CRYSTAL STRUCTURE AT 2.55 A RESOLUTION OF A TWO FIBRONECTION TYPE-III DOMAIN SEGMENT FROM CHICKEN TENASCIN
D. Bisig, P. Weber, L. Vaughan, K.H. Winterhalter, and K. Piontek
Laboratory of Biochemistry, Swiss Federal Institute of
Technology (ETH), Universitaetstrasse 16, CH-8092 Zuerich,
Switzerland
Tenascin is a large multidomain extracellular matrix glycoprotein which exhibits a dynamic spatial and temporal expression both during embryogenesis and in adult tissues. By interaction with various protein and glycosaminoglycan ligands, tenascin-C is thought to mediate diverse biological events such as neural pattern formation, wound healing, and carcinogenesis.
A fragment of chicken tenascin consisting of fibronectin type-III domains (fnIII) 5 and 6 (TNfn56) which contains the binding sites for the glycosaminoglycan heparin [1] and for the neural cell receptor contactin/F11 [2], has been expressed in E. coli. The fusion protein was purified to electrophoretic homogeneity and crystallized. These crystals belong to the space group P21 with cell dimensions a=45.2 A, b=57.9 A, c=72.2 A, and b=91.4° and diffract to at least 2.6 A resolution. There are two molecules of TNfn56 in the asymmetric unit. Based on primary and tertiary structure alignments of several fnIII structures, a "consensus" model was constructed which served as a successful search model for solving the TNfn56 structure by molecular replacement. The model was refined to 2.55 A resolution resulting in an R-factor of 20.9 %.
The structure of TNfn56 consists of seven b-strands which form a b-sandwich structure. Superposition of the individuell domains on various other fnIII structures results in Ca root mean square values between 1.0 and 1.9 A. Structural similarity is mainly restricted to aliphatic residues which protrude into the hydrophobic core of the fnIII module and to central parts of b-strands. Loop regions differ considerably in length and conformation between TNfn5, TNfn6, and other fnIII domains.
The mainchain conformation of the hinge region between domains 5 and 6 is mostly very similar in both molecules of the a.u. but it contains one residue which exhibits completely different f/ y angles. The leverage effect of this variation results in a considerable difference between the two TNfn56 molecule conformations. One molecule possesses a bent conformation whereas the other molecule exhibits a more extended, rodlike structure. It can be assumed that if similarly flexible hinge regions are present throughout the entire fnIII region of tenascin this protein is able to adopt very different conformations. This inherent flexibility is probably a prerequisite for multivalent proteins such as tenascin to be able to adopt a conformation which allows the simultaneous interaction with various ligand molecules present in the extracellular matrix and on cell surfaces.
Analysis of the charge distribution of TNfn56 revealed the
presence of a large positively charged surface cluster area on
top of the major b-sheet of TNfn5.
This cluster is formed by basic residues protruding from a loop
which contains the heparin binding consensus motif and from b-strands at the edge of the major b-sheet. The size of this area is sufficient
to bind a hexameric heparin molecule. It has been possible to
model an artificial TNfn5-heparin complex by placing the same
heparin molecule which is present in the crystal structure of
human basic fibroblast growth factor on top of this charged
surface area.