Sergei V. Litvinovich

Cryptic Self-association Sites in Type III Modules of Fibronectin

The first type III module of fibronectin (Fn) contains a cryptic site that binds Fn and its N-terminal 29 kDa fragment and is thought to be important for fibril formation (Morla, A., Zhang, Z., and Ruoslahti, E. (1994) Nature 367, 193-196; Hocking, D. C., Sottile, J., and McKeown-Longo, P. J. (1994) J. Biol. Chem. 269, 19183-19191). A synthetic 31-mer peptide (NAPQ … TIPG) derived from the middle of domain III1 was also shown to bind Fn, but the site of its interaction was not determined (Morla, A., and Ruoslahti, E. (1992) J. Cell Biol. 118, 421-429). By affinity chromatography on peptide-agarose, we tested a set of fragments representing the entire light chain of plasma Fn. Only 40-kDa Hep-2 (III12-15) failed to bind. The concentration of urea required for peak elution of Fn and the other fragments decreased in the order Fn > 42-kDa GBF (I6II1-2I7-9) > 19-kDa Fib-2 (I10-12) > 110-kDa CBF(III2-10) > 29-kDa Fib-1 (I1-I5). Neither Fn nor any of the fragments bound immobilized intact III1, confirming the cryptic nature of this activity. In an effort to detect interactions between other Fn domains, all fragments were coupled to Sepharose, and each fragment was tested on each affinity matrix before and after denaturation. The only interaction detected was that of fluid phase III1 with immobilized denatured 110-kDa CBF and 40-kDa Hep-2, both of which contain type III domains. Analysis of subfragments revealed this activity to be dominated by domains III7 and III15. Fn itself did not bind to the denatured fragments. Thus, domain III1 contains two cryptic “self-association sites,” one that is buried in the core of the fold but recognizes many Fn fragments when presented as a peptide and another that is concealed in Fn but exposed in the native isolated domain and recognizes cryptic sites in two other type III domains. These interactions between type III domains could play an important role in assembly of Fn multimers in the extracellular matrix.

Crystal structure of the central region of bovine fibrinogen (E5 fragment) at 1.4-Å resolution

The high-resolution crystal structure of the N-terminal central region of bovine fibrinogen (a 35-kDa E5 fragment) reveals a remarkable dimeric design. The two halves of the molecule bond together at the center in an extensive molecular “handshake” by using both disulfide linkages and noncovalent contacts. On one face of the fragment, the Aα and Bβ chains from the two monomers form a funnel-shaped domain with an unusual hydrophobic cavity; here, on each of the two outer sides there appears to be a binding site for thrombin. On the opposite face, the N-terminal γ chains fold into a separate domain. Despite the chemical identity of the two halves of fibrinogen, an unusual pair of adjacent disulfide bonds locally constrain the two γ chains to adopt different conformations. The striking asymmetry of this domain may promote the known supercoiling of the protofibrils in fibrin. This information on the detailed topology of the E5 fragment permits the construction of a more detailed model than previously possible for the critical trimolecular junction of the protofibril in fibrin.

Reversible unfolding of an isolated heparin and DNA binding fragment, the first type III module from fibronectin

Several fragments containing all or part of the first type III homology unit of fibronectin were isolated and their folding properties examined by fluorescence spectroscopy and differential scanning calorimetry. Each fragment exhibits a reversible unfolding transition when heated or titrated with guanidinium chloride. This indicates that an isolated type III module can fold independently in the absence of neighboring modules. A comparison of the specific enthalpies of unfolding of these fragments with those of well-studied globular proteins suggests that this type III unit is composed of a stable core flanked by less compact or unstructured regions. Comparison of the heparin-binding properties of these fragments revealed that removal of 12 amino acids from the amino terminus of the largest one (Ile-585 to Val-675) increased its affinity for immobilized heparin such that it now binds at physiological ionic strength.