Dataset Information

Residues W215, E217 and E192 control the allosteric E*-E equilibrium of thrombin.

ABSTRACT: A pre-existing, allosteric equilibrium between closed (E*) and open (E) conformations of the active site influences the level of activity in the trypsin fold and defines ligand binding according to the mechanism of conformational selection. Using the clotting protease thrombin as a model system, we investigate the molecular determinants of the E*-E equilibrium through rapid kinetics and X-ray structural biology. The equilibrium is controlled by three residues positioned around the active site. W215 on the 215-217 segment defining the west wall of the active site controls the rate of transition from E to E* through hydrophobic interaction with F227. E192 on the opposite 190-193 segment defining the east wall of the active site controls the rate of transition from E* to E through electrostatic repulsion of E217. The side chain of E217 acts as a lever that moves the entire 215-217 segment in the E*-E equilibrium. Removal of this side chain converts binding to the active site to a simple lock-and-key mechanism and freezes the conformation in a state intermediate between E* and E. These findings reveal a simple framework to understand the molecular basis of a key allosteric property of the trypsin fold.

SUBMITTER: Pelc LA

PROVIDER: S-EPMC6707225 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Publications

Residues W215, E217 and E192 control the allosteric E*-E equilibrium of thrombin.

Pelc Leslie A LA Koester Sarah K SK Chen Zhiwei Z Gistover Noah E NE Di Cera Enrico E

Scientific reports 20190823 1

A pre-existing, allosteric equilibrium between closed (E*) and open (E) conformations of the active site influences the level of activity in the trypsin fold and defines ligand binding according to the mechanism of conformational selection. Using the clotting protease thrombin as a model system, we investigate the molecular determinants of the E*-E equilibrium through rapid kinetics and X-ray structural biology. The equilibrium is controlled by three residues positioned around the active site. W ...[more]

PMID: 31444378

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Project description:Substrates containing a P3 aspartic residue are in general cleaved poorly by thrombin. This may be partly due to an unfavourable interaction between the P3 aspartate and Glu192 in the active site of thrombin. In Protein C activation and perhaps also thrombin receptor cleavage, binding of ligands at the anion-binding exosite of thrombin seems to improve the activity of thrombin with substrates containing a P3 aspartate. To investigate the importance of Glu192 and exosite-binding in modulating thrombin's interactions with a P3 aspartate, peptidyl chloromethanes based on the sequence of the thrombin receptor (containing a P3 aspartate) have been synthesized and the kinetics of their inactivation of alpha-thrombin and the mutant Glu192-->Gln determined. The values of the inactivation rate constant (ki) for the chloromethanes containing a P3 aspartate were about two-fold higher with the Glu192-->Gln mutant. A peptide based on the sequence of hirudin (rhir52 65), which binds to the anion-binding exosite of thrombin, was an allosteric modulator of the amidolytic activity of the Glu192-->Gln mutant; a 5-fold decrease in the K(m) value for the substrate D-Phe-pipecolyl-Arg-p-nitroanilide was observed in the presence of saturating concentrations of rhir52-65. This exosite-binding peptide also increased the ki values of chloromethanes containing a P3 aspartate with both alpha-thrombin and the Glu192-->Gln mutant. However, the increases in the ki values were greater with the Glu192-->Gln mutant (5-fold compared with 2-fold for alpha-thrombin). Thus exosite binding does not seem to mitigate putative unfavourable interactions between Glu192 and the P3 aspartate. Moreover, increases in the ki caused by exosite binding were not unique to chloromethanes containing a P3 aspartate; increases of the same magnitude were also observed when the P3 position was occupied by the favourable D-phenylalanine in place of the unfavourable aspartate. The results obtained were consistent with exosite binding's causing changes in the conformation of the S2 and/or S1 site of thrombin.

Dataset Information

Residues W215, E217 and E192 control the allosteric E*-E equilibrium of thrombin.

Publications

Residues W215, E217 and E192 control the allosteric E*-E equilibrium of thrombin.

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