Crystal Structure of a Proteolytically Generated Functional Monoferric C-lobe of Bovine Lactoferrin at 1.9 A ˚ Resolution Sujata Sharma, Jayasankar Jasti, Janesh Kumar, Ashok K. Mohanty and Tej P. Singh * Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India This is the first crystal structure of a proteolytically generated functional C-lobe of lactoferrin. The purified samples of iron-saturated C-lobe were crystallized in 0.1 M Mes buffer (pH 6.5) containing 25% (v/v) polyethyl- eneglycol monomethyl ether 550 M and 0.1 M zinc sulphate heptahydrate. The X-ray intensity data were collected with 300 mm imaging plate scan- ner mounted on a rotating anode generator. The structure was determined by the molecular replacement method using the coordinates of the C-terminal half of bovine lactoferrin as a search model and refined to an R-factor of 0.193 for all data to 1.9 A ˚ resolution. The final model comprises 2593 protein atoms (residues 342–676 and 681–685), 124 carbohydrate atoms (from ten monosaccharide units, in three glycan chains), one Fe 3þ , one CO 3 22 , two Zn 2þ and 230 water molecules. The overall folding of the C-lobe is essentially the same as that of C-terminal half of bovine lacto- ferrin but differs slightly in conformations of some of the loops and reveals a number of new interactions. There are 20 Cys residues in the C-lobe forming ten disulphide links. Out of these, one involving Cys481- Cys675 provides an inter-domain link at 2.01 A ˚ while another Cys405- Cys684 is formed between the main C-lobe 342-676 and the hydrolyzed pentapeptide 681-685 fragment. Six inter-domain hydrogen bonds have been observed in the structure whereas only four were reported in the structure of intact lactoferrin, although domain orientations have been found similar in the two structures. The good quality of electron density has also revealed all the ten oligosaccharide units in the structure. The observation of two metal ions at sites other than the iron-binding cleft is another novel feature of the present structure. These zinc ions stabilize the crystal packing. This structure is also notable for extensive inter- molecular hydrogen bonding in the crystals. Therefore, the present structure appears to be one of the best packed crystal structures among the proteins of the transferrin superfamily. q 2003 Elsevier Ltd. All rights reserved Keywords: C-lobe; proteolysis; proteinase K; crystal structure; monoferric *Corresponding author Introduction Lactoferrin is a bilobal glycoprotein that contains two homologous metal-binding sites with high affi- nities for ferric iron. 1 It plays an essential role in iron homeostasis in vertebrates, controlling the levels of free iron in body fluids. 2–4 The iron-binding sites are situated in the inter-domain clefts. The requirement for an anion, bound synergistically with the Fe ion is a unique feature of lactoferrin. Iron release can occur at low pH and is associated with a large-scale 0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved E-mail address of the corresponding author: tps@aiims.aiims.ac.in Abbreviations used: HLF, human lactoferrin; CLF, bovine lactoferrin; HALF, human apolactoferrin; ELF, equine lactoferrin; EALF, equine apolactoferrin; BLF, buffalo lactoferrin; UALF, camel apolactoferrin; MALDI- TOF, matrix assisted laser desorption/ionization-time-of- flight. doi:10.1016/S0022-2836(03)00717-4 J. Mol. Biol. (2003) 331, 485–496