Lindsay Sawyer

The structure of β-lactoglobulin and its similarity to plasma retinol-binding protein

Since its first isolation1, bovine β-lactoglobulin (BLG) has been an enigma: although it is abundant in the whey fraction of milk, its function is still not clear. The results of the many physicochemical studies on the protein need a structural interpretation. We report here the structure of the orthorhombic crystal form of cow BLG at pH 7.6, at a resolution of 2.8 Å. It has an unusual protein fold, composed of two slabs of antiparallel β-sheet, which shows a remarkable similarity to plasma retinol-binding protein. A possible binding site for retinol in BLG has been identified by model-building. This suggests a role for BLG in vitamin A transport and we have discovered specific receptors for the BLG–retinol complex in the intestine of neonate calves.

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

Abstract 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 A 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 A 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.

The core lipocalin, bovine β-lactoglobulin.

Abstract The lipocalin family became established shortly after the structural similarity was noted between plasma retinol binding protein and the bovine milk protein, β-lactoglobulin. During the past 60 years, β-lactoglobulin has been studied by essentially every biochemical technique available and so there is a huge literature upon its properties. Despite all of these studies, no specific biological function has been ascribed definitively to the protein, although several possibilities have been suggested. During the processing of milk on an industrial scale, the unpredictable nature of the process has been put down to the presence of β-lactoglobulin and certainly the whey protein has been implicated in the initiation of aggregation that leads to the fouling of heat exchangers. This short review of the properties of the protein will concentrate mainly on studies carried out under essentially physiological conditions and will review briefly some of the possible functions for the protein that have been described.

Crystal structure of the trigonal form of bovine beta-lactoglobulin and of its complex with retinol at 2.5 A resolution.

The structure of the trigonal crystal form of bovine beta-lactoglobulin has been determined by X-ray diffraction methods. An electron density map, calculated with phases obtained by the multiple isomorphous replacement method, served as a starting point for alternate cycles of model building and restrained least-squares refinement. The model of the molecule fitted to the initial Fourier map was the one built for the orthorhombic crystal form of beta-lactoglobulin, solved at 2.8 A resolution (1 A = 0.1 nm). The final R factor for 1456 atoms (1276 non-hydrogen protein atoms and 180 solvent atoms) is 0.22, including 5245 reflections from 6.0 to 2.5 A. The molecule shows significant differences in the two crystal forms mentioned, mainly due to different packing. In the trigonal form, the species crystallized does not appear to be dimeric, but a linear polymer with tight intermolecular contacts. A difference electron density map between the complex of beta-lactoglobulin with retinol and the native protein shows no significant peaks in the cavity which, in the similar retinol-binding protein, binds the chromophore. Instead, differences are found at a surface pocket, which is limited almost completely by hydrophobic residues.

The Ligand-binding Site of Bovine β-Lactoglobulin: Evidence for a Function?

Ever since the fortuitous observation that beta-lactoglobulin (beta-Lg), the major whey protein in the milk of ruminants, bound retinol, the details of the binding have been controversial. beta-Lg is a lipocalin, like plasma retinol-binding protein, so that ligand association was expected to make use of the central cavity in the protein. However, an early crystallographic analysis and some of the more recent solution studies indicated binding elsewhere. We have now determined the crystal structures of the complexes of the trigonal form of beta-Lg at pH 7.5 with bound retinol (R=21.4% for 7329 reflections between 20 and 2.4 A resolution, R(free)=30.6%) and with bound retinoic acid (R=22.7% for 7813 reflections between 20 and 2.34 A resolution, R(free)=29.8%). Both ligands are found to occupy the central calyx in a manner similar to retinol binding in retinol-binding protein. We find no evidence of binding at the putative external binding site in either of these structural analyses. Further, competition between palmitic acid and retinol reveals only palmitate bound to the protein. An explanation is provided for the lack of ligand binding to the orthorhombic crystal form also obtained at pH 7.5. Finally, the possible function of beta-Lg is discussed in the light of its species distribution and similarity to other lipocalins.

Caseins as rheomorphic proteins: interpretation of primary and secondary structures of the αS1-, β- and κ-caseins

Caseins are members of a class of proteins with extremely open and flexible conformations. Here, we consider what features of their sequences are important in maintaining such a structure. Primary structures of the αS1-, β- and κ-caseins from species including the cow, sheep, rat, mouse, rabbit and guinea pig were aligned both by a variety of automatic multiple alignment procedures, and manually, to identify conserved features. Fully conserved residues in the mature proteins were unusually rare and involved mainly residues that have high mutation rates in conventional alignment scoring schemes. Autocorrelation of sequences using residue mutation rate scores as measures of similarity revealed that around the PQNI conserved sequence of β-caseins there appear to be repeated sequences similar to Pro-rich domain-linking peptides found in a number of other proteins. Other cryptic repeats were found in αS1-caseins. Predicted secondary structures were calculated and it is argued that apart from the regions around the centres of phosphorylation, the caseins are essentially of the all-β-strand type. However, condensation into β-sheets is inhibited by certain of the conserved features of the primary structure, allowing the proteins to maintain an open and mobile (rheomorphic) conformation.

The two types of 3-dehydroquinase have distinct structures but catalyze the same overall reaction.

The structures of enzymes catalyzing the reactions in central metabolic pathways are generally well conserved as are their catalytic mechanisms. The two types of 3-dehydroquinate dehydratase (DHQase) are therefore most unusual since they are unrelated at the sequence level and they utilize completely different mechanisms to catalyze the same overall reaction. The type I enzymes catalyze a cis-dehydration of 3-dehydroquinate via a covalent imine intermediate, while the type II enzymes catalyze a trans-dehydration via an enolate intermediate. Here we report the three-dimensional structures of a representative member of each type of biosynthetic DHQase. Both enzymes function as part of the shikimate pathway, which is essential in microorganisms and plants for the biosynthesis of aromatic compounds including folate, ubiquinone and the aromatic amino acids. An explanation for the presence of two different enzymes catalyzing the same reaction is presented. The absence of the shikimate pathway in animals makes it an attractive target for antimicrobial agents. The availability of these two structures opens the way for the design of highly specific enzyme inhibitors with potential importance as selective therapeutic agents.

Homozygous SLC2A9 Mutations Cause Severe Renal Hypouricemia

Hereditary hypouricemia may result from mutations in the renal tubular uric acid transporter URAT1. Whether mutation of other uric acid transporters produces a similar phenotype is unknown. We studied two families who had severe hereditary hypouricemia and did not have a URAT1 defect. We performed a genome-wide homozygosity screen and linkage analysis and identified the candidate gene SLC2A9, which encodes the glucose transporter 9 (GLUT9). Both families had homozygous SLC2A9 mutations: A missense mutation (L75R) in six affected members of one family and a 36-kb deletion, resulting in a truncated protein, in the other. In vitro, the L75R mutation dramatically impaired transport of uric acid. The mean concentration of serum uric acid of seven homozygous individuals was 0.17 +/- 0.2 mg/dl, and all had a fractional excretion of uric acid >150%. Three individuals had nephrolithiasis, and three had a history of exercise-induced acute renal failure. In conclusion, homozygous loss-of-function mutations of GLUT9 cause a total defect of uric acid absorption, leading to severe renal hypouricemia complicated by nephrolithiasis and exercise-induced acute renal failure. In addition to clarifying renal handling of uric acid, our findings may provide a better understanding of the pathophysiology of acute renal failure, nephrolithiasis, hyperuricemia, and gout.

Arachidonic acid binds to apolipoprotein D: implications for the protein's function

The lipocalin apolipoprotein D (ApoD) is associated in human plasma with lecithin‐cholesterol acyl transferase. It has also been found in high concentration in the fluid of gross cystic disease of the mammary gland. Using protein fluorescence quenching, it is shown that ApoD binds arachidonic acid (K aof 1.6 × 108M−1) and as previously known progesterone (K aof 2.5 × 106M−1), but neither cholesterol nor any of the other prostanoid molecules examined had measurable affinity. This specific binding of arachidonate, also observable directly, suggests a role for ApoD in the mobilisation of Arachidonic acid, and hence prostaglandin synthesis.

β-lactoglobulin : Structural studies, biological clues

Bovine β-lactoglobulin (β-Lg) is a much studied and commercially important whey protein with an as yet undetermined function, although it is of obvious nutritional value. β-Lg binds a variety of ligands and by comparison of the general structures of these molecules together with several competition studies, it appears that there are at least 3 independent binding sites. In the absence of direct crystallographic evidence, a preliminary modelling study reveals that there is an internal cavity which can readily accommodate retinol in a manner similar to the related lipocalin, retinol-binding protein. On the outer surface, a solvent-accessible hydrophobic cleft runs between the 3-turn α-helix that is packed against the outer surface of the β-barrel. This cleft can accommodate fatty acids like palmitate and stearate. There is difference electron density observed in a soaking experiment with p-nitrophenol at a third site, on the outer surface close to the conserved Trp19/Arg124. This site is large enough to accommodate larger aromatic ligands such as ellipticine, although there is yet no independent evidence for this.