ALTERED DOMAIN CLOSURE AND IRON-BINDING IN TRANSFERRINS - THE CRYSTAL-STRUCTURE OF THE ASP60SER MUTANT OF THE AMINO-TERMINAL HALF-MOLECULE OF HUMAN LACTOFERRIN

The crystal structure of a site-specific mutant of the N-terminal half -molecule of human lactoferrin, Lf(N), in which the iron ligand Asp60 has been mutated to Ser, has been determined at 2.05 Angstrom resoluti on in order to determine the effects of the mutation on iron binding a nd domain closure. Yellow monoclinic crystals of the D60S mutant, in i ts iron-bound form, were prepared, and have unit cell dimensions a = 1 10.2 Angstrom, b = 57.0 Angstrom, c = 55.2 Angstrom, beta = 97.6 degre es, space group C2, with one molecule of 333 residues in the asymmetri c unit. The structure was determined by molecular replacement, using t he wild-type Lf(N) as search model, and was refined by restrained leas t-squares methods. The final model, comprising 2451 protein atoms (fro m residues 2 to 315) one Fe3+ and one CO32-, and 107 water molecules, gives an R-factor of 0.175 for all data in the resolution range 20.0 t o 2.05 Angstrom. The model conforms well with standard geometry, havin g root-mean-square deviations of 0.014 Angstrom and 1.2 degrees from s tandard bond lengths and angles. The structure of the D60S mutant devi ates in two important respects from the parent Lf(N) molecule. At the mutation site the Ser side-chain neither binds to the iron atom nor ma kes any interdomain contact as the substituted Asp does; instead a wat er molecule fills the iron coordination site and participates in inter domain hydrogen bonding. The domain closure is also changed, with the D60S mutant having a more closed conformation. Consideration of crysta l packing suggests that the altered domain closure is a genuine molecu lar property but both the iron coordination and interdomain contacts a re consistent with weakened iron binding in the mutant. The implicatio ns for iron binding in transferrins generally are discussed. (C) 1996 Academic Press Limited