Flexibility of the Thrombin-activatable Fibrinolysis Inhibitor Pro-domain Enables Productive Binding of Protein Substrates

Joan L. Arolas,Christine Schar,Steen V. Petersen,F. Xavier Gomis-Rüth,Laura Sanglas,Daniel Otzen,Zuzana Valnickova,Francesc X. Aviles,Jan J. Enghild

doi:10.1074/jbc.m110.150342

Abstract

We have previously reported that thrombin-activatable fibrinolysis inhibitor (TAFI) exhibits intrinsic proteolytic activity toward large peptides. The structural basis for this observation was clarified by the crystal structures of human and bovine TAFI. These structures evinced a significant rotation of the pro-domain away from the catalytic moiety when compared with other pro-carboxypeptidases, thus enabling access of large peptide substrates to the active site cleft. Here, we further investigated the flexible nature of the pro-domain and demonstrated that TAFI forms productive complexes with protein carboxypeptidase inhibitors from potato, leech, and tick (PCI, LCI, and TCI, respectively). We determined the crystal structure of the bovine TAFI-TCI complex, revealing that the pro-domain was completely displaced from the position observed in the TAFI structure. It protruded into the bulk solvent and was disordered, whereas TCI occupied the position previously held by the pro-domain. The authentic nature of the presently studied TAFI-inhibitor complexes was supported by the trimming of the C-terminal residues from the three inhibitors upon complex formation. This finding suggests that the inhibitors interact with the active site of TAFI in a substrate-like manner. Taken together, these data show for the first time that TAFI is able to form a bona fide complex with protein carboxypeptidase inhibitors. This underlines the unusually flexible nature of the pro-domain and implies a possible mechanism for regulation of TAFI intrinsic proteolytic activity in vivo.

Highlights

The 92-residue pro-domain of thrombin-activatable fibrinolysis inhibitor (TAFI) contains four N-linked glycan structures [18]
Protein inhibitors of MCPs described to date include: potato carboxypeptidase inhibitor (PCI), leech carboxypeptidase inhibitor (LCI), tick carboxypeptidase inhibitor (TCI), Ascaris carboxypeptidase inhibitor (ACI), and latexin (46 –50)
This was substantiated by the crystal structure of bovine TAFI in complex with TCI, which revealed that the prodomain was relocated from its original position, no longer shielding the access to the active site

Summary

EXPERIMENTAL PROCEDURES

Reagents and Proteins—Hippuryl-Arg, 1,10-phenantroline, cyanuric chloride, and 1,4-dioxane were purchased from Sigma. Preparation of TAFI-inhibitor Complexes—TAFI (2.5 ␮g) was incubated with 1 ␮g of PCI, LCI, or TCI for 1 h at 22 °C in a final volume of 25 ␮l of 20 mM Tris-HCl, 150 mM NaCl, pH 7.5. Purified human TAFI (0.1 mg) in 50 mM Tris-HCl, 150 mM NaCl, pH 7.5 (binding buffer) was slowly applied onto each column at 22 °C. 1 ml of pooled human plasma (from 10 individuals) diluted 5 times in binding buffer containing 5 mM EDTA was applied onto the TCI column in a separate experiment. Analysis of TAFI-Protein Inhibitor Complexes by Matrix-assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry (MALDI-TOF MS)—PCI, LCI, or TCI were added to 10 ␮l of human TAFI (0.1 mg/ml) dissolved in 50 mM Tris-HCl, 150 mM NaCl, pH 7.5, using a protease/inhibitor molar ratio of 1:1.

Binding of TAFI to Immobilized

RESULTS

Average multiplicity

Rmsd from target values

Findings

DISCUSSION