PDBsum entry 6v5t

Hydrolase

PDB id: 6v5t

Contents

Protein chain

290 a.a.

Ligands

GOL

SO4

Waters ×103

PDB id:

6v5t

Name:

Hydrolase

Title:

Crystal structure of human prethrombin-2 with tryptophans replaced by 5-f-tryptophan

Structure:

Prothrombin. Chain: e. Fragment: light and heavy chain, residues 333-622. Synonym: coagulation factor ii. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: f2. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.10Å R-factor: 0.180 R-free: 0.230

Authors:

E.A.Ruben,Z.Chen,E.Di Cera

Key ref:

E.A.Ruben et al. (2020). ¹⁹F NMR reveals the conformational properties of free thrombin and its zymogen precursor prethrombin-2. J Biol Chem, 295, 8227-8235. PubMed id: 32358061 DOI: 10.1074/jbc.RA120.013419

Date:

04-Dec-19 Release date: 13-May-20

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 290 a.a.*

* PDB and UniProt seqs differ at 9 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.4.21.5 - thrombin.
• Reaction: Preferential cleavage: Arg-|-Gly; activates fibrinogen to fibrin and releases fibrinopeptide A and B.

DOI no: 10.1074/jbc.RA120.013419

J Biol Chem 295:8227-8235 (2020)

PubMed id: 32358061

¹⁹F NMR reveals the conformational properties of free thrombin and its zymogen precursor prethrombin-2.

E.A.Ruben, P.S.Gandhi, Z.Chen, S.K.Koester, G.T.DeKoster, C.Frieden, E.Di Cera.

ABSTRACT

The conformational properties of trypsin-like proteases and their zymogen forms remain controversial because of a lack of sufficient information on their free forms. Specifically, it is unclear whether the free protease is zymogen-like and shifts to its mature form upon a ligand-induced fit or exists in multiple conformations in equilibrium from which the ligand selects the optimal fit via conformational selection. Here we report the results of ¹⁹F NMR measurements that reveal the conformational properties of a protease and its zymogen precursor in the free form. Using the trypsin-like, clotting protease thrombin as a relevant model system, we show that its conformation is quite different from that of its direct zymogen precursor prethrombin-2 and more similar to that of its fully active Na⁺-bound form. The results cast doubts on recent hypotheses that free thrombin is zymogen-like and transitions to protease-like forms upon ligand binding. Rather, they validate the scenario emerged from previous findings of X-ray crystallography and rapid kinetics supporting a pre-existing equilibrium between open (E) and closed (E*) forms of the active site. In this scenario, prethrombin-2 is more dynamic and exists predominantly in the E* form, whereas thrombin is more rigid and exists predominantly in the E form. Ligand binding to thrombin takes place exclusively in the E form without significant changes in the overall conformation. In summary, these results disclose the structural architecture of the free forms of thrombin and prethrombin-2, consistent with an E*-E equilibrium and providing no evidence that free thrombin is zymogen-like.