Altered Domain Closure and Iron Binding in Lactoferrin Mutants
Two features of the functional properties of lactoferrin are its ability to bind iron exceptionally tightly and the coupling of rigid-body domain movements to iron binding and release. The latter cause transitions between open and closed forms of the protein. Using site-directed mutagenesis and X-ray crystallography we have examined the importance of selected residues, including the iron ligands Asp 60 and His 253, the anion-binding Arg 121, and Pro 251 in the hinge region. Five mutants, D60S, R121S, R121E, H253M, and P251 A, have been prepared in the context of the N-terminal half-molecule of human lactoferrin, Lfn, and three-dimensional structures have been determined in each case. In D60S the mutation leads to weakened iron binding because a water molecule binds to the iron atom in place of Asp 60. Interdomain interactions are also weakened, and the loss of the Asp side-chain causes a significant change in domain closure; the domains move closer together by 7° in the mutant. The R121S and R121E mutants show altered anion binding and very small changes in domain orientations. The H253M and P251A mutants show identical domain closure to wild-type LfN, but the iron site is altered in H253M; the Met 253 side-chain is not bound to iron, leaving a 5-coordinate site. These results are interpreted in terms of the roles of each of the residues in iron binding and release.
KeywordsCrystallization Filtration Hydroxyl Carboxylate Polypeptide
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