Altered Domain Closure and Iron Binding in Transferrins: The Crystal Structure of the Asp60Ser Mutant of the Amino-terminal Half-molecule of Human Lactoferrin

• Published in: Journal of molecular biology 1996-02, Vol.256 (2), p.352-363
• Main Authors: Faber, Rick H., Bland, Tony, Day, Catherine L., Norris, Gillian E., Tweedie, John W., Baker, Edward N.
• Format: Article
• Language: English
• Subjects: Animals; Anions - metabolism; Aspartic Acid - chemistry; Binding Sites; Cell Line; Cricetinae; crystal structure; Crystallography, X-Ray; domain closure; Humans; Iron - metabolism; iron binding; lactoferrin; Lactoferrin - chemistry; Lactoferrin - metabolism; Metals - metabolism; mutagenesis; Mutagenesis, Site-Directed; Protein Conformation; Serine - chemistry; Structure-Activity Relationship; Transferrin - chemistry; Transferrin - metabolism
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1996.0091

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 Å resolution in order to determine the effects of the mutation on iron binding and domain closure. Yellow monoclinic crystals of the D60S mutant, in its iron-bound form, were prepared, and have unit cell dimensions a=110.2 Å, b=57.0 Å, c=55.2 Å, β=97.6°, space group C2, with one molecule of 333 residues in the asymmetric unit. The structure was determined by molecular replacement, using the wild-type Lf Nas search model, and was refined by restrained least-squares methods. The final model, comprising 2451 protein atoms (from residues 2 to 315) one Fe 3 +and one CO 3 2−, and 107 water molecules, gives an R-factor of 0.175 for all data in the resolution range 20.0 to 2.05 Å. The model conforms well with standard geometry, having root-mean- square deviations of 0.014 Å and 1.2° from standard bond lengths and angles. The structure of the D60S mutant deviates in two important respects from the parent Lf Nmolecule. At the mutation site the Ser side-chain neither binds to the iron atom nor makes any interdomain contact as the substituted Asp does; instead a water molecule fills the iron coordination site and participates in interdomain hydrogen bonding. The domain closure is also changed, with the D60S mutant having a more closed conformation. Consideration of crystal packing suggests that the altered domain closure is a genuine molecular property but both the iron coordination and interdomain contacts are consistent with weakened iron binding in the mutant. The implications for iron binding in transferrins generally are discussed.