Hale Nicholson

Structure of a hinge-bending bacteriophage T4 lysozyme mutant, Ile3-->Pro.

The mutant T4 phage lysozyme in which isoleucine 3 is replaced by proline (I3P) crystallizes in an orthorhombic form with two independent molecules in the asymmetric unit. Relative to wild-type lysozyme, which crystallizes in a trigonal form, the two I3P molecules undergo large hinge-bending displacements with the alignments of the amino-terminal and carboxy-terminal domains changed by 28.9 degrees and 32.9 degrees, respectively. The introduction of the mutation, together with the hinge-bending displacement, is associated with repacking of the side-chains of Phe4, Phe67 and Phe104. These aromatic residues are clustered close to the site of the mutation and are at the junction between the amino and carboxyl-terminal domains. As a result of this structural rearrangement the side-chain of Phe4 moves from a relatively solvent-exposed conformation to one that is largely buried. Mutant I3P also crystallizes in the same trigonal form as wild-type and, in this case, the observed structural changes are restricted to the immediate vicinity of the replacement. The main change is a shift of 0.3 to 0.5 A in the backbone of residues 1 to 5. The ability to crystallize I3P under similar conditions but in substantially different conformations suggests that the molecule undergoes large-scale hinge-bending displacements in solution. It is also likely that these conformational excursions are associated with repacking at the junction of the N-terminal and C-terminal domains. On the other hand, the analysis is complicated by possible effects of crystal packing. The different I3P crystal structures show substantial differences in the binding of solvent, both at the site of the Ile3-->Pro replacement and at other internal sites.

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.

Crystallographic and Genetic Approaches Toward the Design of Proteins of Enhanced Thermostability

The advent of directed mutagenesis has made it possible to alter protein structures at will. For the first time it is possible to design and to introduce modifications into a protein that are intended to change its behavior in predictable ways.

Mutagenesis of Human Lactoferrin and Expression in Baby Hamster Kidney Cells

We have previously reported the expression of both full-length recombinant lactoferrin and the recombinant N-lobe half-molecule in baby hamster kidney (BHK) cells (Stowell et al, 1991; Day et al., 1992). The properties of the full-length recombinant protein produced in this system were virtually indistinguishable from those of the native protein isolated from human milk, except for an increased resistance of a minor fraction of the protein to deglycosylation by PNGase. The N-lobe recombinant protein has been characterized (Day et al., 1992) and the structure determined by X-ray crystallography (Day et al., 1993).