CRYSTAL-STRUCTURE OF A COMPLEX FORMED BETWEEN PROTEOLYTICALLY-GENERATED LACTOFERRIN FRAGMENT AND PROTEINASE-K

Lactoferrin is an iron binding glycoprotein with a molecular weight of 80 kDa. The molecule is divided into two lobes representing the N-ter minal and C-terminal halves of the polypeptide chain, each containing an iron binding site. The serine proteinases such as trypsin, chymotry psin, and pepsin hydrolyze lactoferrin into two unequal halves while p roteinase K divides this protein into two equal halves. In the first s tep of hydrolysis by proteinase K, the C- and N-lobes, each having a m olecular weight of approximately 40 kDa, are generated. In the next st ep, the lobes are further hydrolyzed into small molecular weight pepti des. The proteinase K isolated from the hydrolyzed product does not sh ow enzymatic activity suggesting that the enzyme is inhibited. Further more, the hydrolysis experiments on N-lobe and C-lobe showed that the inhibitory fragment came fi om the C-lobe. The purified lactoferrin fr agment was found to be a decapeptide with an amino acid sequence of H2 N-Val-Ala-Gln-Gly-Ala-Ala-Gly-Leu-Ala-COOH. The complex formed between proteinase K and lactoferrin fragment was crystallized by microdialys is. The crystals belonged to the monoclinic space group P2(1) with cel l dimensions a = 44.4 Angstrom,b = 38.6 Angstrom,c = 79.2 Angstrom, be ta = 105.8 degrees and Z = 2. The crystal structure has been determine d at 2.4 Angstrom resolution. It has been refined to an R factor of 0. 163 for 9044 reflections. The Lf-fragment forms several intermolecular interactions with proteinase K. The Ser-224 O gamma and His-57 N epsi lon 2 move away to a distance of 3.68 Angstrom in the complex. In the crystal structure, Gln-3I (I indicates inhibitor i.e., lactoferrin fra gment) is involved in a direct intermolecular interaction with a symme try related proteinase K molecule through a strong hydrogen bond with Asp-254. The mode of intermolecular interactions in the complex confor mational features of the enzyme and placement of the fragment with res pect to the enzyme resemble with the molecular complex of proteinase K with its natural inhibitor PKI3 from wheat. (C) 1998 Wiley-Liss, Inc.