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Crystal structure of a complex formed between proteolytically‐generated lactoferrin fragment and proteinase K
Author(s) -
Singh Tej P.,
Sharma Sujata,
Karthikeyan S.,
Betzel Christian,
Bhatia Krishan L.
Publication year - 1998
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/(sici)1097-0134(19981001)33:1<30::aid-prot3>3.0.co;2-p
Subject(s) - lactoferrin , chemistry , trypsin , chymotrypsin , serine , biochemistry , stereochemistry , proteinase k , peptide sequence , subtilisin , glycoprotein , hydrolysis , enzyme , gene
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 H 2 N‐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 Å, b = 38.6 Å, c = 79.2 Å, β = 105.8 o and Z = 2. The crystal structure has been determined at 2.4 Å 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 Oγ and His‐57 Nϵ2 move away to a distance of 3.68 Å 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. Proteins 33:30–38, 1998. © 1998 Wiley‐Liss, Inc.