D.C. Phillips

Crystallographic Studies of the Activity of Hen Egg-White Lysozyme

The chemical evidence for the enzymic activity of lysozyme will be discussed in detail by other speakers at this meeting, but in order to describe our crystallographic studies of the interactions between the enzyme and its substrates it is necessary to summarize briefly what was known about them at the beginning of our work. Simultaneously with his discovery of lysozyme Fleming (1922) discovered a Gram-positive species of bacteria, Micrococcus lysodeikticus, which is particularly susceptible to the action of the enzyme. It was not until much later, however, that Salton (1952) demonstrated that the substrate is located entirely within the bacterial cell wall and it is only very recently that its chemical constitution has been established. Valuable early experiments (for example, by Meyer, Palmer, Thomson & Khorazo 1936; Meyer, Hahnel & Steinberg 1946; and by Epstein & Chain 1940) showed that lysozyme releases N-acetyl-amino sugars from M. lysodeikticus, but the first indication of the type of linkage attacked by lysozyme came when Berger & Weiser (1957) showed that lysozyme also degrades chitin, the linear polymer of N-acetylghicosamine.

A possible three-dimensional structure of bovine α-lactalbumin based on that of hen's egg-white lysozyme

Abstract Bovine α-lactalbumin and hen egg-white lysozyme have closely similar amino acid sequences. A model of α-lactalbumin has been constructed on the basis of the main chain conformation established for lysozyme. The side chain interactions of lysozyme are listed (Table 2) and the consequences of the side chain replacements in α-lactalbumin examined. Changes in internal side chains are generally interrelated in a convincing manner, suggesting that the model is largely correct, but there are some regions where it has not been possible to deduce the conformation unequivocally. Glu 35, which acts as a proton donor in lysozyme, is absent in α-lactalbumin, in which a neighbouring histidine residue may assume a similar function. The surface cleft, which is the site of substrate binding in lysozyme, is shorter in α-lactalbumin. While this would be consistent with α-lactalbumin having a mono- or disaccharide as substrate, the biochemical evidence shows that the role of α-lactalbumin in the synthesis of lactose is a complex one requiring direct interaction with the A protein.

On the conformation of the hen egg-white lysozyme molecule

Crystals of hen egg-white lysozyme, grown at pH 4.7 (Alderton & Fevold 1946), are tetragonal with a = b = 79.1 Å, c = 37.9 Å, space group P43212 (Palmer, Ballantyre & Galvin 1948; Blake, Fenn, North, Phillips & Poljak 1962). Each of the eight asymmetric units in the cell comprises a single lysozyme molecule, molecular weight about 14 600, together with 1 M sodium chloride solution which constitutes some 33.5% of the weight of the crystal (Steinrauf 1959). The structure of these crystals has been determined by X-ray analysis by the method of multiple isomorphous replacement developed in the studies of haemoglobin (Green, Ingram & Perutz 1954; Blow 1958; Perutz, Rossmann, Cullis, Muirhead, Will & North 1960) and myoglobin (Kendrew, Dickerson, Strandberg, Hart, Davies, Phillips & Shore 1960). Anomalous scattering data were used in conjunction with the isomorphous replacement intensity differences (North 1965) to form a joint probability distribution for the phase of each reflexion. The position of the centroid of each probability distribution gave a phase angle and weighting factor for each reflexion from which the electron density map with minimum r.m.s. error was calculated (Blow & Crick 1959). A large number of different heavy atom derivatives were studied (Poljak 1963; Blake, Koenig, Mair, North, Phillips & Sarma 1965) and three proved satisfactory for calculating an electron density map at 2 Å resolution. They contained respectively ortho-mercuri hydroxytoluene para-sulphonic acid, UO2F53- and an ion derived from UO2(NO3)2, probably UO2(OH)n(n-2)-

Crystal structure of human α-lactalbumin at 1.7 Å resolution

Abstract The three-dimensional X-ray structure of human α-lactalbumin, an important component of milk, has been determined at 1·7 A (0·17 nm) resolution by the method of molecular replacement, using the refined structure of baboon α-lactalbumin as the model structure. The two proteins are known to have more than 90% amino acid sequence identity and crystallize in the same orthorhombic space group, P21212. The crystallographic refinement of the structure using the simulated annealing method, resulted in a crystallographic R-factor of 0·209 for the 11,373 observed reflections (F ≥ 2σ(F)) between 8 and 1·7 A resolution. The model comprises 983 protein atoms, 90 solvent atoms and a bound calcium ion. In the final model, the root-mean-square deviations from ideality are 0·013 A for covalent bond distances and 2·9° for bond angles. Superposition of the human and baboon α-lactalbumin structures yields a root-mean-square difference of 0·67 A for the 123 structurally equivalent Cα atoms. The C terminus is flexible in the human α-lactalbumin molecule. The striking structural resemblance between α-lactalbumins and C-type lysozymes emphasizes the homologous evolutionary relationship between these two classes of proteins.

|[alpha]|-Lactalbumin possesses a novel calcium binding loop

Calcium performs a unique role in biology, achieving biological effects through highly specific interactions with and modulation of target proteins1. It has been proposed that calcium-modulated proteins possess a characteristic, evolutionarily related, binding fold, known as the EF-hand2. The high-resolution X-ray structure of α-lactalbumin reveals a Ca2+ binding fold that resembles an EF-hand only superficially and presumably has no evolutionary relationship with it. However, there is clear homology with the corresponding loop in c-type lysozyme (the ‘parent’ molecule of α-lactalbumin). This study, at 1.7 Å resolution, represents one of the most accurate analyses of a calcium binding protein yet reported.

Refinement of an enzyme complex with inhibitor bound at partial occupancy. Hen egg-white lysozyme and tri-N-acetylchitotriose at 1.75 A resolution.

The structure of the tri-N-acetylchitotriose inhibitor complex of hen egg-white lysozyme has been refined at 1.75 A resolution, using data collected from a complex crystal with ligand bound at less than full occupancy. To determine the exact value of the inhibitor occupancy, a model comprising unliganded and sugar-bound protein molecules was generated and refined against the 1.75 A data, using a modified version of the Hendrickson & Konnert least-squares procedure. The crystallographic R-factor for the model was found to fall to a minimum at 55% bound sugar. Conventional refinement assuming unit occupancy was found to yield incorrect thermal and positional parameters. Application of the same refinement procedures to an earlier 2.0 A data set, collected independently on different complex crystals by Blake et al. gave less consistent results than the 1.75 A refinement. From an analysis of the high resolution structure a detailed picture of the protein-carbohydrate interactions in the non-productive complex has emerged, together with the conformation and mobility changes that accompany ligand binding. The specificity of interaction between the protein and inhibitor, bound in subsites A to C of the active site, is seen to be generated primarily by an extensive network of hydrogen bonds, both to the protein itself and to bound solvent molecules. The latter also play an important role in maintaining the structural integrity of the active site cleft in the apo-protein.

High resolution1H- and13C-N.M.R spectra ofD-glucopyranose, 2-acetamido-2-deoxy-D-glucopyranose, and related compounds in aqueous media

Abstract The 1 H- and 13 C-n.m.r spectra of D -glucopyranose and 2-acetamido-2-deoxy- D -glucopyranose and its derivatives in D 2 O at 25° have been completely interpreted. Iterative analysis allowed accurate determination of the chemical shifts and coupling constants in the 270-MHz 1 H-spectra, and these are used to correlate the chemical shift changes with substitution patterns. The implications of the systematic errors from assuming first-order conditions for the p.m.r spectra of sugars are discussed in relation to measuring shift changes of sugar-enzyme complexes.

Molecular dynamics simulations of native and substrate-bound lysozyme: A study of the average structures and atomic fluctuations

Molecular dynamics simulations of hen egg-white lysozyme in the free and substrate-bound states are reported and the nature of the average structures and atomic fluctuations are analyzed. Crystallographic water molecules of structural importance, as determined by hydrogen-bonding, were included in the simulations. Comparisons are made between the dynamics and the X-ray results for the atomic positions, the main-chain and side-chain dihedral angles, and the hydrogen-bonding geometry. Improvements over earlier simulations in the potential energy function and methodology resulted in stable trajectories with the C alpha co-ordinates within 1.5 A of the starting X-ray structure. Structural features analyzed in the simulations agreed well with the X-ray results except for some surface residues. The Asx chi 2 dihedral distribution and the geometry of hydrogen bonding at reverse turns show differences; possible causes are discussed. The relation between the magnitudes and time-scales of the residue fluctuations and secondary structural features, such as helices beta-sheets and coiled loops, is examined. Significant differences in the residue mobilities between the simulations of the free and substrate-bound states were found in a region of the enzyme that is in direct contact with the substrate and in a region that is distant from the active-site cleft. The dynamic behavior of the structural water molecules is analyzed by examining the correlation between the fluctuations of the water oxygens and the lysozyme heavy-atoms to which they are hydrogen-bonded.

Studies of the histidine residues of triose phosphate isomerase by proton magnetic resonance and x-ray crystallography

A high resolution proton magnetic resonance study of triose phosphate isomerase (E.C. 5.3.1.1) from both rabbit and chicken is reported. The He1 and Hδ2 proton resonances of five histidines in the chicken enzyme, and one histidine in the rabbit enzyme, are observed to titrate in the pH range 5·4 to 9. The known amino acid sequences and the crystal structure determined by Banner et al. (1975), have now been used to assign the resonances of histidine 100 in both enzymes and histidine 195 in the chicken enzyme. Details of the environments of the histidine residues are presented. It is concluded that three conserved histidines, 95, 115 and 185 do not titrate in the pH range studied. Histidine 100 (and to a lesser extent histidine 195 in the chicken enzyme) is perturbed by the addition of ligands such as 2-phosphoglycollate, d -glycerol-3-phosphate and dihydroxyacetone phosphate but is not perturbed by known inhibitors such as orthophosphate, pyrophosphate and phenyl phosphate. Members of the former set of ligands thus may bring about a change in conformation. Slowly exchanging peptide NH protons which resonate in the same region as the He1 and Hδ2 histidine resonances could be eliminated by unfolding the protein and refolding in 2H2O. This procedure simplifies the spectrum and also shows that peptide NH resonances cover a large range and extend throughout the aromatic region.

A critical evaluation of the predicted and X-ray structures of α-lactalbumin.

The rapidly increasing availability of protein amino-acid sequences, many of which have been determined from the corresponding gene sequences, has intensified interest in the prediction of related protein structures when the three-dimensional structure of another member of the family is known. The study of bovine α-Lactalbumin provides a classic example in which the three-dimensional structure was predicted, first by Browneet al. (1969) and later by Warmeet al. (1974), from the three-dimensional structure of hen-egg-white lysozyme (Blakeet al., 1965), taking into account the striking relationship between the amino acid sequences of the two proteins. A comprehensive comparison of these models with the structure of baboon α-Lactalbumin derived from X-ray crystallography (Acharyaet al., 1989) is presented. The models mostly compare well with the experimentally determined structure except in the flexible C-terminal region of the molecule (rms deviation on Cα of residues 1–95, 1.1 Å).