Structure, Stability, and Interaction of the Fibrin(ogen) αC-Domains

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Abstract
Our recent study established the NMR structure of the recombinant bAα406−483 fragment corresponding to the NH2-terminal half of the bovine fibrinogen αC-domain and revealed that at increasing concentrations this fragment forms oligomers (self-associates). The major goals of the study presented here were to determine the structure and self-association of the full-length human fibrinogen αC-domains. To accomplish these goals, we prepared a recombinant human fragment, hAα425−503, homologous to bovine bAα406−483, and demonstrated using NMR, CD, and size-exclusion chromatography that its overall fold and ability to form oligomers are similar to those of bAα406−483. We also prepared recombinant hAα392−610 and bAα374−568 fragments corresponding to the full-length human and bovine αC-domains, respectively, and tested their structure, stability, and ability to self-associate. Size-exclusion chromatography revealed that both fragments form reversible oligomers in a concentration-dependent manner. Their oligomerization was confirmed in sedimentation equilibrium experiments, which also established the self-association affinities of these fragments and revealed that the addition of each monomer to assembling αC-oligomers substantially increases the stabilizing free energy. In agreement, unfolding experiments monitored by CD established that self-association of both fragments results in a significant increase in their thermal stability. Analysis of CD spectra of both fragments revealed that αC self-association results in an increase in the level of regular structure, implying that the COOH-terminal half of the αC-domain adopts an ordered conformation in αC-oligomers and that this domain contains two independently folded subdomains. Altogether, these data further clarify the structure of the human and bovine αC-domains and the molecular mechanism of their self-association into αC-polymers in fibrin.

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