According to the current hypothesis, in fibrinogen, the COOH-terminal portions of two Aα chains are folded into compact αC-domains that interact intramolecularly with each other and with the central region of the molecule; in fibrin, the αC-domains switch to an intermolecular interaction resulting in αC polymers. In agreement, our recent NMR study identified within the bovine fibrinogen Aα374-538 αC-domain fragment an ordered compact structure including a β-hairpin restricted at the base by a 423-453 disulfide linkage. To establish the complete structure of the αC-domain and to further test the hypothesis, we expressed a shorter αC-fragment, Aα406-483, and performed detailed analysis of its structure, stability, and interactions. NMR experiments on the Aα406-483 fragment identified a second loose β-hairpin formed by residues 459-476, yielding a structure consisting of an intrinsically unstable mixed parallel/anti-parallel β-sheet. Size-exclusion chromatography and sedimentation velocity experiments revealed that the Aα406-483 fragment forms soluble oligomers whose fraction increases with increasing concentration. This was confirmed by sedimentation equilibrium analysis, which also revealed that the addition of each monomer to an assembling αC oligomer substantially increases its stabilizing free energy. In agreement, unfolding experiments monitored by CD established that oligomerization of Aα406-483 results in increased thermal stability. Altogether, these experiments establish the complete NMR solution structure of the Aα406-483 αC-domain fragment, provide direct evidence for the intra-and intermolecular interactions between the αC-domains, and confirm that these interactions are thermodynamically driven.Fibrinogen is a multidomain plasma protein whose major function is to form fibrin clots that prevent the loss of blood upon vascular injury. In addition to its prominent role in haemostasis, fibrinogen also contributes to wound healing and participates in a number of other physiological and pathological processes through the interaction of its multiple regions/ domains with various proteins and cell types. The three-dimensional structure of most of these regions has been established by crystallographic studies of proteolytically derived and recombinant fragments of human and bovine fibrinogen (1-4). The crystal structures of a proteolytically truncated bovine fibrinogen and intact chicken fibrinogen have also been solved † This work was supported by National Institutes of Health Grant HL-56051 to L.M., American Heart Association, Mid-Atlantic Affiliate Grant-in-Aid 055527U to R.R.H., and by the Intramural Research Program of the NIH, National Heart, Lung, and Blood Institute to N.T. *To whom correspondence should be addressed. Leonid Medved. 402-3404. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 December 8. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript (5,6). However, the COOH-terminal regions of two fibrin(ogen) Aα ch...
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 present study 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-domain, 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 of their thermal stability. Analysis of CD spectra of both fragments revealed that αC self-association results in the increase of regular structures implying that the COOH-terminal half of the αC-domain adopts ordered conformation in αC-oligomers and that this domain contains two independently folded sub-domains. 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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