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

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 prodomain 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 prodomain and implies a possible mechanism for regulation of TAFI intrinsic proteolytic activity in vivo.Pancreatic carboxypeptidase A1 is the archetype of the M14 metallocarboxypeptidase (MCP) 3 family (1), also referred to as the funnelin tribe of metallopeptidases (2). The members of this family are subdivided into A/B or N/E MCPs on the basis of primary structure and structural homologies (3). They are also classified according to their substrate preference for C-terminal hydrophobic (A-type) or basic (B-type) amino acid residues. Thrombin-activatable fibrinolysis inhibitor (TAFI) (EC 3.4.17.20; UniProt Q96IY4) is a paralogue of pancreatic procarboxypeptidase B1 (pro-CPB1), pro-carboxypeptidases A1 (pro-CPA1), and A2 (pro-CPA2) and is also known as plasma pro-carboxypeptidase B2, R, or U (4 -9). Similarly to other B-type MCPs, TAFI possesses an N-terminal pro-domain that functions as a gatekeeper preventing substrate access to a preformed active site (for a review, see Refs. 2, 3). In human and porcine pancreatic pro-CPB1, access to the active site is completely shielded, resulting in no detectable intrinsic enzymatic activity of the zymogen (10). This is in contrast to human pro-CPA1 and pro-CPA2, which allow cleavage of small peptide substrates (11). Equally, pro-carboxypeptidase B from Pacific dogfish and African lungfish exhibit intrinsic activity toward small substrate molecules (12, 13). Likewise, TAFI interferes with carboxypetidase N assays (14), and is capable of cleaving larger peptide substrates due to its low but continuous carboxypeptidase activity...

Structure‐function relationships in thrombin‐activatable fibrinolysis inhibitor

“…Furthermore, the structure of bovine TAFI in complex with TCI revealed that the activation peptide in bovine TAFI is flexible enough to allow binding to TCI. It is not known if this is also true for human TAFI .The structures of human TAFIa in complex with TCI , and human TAFI‐YQ (H333Y/H335Q) in complex with the experimental inhibitor ‘compound 5’ , provided new insights into the molecular mechanism of TAFIa inactivation and showed that the crystal structure of TAFI can be used to design TAFIa inhibitors. Recently, three human TAFI structures in complex with TAFI activation‐inhibiting nanobodies were presented at the 2015 ISTH meeting by Zhou et al .…”

Structure of Aedes aegypti carboxypeptidase B1‐inhibitor complex uncover the disparity between mosquito and non‐mosquito insect carboxypeptidase inhibition mechanism

“…Apart from PCI, other well‐studied inhibitors include Lycium barbarum carboxypeptidase inhibitor (WCI), 30 the tick carboxypeptidase inhibitor (TCI), 22 and the leech carboxypeptidase inhibitor (LCI) 31 . WCI is the closest to PCI in terms of size (4 kDa) and amino acid residue conservation (64% identity; Figure S3).…”

Structure of Aedes aegypti procarboxypeptidase B1 and its binding with Dengue virus for controlling infection

“…Currently, there is no function ascribed to this cysteine residue. Moreover, CPBAe1 displays two of the three cis-peptide bonds (Ser 193 –Tyr 194 and Pro 201 –Trp 202 ) present in mammalian MCPs ( Figs 2A and S2 ) ( 10 , 11 , 20 , 23 , 25 ).…”