, as recently revealed by the x-ray crystal structure (5). Evidence of a direct interaction of histone H4 with prothrombin comes from fluorescence titrations using the prothrombin mutant S101C/S210C engineered recently for quantitative LRET measurements (5). The mutant labeled with the dye Alexa Fluor 647 at both Cys residues was used first for direct fluorescence titrations (Fig.…”

“…Fluorescence Titration Studies-The prothrombin mutant S101C/S210C was labeled with Alexa Fluor 647 after selective reduction of the engineered cysteines as described elsewhere (5). The labeled protein was separated from the unreacted dye and aggregates by size exclusion chromatography.…”