title: Crystal structure of a complex formed between proteolytically-generated lactoferrin fragment and proteinase K.
abstract: Lactoferrin is an iron binding glycoprotein with a molecular weight of 80 kDa. The molecule is divided into two lobes representing the N-terminal and C-terminal halves of the polypeptide chain, each containing an iron binding site. The serine proteinases such as trypsin, chymotrypsin, and pepsin hydrolyze lactoferrin into two unequal halves while proteinase K divides this protein into two equal halves. In the first step of hydrolysis by proteinase K, the C- and N-lobes, each having a molecular weight of approximately 40 kDa, are generated. In the next step, the lobes are further hydrolyzed into small molecular weight peptides. The proteinase K isolated from the hydrolyzed product does not show enzymatic activity suggesting that the enzyme is inhibited. Furthermore, the hydrolysis experiments on N-lobe and C-lobe showed that the inhibitory fragment came from the C-lobe. The purified lactoferrin fragment was found to be a decapeptide with an amino acid sequence of H2N-Val-Ala-Gln-Gly-Ala-Ala-Gly-Leu-Ala-COOH. The complex formed between proteinase K and lactoferrin fragment was crystallized by microdialysis. The crystals belonged to the monoclinic space group P2(1) with cell dimensions a = 44.4 A, b = 38.6 A, c = 79.2 A, beta = 105.8 degrees and Z = 2. The crystal structure has been determined at 2.4 A 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 Ogamma and His-57 N epsilon2 move away to a distance of 3.68 A in the complex. In the crystal structure, Gln-3I (I indicates inhibitor i.e., lactoferrin fragment) is involved in a direct intermolecular interaction with a symmetry related proteinase K molecule through a strong hydrogen bond with Asp-254. The mode of intermolecular interactions in the complex conformational features of the enzyme and placement of the fragment with respect to the enzyme resemble with the molecular complex of proteinase K with its natural inhibitor PKI3 from wheat.
authors.affiliation: Department of Biophysics, All India Institute of Medical Sciences, New Delhi. tps@medinst.ernet.in
authors.forename: T P
authors.initials: TP
authors.lastname: Singh
authors.forename: S
authors.initials: S
authors.lastname: Sharma
authors.forename: S
authors.initials: S
authors.lastname: Karthikeyan
authors.forename: C
authors.initials: C
authors.lastname: Betzel
authors.forename: K L
authors.initials: KL
authors.lastname: Bhatia
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray; D019286:Endopeptidase K / Q000378:metabolism*; D007781:Lactoferrin / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation
chemical_list: D010446:Peptide Fragments; D007781:Lactoferrin; D019286:Endopeptidase K