# Allosteric equilibria of thrombin and its precursors

> **NIH NIH R01** · SAINT LOUIS UNIVERSITY · 2022 · $378,750

## Abstract

The proposed research project focuses a recently uncovered, paradigm-shifting structure-function link relevant
to the entire family of trypsin-like enzymes to which thrombin belongs. A pre-existing, allosteric equilibrium
between ensembles of closed (E*) and open (E) conformations of the active site influences the level of activity
and mechanism of binding in the protease. The equilibrium also exists in the zymogen and explains the
spontaneous autoactivation observed with several proteins involved in blood coagulation, immune response,
fibrinolysis and development. Observational evidence of the E*-E equilibrium comes from a large body of
structures currently deposited in the Protein Data Bank. Additional independent evidence comes from rapid
kinetics measurements of ligand binding to the active site of protease and zymogen that support conformational
selection as a general mechanism of recognition in the trypsin fold. Studies under specific aim 1 will test the
hypothesis that protease and zymogen undergo the E*-E equilibrium in solution and that the relative distribution
of E* and E influences activity in the protease and the mechanism of activation in the zymogen. A significant
component of these studies will involve pioneering NMR (2D and 19F) measurements of thrombin and
prethrombin-2 with the goal of elucidating, for the first time, the structure and dynamics of their free
conformation(s) in solution. We will focus on the likely structural determinants of the E*-E equilibrium and critical
residues that decorate the entrance to the active site region in the 215-217 segment (W215, G216, E217), the
60-loop (W60d), the autolysis loop (W148) and the 190-193 corridor (E192). The functional role of these residues
will be tested by rapid kinetics measurements of ligand binding to the active site of wild-type and mutants of
thrombin and its direct zymogen precursor prethrombin-2. These studies will advance our understanding of a
basic structure-function link of the trypsin fold and will provide background for studies to be carried out under
specific aim 2. Members of the trypsin family of proteases, to which thrombin belongs, are expressed as inactive
zymogens and irreversibly converted to the mature protease by proteolytic cleavage at R15 in the activation
domain. The cleavage generates a new N-terminus that inserts into the protein core and H-bonds to the side
chain of residue D194. Elucidating how the Huber-Bode mechanism of zymogen activation described above is
linked to the allosteric E*-E equilibrium will be center stage in our investigation. We will perturb the critical I16-
D194 H-bond with several substitutions that weaken or abolish the interaction. Each mutant will be studied by
rapid kinetics to directly measure the E*-E distribution in solution. Additionally, key mutants such as D914A will
be characterized structurally for the first time by X-ray and NMR to complement studies of prethrombin-2 and
thrombin under specific aim 1. Development...

## Key facts

- **NIH application ID:** 10429976
- **Project number:** 5R01HL147821-04
- **Recipient organization:** SAINT LOUIS UNIVERSITY
- **Principal Investigator:** Enrico Di Cera
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $378,750
- **Award type:** 5
- **Project period:** 2019-06-01 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10429976

## Citation

> US National Institutes of Health, RePORTER application 10429976, Allosteric equilibria of thrombin and its precursors (5R01HL147821-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10429976. Licensed CC0.

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