1.2 A refinement of the Kunitz-type domain from the alpha3 chain of human type VI collagen.

The recombinant Kunitz-type domain (C5) of human collagen alpha3(VI) chain was previously described at 1.6 A resolution at room temperature. By changing the crystallization conditions and using synchrotron radiation, we are able to record diffraction data to 1.2 A resolution for crystals of the same space group at 291 K. The protein-water-ion model has been refined anisotropically against these new data using the program SHELXL93; the results converged to an R factor of 15.0%, with all data between 7 and 1.2 A. The final electron-density map reveals a clear chain tracing with a few disordered residues and five residues out of 58 that present alternate conformations. The Cys14-Cys38 bond presents the less frequently observed left-hand conformation (chi1 = -60 degrees). The solvent molecules and a phosphate ion are well ordered with an average B of 38 A2. The high-resolution structure reveals the N and C termini which were missing from the 1.6 A structure.

The 1.6 A structure of Kunitz-type domain from the alpha 3 chain of human type VI collagen.

The C-terminal Kunitz-type domain from the alpha 3 chain of human type VI collagen (C5), a single 58 amino acid residue chain with three disulfide bridges, was cloned, expressed and crystallized in a monoclonic form, space group P2(1), with a = 25.7 A, b = 38.2 A, c = 28.8 A and beta = 109 degrees. The structure was resolved by molecular replacement, using Alzheimer's protein precursor inhibitor and bovine pancreatic trypsin inhibitor three-dimensional structures as search models. The molecule with one sulfate ion and 43 associated water molecules was refined by XPLOR to an R-factor of 18.9% at 1.6 A. The molecule was not degraded by trypsin and did not inhibit trypsin or tested serine proteases. As opposed to the other Kunitz family members, C5 demonstrates left-handed chirality of the Cys14-Cys38 disulfide bond. Inversion of the Thr13 carbonyl and bulky side-chains at the interface with trypsin in a model of the C5-trypsin complex may explain the lack of inhibition of trypsin.