Characterizing the Tick Carboxypeptidase Inhibitor

Abstract

Tick carboxypeptidase inhibitor (TCI) is a small, disulfiderich protein that selectively inhibits metallocarboxypeptidases and strongly accelerates the fibrinolysis of blood clots. TCI consists of two domains that are structurally very similar, each containing three disulfide bonds arranged in an almost identical fashion. The oxidative folding and reductive unfolding pathways of TCI and its separated domains have been characterized by kinetic and structural analysis of the acid-trapped folding intermediates. TCI folding proceeds through a sequential formation of 1-, 2-, 3-, 4-, 5-, and 6-disulfide species to reach the native form. Folding intermediates of TCI comprise two predominant 3-disulfide species (named IIIa and IIIb) and a major 6-disulfide scrambled isomer (Xa) that consecutively accumulate along the reaction and are strongly prevented by the presence of protein disulfide isomerase. This study demonstrates that IIIa and IIIb are 3-disulfide species containing the native disulfide pairings of the N-and C-terminal domains of TCI, respectively, and explains why the two domains of TCI fold sequentially and independently. Also, we show that the reductive unfolding of TCI undergoes two main independent unfolding events through the formation of IIIa and IIIb intermediates. Together, the comparison of the folding, stability, and inhibitory activity of TCI with those of the isolated domains reveals the reasons behind the two-domain nature of this protein: both domains contribute to the specificity and high affinity of its double-headed binding to carboxypeptidases. The results obtained herein provide valuable information for the design of more potent and selective TCI molecules.The mechanism by which an unfolded protein achieves its native state is one of the most complex problems in structural biology. Understanding the sequence of folding events in proteins may not only help to predict protein structures from amino acid sequences but also provide invaluable information to a variety of related fields, such as protein design or protein misfolding associated with pathological diseases (1, 2). A number of studies concerning folding have been focused on small, disulfide-rich proteins, given that the chemistry of disulfide bond formation allows the trapping and subsequent characterization of the folding intermediates that accumulate, something difficult to attain with disulfide-free proteins (3, 4). In oxidative folding, reduced and denatured proteins are allowed to recover both its native disulfide bonds and native structures in the absence or presence of redox agents. Bovine pancreatic trypsin inhibitor, ribonuclease A, lysozyme, ␣-lactalbumin, and hirudin have been extensively investigated using this methodology (5-9). The heterogeneity and structures of the intermediates that occur during the process define their respective folding landscapes (10).Although most of the analyzed proteins comprise a small, single-domain fold containing three or four disulfide bonds, a great diversity of folding mechanisms is observ...

The NMR Structures of the Major Intermediates of the Two-domain Tick Carboxypeptidase Inhibitor Reveal Symmetry in Its Folding and Unfolding Pathways

“…of 1.8 Å for backbone atoms) and identical disulfide bond pattern, the two domains of TCI fold at very different rates and through distinct mechanisms (32). This behavior is reminiscent of the cases of BPTI and tick anticoagulant peptide, two small, single domain proteins with similar structures and disulfide bonds but with completely different folding pathways (49).…”

“…This is explained by the lower working pH, which causes reductive reactions to prevail over disulfide reshuffling, slowing down and hindering the conversion of IIIb into IIIa. Previous Stop/Go experiments demonstrated the fluctuation of IIIb into IIIa at alkaline pH (32). On the other hand, both intermediates reduce their three native disulfide bonds in a cooperative manner following an "all-or-none" mechanism, with IIIb resisting slightly higher concentrations of reducing agent than IIIa (data not shown).…”

“…The conformation of this short segment is defined only by local constraints and displays an increased flexibility as compared with the two protein domains. In addition, we have previously characterized the oxidative folding and reductive unfolding of TCI and its dissected domains (32). The comparative folding analysis of the whole protein and its individual domains suggested that the folding pathway of this two-domain protein results from the additive contribution of their individual N-and C-terminal domains, which appear to fold autonomously.…”

“…As previously shown (32), TCI refolds through the sequential formation of 1-, 2-, 3-, 4-, 5-, and 6-disulfide intermediates that finally render two products: the Xa scrambled isomer and the native form. This sequential oxidation of disulfide bonds is thought to take place autonomously for each domain, leading to the predominant accumulation of two major intermediates (IIIa and IIIb), which contain three native disulfide bonds in one domain and six free cysteines in the other one (32 . The reductive unfolding of TCI also seems to proceed autonomously through the formation of the IIIa and IIIb intermediates (Fig.…”

“…Each domain comprises three disulfide bonds of identical pattern that constrain the TCI structure contributing to its extremely high stability against temperature and denaturing agents. The oxidative folding and reductive unfolding pathways of TCI and its individual domains have been examined in a previous study by acid-trapping and further chromatographic analysis of the occurring intermediates (32). TCI folding proceeds through a sequential oxidation of cysteines that leads to the formation of a complex population of non-native 6-disulfide (scrambled) isomers.…”