Numerous data indicate that the αC domains are inactive in fibrinogen and adopt a physiologically active conformation only upon their polymerization in fibrin.6,8,12,20,22
Our previous study suggests that formation of αC polymers in fibrin occurs through self-association of the αC-domains,20
which are then covalently cross-linked to the αC-connectors of neighboring molecules by factor XIIIa.39
Thus, αC polymers are actually formed by fibrin αC regions that include the αC-domains and αC-connectors. Our recent study with the recombinant αC regions revealed that they self-associate in solution into highly ordered oligomers that mimic the structure and functional properties of fibrin αC polymers.25
In the present study, we tested the interaction between different portions of the αC regions, performed molecular modeling and site-directed mutagenesis of one of these portions, the N-terminal subdomain, and evaluated the size of fibrin α chain polymers to further clarify the structure of αC polymers in fibrin and molecular mechanism of their formation.
We have previously found that the N-terminal subdomains of the αC-domains interact with each other (self-associate).20,22
This finding has been confirmed in the present study using the optical trap-based force spectroscopy. Furthermore, the results of the present study clearly indicate that their C-terminal subdomains do not interact in the same manner, leaving the interaction between the N-terminal subdomains to be responsible for self-association of the αC-domains. We have also found that the αC-domain interacts with the αC-connector.24
The present study has revealed that this interaction occurs exclusively through the C-terminal subdomain. Thus, in fibrin these two subdomains carry out different functions: the N-terminal subdomains are responsible for self-association of the αC-domains while the C-terminal subdomains interact with the αC-connectors of neighboring αC-regions, thereby contributing to self-association of the αC regions and further reinforcement of αC polymers. Taking into account that the reactive Lys and Gln residues that are cross-linked by factor XIIIa were localized in the C-terminal subdomain and the αC-connector, respectively,39,44,45
the interaction between the C-terminal subdomains and αC-connectors may also provide the proper positioning of these residues to facilitate their covalent cross-linking by factor XIIIa.
The data presented in this study together with our previous data on the structure and interaction of the αC-domains and αC regions16,20,22–25,39
are summarized in , which represents schematically our current view on the structure of fibrin αC polymers and the mechanism of their formation. The model presented is based on the following findings: (i) self-association of the αC-domains occurs through the interaction between their N-terminal subdomains (present study and refs 20
); (ii) the C-terminal subdomains interact with the αC-connectors (present study); (iii) factor XIIIa covalently cross-links the C-terminal subdomains to the αC-connectors.39
Such interactions and cross-linking require antiparallel arrangement of the αC regions that are coming from the neighboring fibrin molecules. It should be noted that this two-dimensional model shows the established interactions between monomeric αC regions in αC polymers and their cross-linking; however, it does not reflect the exact spatial arrangement of individual subdomains in the polymers. A recent electron microscopy study of cross-linked αC region oligomers revealed that they have a width of 7.8 ± 0.9 nm, which is slightly less than twice the diameter of the monomeric αC region (4.5 ± 0.7 nm).25
The length of the N-terminal subdomain (4.5 nm)22
is also comparable with the diameter of the αC region monomer. Thus, to make a polymer with such a width, their N- and C-terminal subdomains should overlap in the real 3D structure.
Figure 9 Schematic presentation of fibrin αC polymers. Panel A shows monomeric αC regions (top), whose interaction through the N-terminal subdomains (denoted by “N”) results in formation of a polymer; the C-terminal subdomains (denoted (more ...)
Our DLS experiments revealed that the size of α chain polymers from cross-linked fibrin is restricted. The major fraction of α chain polymers includes species whose size varies from about 60 to 90 monomeric units. This suggests that this many αC regions may form linear αC polymers in native fibrin. Since the widths of αC oligomers is about twice the diameter of the monomeric αC region,25
as mentioned above, one can estimate the length of such αC polymers at ~130–200 nm. This length is much shorter than that of individual fibrin fibers, excluding a possibility that fiber length may restrict the size of αC polymers in fibrin. At the same time, one cannot exclude the likelihood that their size may be restricted by the length of the αC-connectors that allow individual αC-domains to move from the bulk of the molecules and cluster into polymers. This is shown schematically in . It should be noted that the amount of the observed larger polymers consisting of up to several hundred monomers should be relatively low because light scattering from them is significantly higher than that from smaller polymers of the major fraction.30,43
The larger polymers could be formed by cross-linking of smaller overlapped αC polymers that may come from neighboring fibers of fibrin network. This speculation is based on our previous finding that branched αC oligomer formed by the recombinant αC regions in solution may result from cross-linking of smaller oligomers to each other.25
As to the molecular mechanism of αC polymer formation, we have previously hypothesized that the interaction between the N-terminal subdomains upon their self-association may involve swapping of the β-hairpins between neighboring subdomains.22
In the present study, we tested this hypothesis using molecular modeling and site-directed mutagenesis. Molecular modeling of oligomers made of the bovine Aα406–483 fragment (N-terminal subdomain) confirmed that the hypothesized swapping can occur without any steric hindrance. It should be noted that among the two alternative models demonstrating possible β-hairpin swapping mechanism () the model shown in is more reasonable. Although this model may not reflect the real structure of the N-terminal subdomains in fibrin αC polymers, it demonstrates how β-hairpin swapping to form antiparallel β-sheet structures may occur without significant structural rearrangement in the monomeric units and thereby without significant energy toll. Furthermore, the width of such structure is 4.8 nm, i.e., only slightly higher than the length of the monomeric N-terminal subdomain. This leaves more space for accommodation of the C-terminal subdomains in the 7.8 nm wide αC polymer.25
Thus, our molecular modeling study supports the hypothesized mechanism of formation of antiparallel β-sheet polymers by β-hairpin swapping.
To test experimentally whether self-association of the N-terminal subdomains may occur through a β-hairpin swapping mechanism, we introduced an extra disulfide bond in between two neighboring β-hairpins of the Aα406–483 fragment (“disulfide lock”) by site-directed mutagenesis (). We expected that the newly formed disulfide should prevent β-hairpin swapping and thereby affect oligomerization of Aα406–483 mutant and its oligomerization-induced stability. Indeed, the experiments revealed that although this mutant had a tendency to form oligomers, the amount of such oligomers was lower than that formed by wild-type Aα406–483, and the kinetics of their formation was slower. Furthermore, while oligomerization of wild-type Aα406–483 resulted in a significant increase of its thermal stability, no such effect was observed with the mutant. These results can be interpreted based on the models of oligomers presented in . For all three models, oligomerization process is driven mainly by formation of hydrogen bond network between interacting antiparallel β-strands of the first and second β-hairpins of the neighboring monomeric units. Hence, oligomerization of the mutant fragment may proceed without β-hairpin swapping, resulting in parallel/antiparallel β-sheet oligomers structurally similar to those shown in ; however, the swapping should result in antiparallel β-sheet formation, as shown in , thereby increasing the stability of the final oligomers. Therefore, in our experiments, when β-hairpin swapping in Aα406–483 mutant was prevented by the “disulfide lock”, the monomers were still able to form oligomers, but this process was less efficient and the oligomers were not stabilized. Thus, the results of our experiments with Aα406–483 mutant are in agreement with the hypothesized β-hairpin swapping mechanism, albeit they do not provide direct evidence for it.
Although the three-dimensional structure of the C-terminal subdomain is not established yet, our previous study revealed that it acquires mainly regular β-conformation in oligomers formed by the αC-domains.20
Moreover, on the basis of the CD analysis of oligomers formed by the recombinant αC region, we previously hypothesized that a portion of the αC-connectors in αC polymers may also acquire β-strand conformation.25
This hypothesis is in agreement with the observed significant increase of β-sheet content upon fibrin polymerization, which was suggested to occur upon interaction between the α chains forming αC regions.46,47
In this connection, it should be noted that fibronectin Fib-1 region, which includes five finger domains, interacts with bacterial fibronectin binding proteins (FnBPs) through their unstructured regions of tandem repeats, and this interaction occurs through a so-called tandem β-zipper mechanism.48–52
This mechanism includes formation of additional antiparallel β-strands by the unstructured regions of FnBPs that interact with the edges of the triple-stranded β-sheets of adjacent finger domains of Fib-1.48,49
Since the C-terminal subdomain of the αC-domain contains β-sheets20
and the αC-connector of the αC region is unstructured16,23
and contain several tandem repeats,53
one can hypothesize that the interaction between them occurs through the same tandem β-zipper mechanism. Namely, upon this interaction, a portion of the αC-connector acquires β-strand conformation and this β-strand(s) forms hydrogen bonds with the antiparallel β-strand(s) of the C-terminal subdomain, as shown schematically in , bottom.
In summary, our binding study revealed that self-association of the αC-domains into αC polymers occurs through their N-terminal subdomains while their C-terminal subdomains interact with the αC connectors, reinforcing the structure of the polymers and providing the proper orientation of their reactive residues for efficient cross-linking by factor XIIIa. The results of our molecular modeling and site-directed mutagenesis support in part the previously proposed hypothesis that interaction between the N-terminal subdomains occurs through the β-hairpin swapping mechanism. On the basis of the results of our present and previous studies, we propose a model that describes the structure of fibrin αC polymers and aspects of the molecular mechanism of their formation.