Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation

Abstract

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-metallocarboxypeptidase that can be proteolytically activated (TAFIa). TAFIa is unique among carboxypeptidases in that it spontaneously inactivates with a short half-life, a property that is crucial for its role in controlling blood clot lysis. We studied the intrinsic instability of TAFIa by solving crystal structures of TAFI, a TAFI inhibitor (GEMSA) complex and a quadruple TAFI mutant (70-fold more stable active enzyme). The crystal structures show that TAFIa stability is directly related to the dynamics of a 55-residue segment (residues 296-350) that includes residues of the active site wall. Dynamics of this flap are markedly reduced by the inhibitor GEMSA, a known stabilizer of TAFIa, and stabilizing mutations. Our data provide the structural basis for a model of TAFI auto-regulation: in zymogen TAFI the dynamic flap is stabilized by interactions with the activation peptide. Release of the activation peptide increases dynamic flap mobility and in time this leads to conformational changes that disrupt the catalytic site and expose a cryptic thrombincleavage site present at Arg302. This represents a novel mechanism of enzyme control that enables TAFI to regulate its activity in plasma in the absence of specific inhibitors. (Blood. 2008;112: 2803-2809) Introduction TAFI 1,2 is a pro-metallocarboxypeptidase that links the coagulation and fibrinolytic systems. TAFI is activated by thrombin, the thrombin-thrombomodulin complex or plasmin. 3 Activated TAFI (TAFIa) inhibits plasmin-mediated blood clot lysis by removing C-terminal lysine residues from partially degraded fibrin that are required for positive feedback in tissue plasminogen-activator dependent plasmin generation. In addition, TAFIa has been implicated in modulation of the inflammatory response by inactivating bradykinin and the anaphylatoxins C3a and C5a. 4,5 Although it is a powerful antifibrinolytic agent, there are no known physiologic inhibitors of TAFIa. Instead, the half-life of TAFIa activity is regulated by its intrinsic instability. The inactivation rate, 5 to 10 minutes at 37°C, is highly temperature-dependent, suggesting that inactivation involves a large conformational change. 6 This is also suggested by the susceptibility of the inactive enzyme, TAFIai to proteolytic cleavage by thrombin at Arg302, a site that is cryptic in TAFI and TAFIa. 6,7 The stability of TAFIa is an important determinant for its antifibrinolytic potential because TAFIa inhibits fibrinolysis through a threshold-dependent mechanism. [8][9][10] Full-length TAFI consists of 401 amino acids divided into 2 domains: the first 92 amino acids form the activation peptide; the next 309 amino acids form the catalytic domain. The activation peptide restricts substrate access to the catalytic cleft in the zymogen. TAFI is activated through cleavage at Arg92, which releases the activation peptide.TAFI is highly homologous to the pancreatic procarboxypeptidases with 42% sequence identity to human procarboxypeptidase B (pro...

Low thrombin activatable fibrinolysis inhibitor activity levels are associated with an increased risk of a first myocardial infarction in men

“…The TAFI crystal structure shows that the amino acid at position 147 is solvent exposed. 35 Thus, theoretically, this residue, which is located close to the Arg145 that is known to be one of the essential residues for substrate peptide anchoring, 36 might contribute to ligand binding and the 505G/A polymorphism might affect this process. Although the 505G/A polymorphism does not affect TAFI fibrinolytic activity, 34 the effect of the polymorphism on the catalytic activity towards other molecular substrates such as bradykinin and anaphylatoxins has, to our knowledge, not yet been tested.…”

Plasma carboxypeptidase B downregulates inflammatory responses in autoimmune arthritis

“…No physiological inhibitors of CPB have been identified, and inactivation of CPB is achieved only through spontaneous structural destabilization of CPB after release of its activation peptide (53). Therefore, the stability of CPB is an important determinant of its functional activity.…”

Substrate Specificity of Human Carboxypeptidase A6

“…In recent years more has been learned about the enzymatic specificity, gene expression, and physiological roles of other members of this subfamily. X-ray crystal structures have been solved for five of the nine members of this subfamily (3,(12)(13)(14)(15)(16)(17). CPA2 and CPB1 are pancreatic metallocarboxypeptidases that function in the digestion of food (18).…”