title: Structure of the iron-free true C-terminal half of bovine lactoferrin produced by tryptic digestion and its functional significance in the gut.
abstract: Bovine lactoferrin, a 76-kDa glycoprotein (Ala1-Arg689) consists of two similar N- and C-terminal molecular halves with the ability to bind two Fe(3+) ions. The N-terminal half, designated as the N-lobe (Ala1-Arg341) and the C-terminal half designated as the C-lobe (Tyr342-Arg689) have similar iron-binding properties, but the resistant C-lobe prolongs the physiological role of bovine lactoferrin in the digestive tract. Here, we report the crystal structure of true C-lobe, which was produced by limited proteolysis of bovine lactoferrin using trypsin. In the first proteolysis step, two fragments of 21 kDa (Glu86-Lys282) and 45 kDa (Ser283-Arg689) were generated because two lysine residues, Lys85 and Lys282, in the structure of iron-saturated bovine lactoferrin were fully exposed. The 45-kDa fragment was further digested at the newly exposed side chain of Arg341, generating a 38-kDa perfect C-lobe (Tyr342-Arg689). By contrast, the apo-lactoferrin was cut by trypsin only at Arg341, which was exposed in the structure of apo-lactoferrin, whereas the other two sites with Lys85 and Lys282 are inaccessible. The purified iron-saturated C-lobe was crystallized at pH 4.0. The structure was determined by the molecular replacement method using coordinates of the C-terminal half (Arg342-Arg689) of intact camel apo-lactoferrin. The structure determination revealed that the iron atom was absent and the iron-binding cleft was found in a wide-open conformation, whereas in the previously determined structure of iron-saturated C-lobe of bovine lactoferrin, the iron atom was present and the iron-binding site was in the closed confirmation.
authors.affiliation: Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: Nilisha
authors.initials: N
authors.lastname: Rastogi
authors.forename: Avinash
authors.initials: A
authors.lastname: Singh
authors.forename: Sada Nand
authors.initials: SN
authors.lastname: Pandey
authors.forename: Mau
authors.initials: M
authors.lastname: Sinha
authors.forename: Asha
authors.initials: A
authors.lastname: Bhushan
authors.forename: Punit
authors.initials: P
authors.lastname: Kaur
authors.forename: Sujata
authors.initials: S
authors.lastname: Sharma
authors.forename: Tej P
authors.initials: TP
authors.lastname: Singh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D007413:Intestinal Mucosa / Q000378:metabolism*; D007781:Lactoferrin / Q000096:biosynthesis / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000378:metabolism*
chemical_list: D007781:Lactoferrin; D014357:Trypsin
keywords: apo C-lobe; crystal structure; lactoferrin; open conformation; trypsin-digestion