CRYSTALLOGRAPHIC ANALYSIS OF A FRAGMENT FROM CHICKEN TENASCIN CONTAINING TWO FIBRONECTIN-TYPE III DOMAINS Klaus Piontek, Daniel Bisig, Peter Weber, Lloyd Vaughan, Kaspar Winterhalter, Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), CH-8092 Zürich, Switzerland.
Tenascin-C is an extracellular glycoprotein composed of six identical polypeptide chains which are linked together at their N-terminal ends to form a hexabrachion structure. Each polypeptide comprises a serie of 13.5 tandem repeats homologous to epidermal growth factor followed by 8-13 fibronectin type III (TNfn)-like domains and finally one fibrinogen-like domain at the C-terminus. Alternative splicing of TNfn domains between the domains 5 and 6 leads to the three major isoforms found in chicken. In the smallest isoform (190 kDa) these two domains are adjacent to each other. Tenascin-C isoforms are differently expressed in development and in response to malignancy, leading to the expectation of isoform-specific functions. TNfn5 binds heparin and other iduronic acid-containing glycosaminoglycans, presumably via a conserved heparin binding motif. Furthermore, TNfn56 binds to the neural cell adhesion molecule contactin/F11, an interaction which is inhibitable by heparin. To explore these interactions in greater detail we have prepared a fusion protein containing domains 5 and 6 and attempt now the crystal structure determination of this 20 kDa protein.
TNfn56 crystallizes from 27% PEG2000, 100mM Na-acetate, pH 4.5 at RT in a monoclinic (P21, a=47.7Å, b=54.9Å, c=97.0Å, ß=104.4deg., Vm=3.08 Å3/Da, 2 mol/a.u.) and an orthorhombic (P2221, a=45.3Å, b=71.9Å, c=58.0Å, Vm=2.36 Å3/Da, 1mol/a.u.) form. Both crystal forms diffract to about 2.6 Å resolution. The majority of the monoclinic crystals is twinned and the diffraction limit of this form frequently lies only between 3.2 and 4.0 Å. Furthermore, the formation of the orthorhombic form can not be directed. Despite the sparse availability of suitable single crystals we have collected a data set of monoclinic crystals to 2.6 Å resolution utilizing synchrotron radiation (DESY/Germany) and a data set to 2.8 Å resolution on a conventional X-ray source. Presently we attempt the structure determination with the molecular replacement technique and, in parallel, with isomorphous replacement.