Bovine β-lactoglobulin at 1.8 Å resolution — still an enigmatic lipocalin

Background:β-Lactoglobulin (β-Lg) is the major whey protein in the milk of ruminants and many other mammals. Its function is not known, but it undergoes at least two pH-dependent conformational changes which may be important. Bovine β-Lg crystallizes in several different lattices, and medium-resolution structures of orthorhombic lattice Y and trigonal lattice Z have been published. Triclinic lattice X and lattice Z crystals grow at pH values either side of the pH at which one of the pH-induced conformational changes occurs. A full understanding of the structure is needed to help explain both the conformational changes and the different denaturation behaviour of the genetic variants.

Results: We have redetermined the structure of β-Lg lattice Z at 3.0 Å resolution by multiple isomorphous replacement and have partially refined it (R factor = 24.8%). Using the dimer from this lattice Z structure as a search model, the triclinic crystal form grown at pH 6.5 (lattice X) has been solved by molecular replacement. Refinement of lattice X at 1.8 Å resolution gave an R factor of 18.1%. The structure we have determined differs from previously published structures in several ways.

Conclusions: Incorrect threading of the sequence in the published structures of β-Lg affects four of the nine β strands. The basic lipocalin fold of the polypeptide chain is unchanged, however. The relative orientation of the monomers in the β-Lg dimer differs in the two lattices. On raising the pH, there is a rotation of approximately 5°, which breaks a number of intersubunit hydrogen bonds. It is not yet clear, however, why the stability of the structure should depend so heavily upon the external loop around residue 64 or the β strand with the free thiol, each of which shows genetic variation.