Role of the β-Strand Insert in the Central Domain of the Fibrinogen γ-Module

Abstract

The crystal structure of the fibrinogen gamma-module (residues gamma143-411) [Yee, V. C., et al. (1997) Structure 5, 125-138] revealed an unusual feature. Namely, residues gamma381-390 in the functionally important COOH-terminal region form a beta-strand that is inserted into an antiparallel beta-sheet of the central domain (gamma192-286), while the rest (gamma393-411) seems to be flexible. To clarify the structural and functional importance of this beta-strand insert, we analyzed the folding status of the plasmin-derived fibrinogen fragment D(3) and several truncated variants of the gamma-module expressed in Escherichia coli. It was found that D(3), in which most of the COOH-terminal domain of the gamma-module (gamma287-379) is removed proteolytically, retains a gamma374-405 peptide that seems to be associated noncovalently with the bulk of the molecule via its beta-strand insert region. A study of the denaturation-renaturation process of D(3) suggested that without this peptide its truncated gamma-module remains folded but is destabilized. This was confirmed directly with the truncated recombinant variants of the gamma-module, including residues gamma148-392, gamma148-373, and gamma148-286. They all were folded, but those devoid of the beta-strand insert were destabilized. The results indicate that although the beta-strand insert contributes to the stabilization of the gamma-module, it can be removed without destroying the compact structure of the latter. On the basis of this finding and some other observations, we propose a mechanism for the function-related conformational changes in the fibrin(ogen) gamma-modules.

Mechanism of Fibrin(ogen) Forced Unfolding

“…Detachment of the C -terminal β strand and its structural consequences observed in simulations confirm the “pull-out hypothesis” (Yakovlev et al, 2000a; Yakovlev et al, 2001), stating that the β strand (residues γ380–392) inserted into the central domain of the γ-nodule, can be removed (“pulled-out”) without destroying its compact structure; yet, such structure without the β strand insert is significantly destabilized. Our results show that the applied force first pulls out the β strand, after which the γ-nodule becomes unstable and falls apart (type 1 transition).…”

The molecular mechanisms of congenital hypofibrinogenaemia

“…Based on the position of the g375 residue and the similarity of the b sheet to that of antitrypsin and other serpins, the hypofibrinogenaemia might be a result of domain destabilization, leading to the intermolecular insertion of strand 2. Interestingly, Yakovlev et al [39] have demonstrated that strand 2 of the gD module can be pulled out of the sheet without destroying the compact structure of the domain, lending support to our suggestion that the new Trp residue might allow intermolecular insertion of this strand. An interesting feature of the fibrinogen Aguadilla and Brescia families is the variation in the severity of the liver disease.…”

Structural Basis of Interfacial Flexibility in Fibrin Oligomers

“…This suggests that the transverse γ-γ crosslinking is not feasible without substantial elongation of the γ-nodules, which is potentially possible provided that elongation of the γ chain due to the C-terminal β strand pull out occurs. According to this “pull-out” hypothesis, the C-terminal β strand of the γ chain (residues γLys381-Asn390) can be drawn out of the γ-nodule without much perturbation of its native fold (Yakovlev et al, 2000). Consequently, the β strand can extend outwards, increasing the reach of the γ-γ crosslinks.…”