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.
KeywordsIron Atom Iron Binding Domain Closure Iron Release Iron Site
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