Dataset Information

Probing prothrombin structure by limited proteolysis.

ABSTRACT: Prothrombin, or coagulation factor II, is a multidomain zymogen precursor of thrombin that undergoes an allosteric equilibrium between two alternative conformations, open and closed, that react differently with the physiological activator prothrombinase. Specifically, the dominant closed form promotes cleavage at R320 and initiates activation along the meizothrombin pathway, whilst the open form promotes cleavage at R271 and initiates activation along the alternative prethrombin-2 pathway. Here we report how key structural features of prothrombin can be monitored by limited proteolysis with chymotrypsin that attacks W468 in the flexible autolysis loop of the protease domain in the open but not the closed form. Perturbation of prothrombin by selective removal of its constituent Gla domain, kringles and linkers reveals their long-range communication and supports a scenario where stabilization of the open form switches the pathway of activation from meizothrombin to prethrombin-2. We also identify R296 in the A chain of the protease domain as a critical link between the allosteric open-closed equilibrium and exposure of the sites of cleavage at R271 and R320. These findings reveal important new details on the molecular basis of prothrombin function.

SUBMITTER: Acquasaliente L

PROVIDER: S-EPMC6467981 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Probing prothrombin structure by limited proteolysis.

Acquasaliente Laura L Pelc Leslie A LA Di Cera Enrico E

Scientific reports 20190416 1

Prothrombin, or coagulation factor II, is a multidomain zymogen precursor of thrombin that undergoes an allosteric equilibrium between two alternative conformations, open and closed, that react differently with the physiological activator prothrombinase. Specifically, the dominant closed form promotes cleavage at R320 and initiates activation along the meizothrombin pathway, whilst the open form promotes cleavage at R271 and initiates activation along the alternative prethrombin-2 pathway. Here ...[more]

PMID: 30992526

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Project description:BACKGROUND: MTMDAT is a program designed to facilitate analysis of mass spectrometry data of proteins and biomolecular complexes that are probed structurally by limited proteolysis. This approach can provide information about stable fragments of multidomain proteins, yield tertiary and quaternary structure data, and help determine the origin of stability changes at the amino acid residue level. Here, we introduce a pipeline between MTMDAT and HADDOCK, that facilitates protein-protein complex structure probing in a high-throughput and highly automated fashion. RESULTS: A new feature of MTMDAT allows for the direct identification of residues that are involved in complex formation by comparing the mass spectra of bound and unbound proteins after proteolysis. If 3D structures of the unbound components are available, this data can be used to define restraints for data-driven docking to calculate a model of the complex. We describe here a new implementation of MTMDAT, which includes a pipeline to the data-driven docking program HADDOCK, thus streamlining the entire procedure. This addition, together with usability improvements in MTMDAT, enables high-throughput modeling of protein complexes from mass spectrometry data. The algorithm has been validated by using the protein-protein interaction between the ubiquitin-binding domain of proteasome component Rpn13 and ubiquitin. The resulting structural model, based on restraints extracted by MTMDAT from limited proteolysis and modeled by HADDOCK, was compared to the published NMR structure, which relied on twelve unambiguous intermolecular NOE interactions. The MTMDAT-HADDOCK structure was of similar quality to structures generated using only chemical shift perturbation data derived by NMR titration experiments. CONCLUSIONS: The new MTMDAT-HADDOCK pipeline enables direct high-throughput modeling of protein complexes from mass spectrometry data. MTMDAT-HADDOCK can be downloaded from http://www.ifm.liu.se/chemistry/molbiotech/maria_sunnerhagens_group/mtmdat/together with the manual and example files. The program is free for academic/non-commercial purposes.

Dataset Information

Probing prothrombin structure by limited proteolysis.

Publications

Probing prothrombin structure by limited proteolysis.

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