Lactoferrin half-molecules

Complementing the structural studies of the intact transferrins, a number of fragments have also been crystallized, including proteolytic N-terminal half-molecules of rabbit serum transferrin (69) and chicken ovotransferrin (70), recombinant N-terminal half-molecules of human lactoferrin (71) and human serum transferrin (72), and a quarter-molecule fragment of duck ovotransferrin (73). All of these have now led to high-resolution structures (74-77). 

The bilobal structure of transferrins means that half-molecules, representing either the N-terminal or C-terminal lobe, can be relatively easily prepared, either by limited proteolysis or by recombinant DNA methods (Section III.A). Relatively high-resolution crystal structures have been determined for three such half-molecules, the proteolytic N-lobes of rabbit transferrin (74) and chicken ovotransferrin (77) at 2.3 A and the recombinant N-lobe of human lactoferrin at 2.0-A resolution (75). These show that both the protein structure and the metal and anion binding sites are the same as in the intact parent structures. In fact comparison of the metal and anion sites of the lactoferrin and transferrin half-molecules with each other and with the N-lobe of lactoferrin shows very close correspondence 92 atoms from the nine residues, plus metal and anion, making up the immediate binding site can be superimposed with an rms deviation of only 0.4 A (75). 

Solution scattering measurements on half-molecules show that they undergo very similar conformational changes, on iron binding or release, to those of the corresponding lobes of intact transferrins (104). This, together with the structural correspondence noted above, makes them valid as models of single-sited transferrins. The lactoferrin half-... 

Fig. 28. The pH dependence of iron release from human serum transferrin (Tf), human lactoferrin (Lf), and the recombinant N-terminal half-molecule of human lactoferrin (Lfm). Also shown is a plot (dashed line) for the release of cerium from Ce4+-substituted lactoferrin, demonstrating the increased difference between the two sites for metal ions other than Fe3+.

Finally the question of whether there is any cooperativity between the sites remains to be addressed. Although evidence of cooperativity from solution studies has mostly been equivocal (3), there are certainly structural interactions between the two lobes, involving helices from each (78, 85), and studies of a half-molecule fragment of lactoferrin have shown that separating the N-lobe from the C-lobe gives it altered... 

The half-molecules differ somewhat, although this is not unexpected, because the properties of the whole molecules are an amalgam of those of their two slightly different sites. Moreover, the lactoferrin halfmolecule shows that its iron release properties are changed as a result of the loss of interactions from the other lobe (Ref. 49 see also Section V.B.2 and Fig. 28). The visible max for the transferrin half-molecule... 

Fig. 15. Polypeptide folding patterns for (a) one-half of a transferrin molecule (the N-lobe of lactoferrin) and (b) the bacterial periplasmic sulfate-binding protein. Adapted from Baker et al. (85), with permission.

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