Identification of an Ordered Compact Structure within the Recombinant Bovine Fibrinogen alphaC-Domain Fragment by NMR.

Fibrinogen is a 340-kDa plasma protein that plays a prominent role in hemostasis, thrombosis, wound healing, inflammation, angiogenesis, and tumorigenesis. This multitude of fibrinogen functions is connected with its complex multidomain structure that provides its homophilic interaction upon fibrin assembly and its interactions with numerous proteins and cell types in other processes. Fibrinogen consists of two identical disulfide-linked subunits each of which is composed of three non-identical polypeptide chains, Aα, Bβ, and γ (1; 2). These chains assemble to form at least 20 distinct domains grouped into several major structural regions (3–6). The disulfide-linked NH2-terminal portions of all six chains form the central E region, the COOH-terminal portions of the Bβ and γ chains and the middle portion of the Aα chain form two distal D regions, and the remaining COOH-terminal two-thirds of two Aα chains form two αC regions. The D-E-D regions account for three nodules observed in numerous electron microscopy studies (7–10), while the fourth nodule observed in some molecules near the central nodule E corresponds to the two interacting αC regions, often referred to as αC-domains (9; 11; 12) (Figure 1A). Figure 1 Schematic representation of the interacting αC-domains in fibrinogen and their dissociation upon its conversion to fibrin. Panel A shows fibrinogen molecule consisting of the D-E-D nodules linked by coiled coil connectors and two αC-domains ... Activation of the coagulation cascade leads to the generation of thrombin which converts fibrinogen into fibrin by cleavage of fibrinopeptides A and B (FPA and FPB1, respectively) from its central region. This cleavage results in spontaneous polymerization of individual fibrin molecules into double-stranded protofibrils (Figure 1B) which then aggregate laterally to produce thicker fibers composing fibrin clots (1; 10). According to our current view, in fibrinogen the αC-domains interact intramolecularly with each other and with the central E region, most probably via its FPB, while upon fibrin assembly they dissociate and switch from intra- to intermolecular interaction thus promoting lateral aggregation of protofibrils (12–16). The functional role of the αC-domains is not restricted to their participation in fibrin assembly. They are involved in controlling activation of plasma transglutaminase factor XIII (17), which subsequently cross-links fibrin clots to increase their mechanical stability. The human fibrinogen αC-domains promote cell adhesion via their Aα572-574 RGD sequence2 and via bound fibronectin (18–21), and may contribute to the development of atherothrombosis (22). They are also involved in regulation of fibrinolysis via binding of plasminogen, its activator tPA, its inhibitor α2-antiplasmin, and via modulating the structure of fibrin clots (16; 23–25). The fibrinogen D and E regions can be separated by limited proteolysis resulting in the D and E fragments, respectively, while the αC regions are vulnerable to proteolytic enzymes, which degrade them into smaller pieces (1; 2). The success in crystallization of these fragments resulted in determination of the three-dimensional structure of individual fibrinogen regions and promoted X-ray studies of the entire molecule. Particularly, the X-ray structures have been established for the human fibrinogen D fragment (26) and for the bovine and human fibrinogen E fragments (6; 27). Further, a low resolution structure of most of the fibrinogen molecule was obtained from a crystallographic study of a proteolytically truncated bovine fibrinogen (28), followed by a higher resolution structure of intact chicken fibrinogen (29). Altogether, these studies have established the three-dimensional structure of more than two-thirds of the molecule including the complete D regions and most of the E region; that of the αC-domains remains to be determined. In the absence of the three-dimensional structure of the αC regions, the current view on their structural organization is based on a number of studies performed in different laboratories mainly by electron microscopy, differential scanning calorimetry (DSC), and sequence analysis (3; 4; 9; 11; 15). These studies revealed that in fibrin(ogen) each αC region forms a compact globular entity, αC-domain, attached to the bulk of the molecule with a flexible connector (15). The full-length αC region and its halves corresponding to αC-connector and the αC-domain have been prepared by recombinant technique (24; 30). A detailed analysis of their folding status by several methods has confirmed that the αC-domain contain a compact cooperative structure while the connector is flexible (30). At the same time, there is also an alternative view suggesting that the αC-domains represent “free-swimming appendages” devoid of any ordered structure (1; 8). This view was reinforced by an X-ray study of intact chicken fibrinogen, in which the αC-domains were not observed in electron density maps (29). Thus, the question whether these domains contain compact ordered structure is still debated (31; 32). In this study, we report the NMR solution structure of the recombinant bovine fibrinogen αC-domain fragment including residues Aα374-5383, which reveals a type I′ β-hairpin and a collapsed hydrophobic cluster, and discuss a possible role of such a structure in the previously observed intra- and intermolecular interactions.