Biomedical subjects
Publications and source records attributed to E N Baker.
The purification of bovine lactoferrin, its crystallization at low ionic strength, and preliminary X-ray crystallographic data are reported. The crystals, which grow from a two-phase system, are radiation-stable and suitable for a medium-resolution X-ray analysis. They are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 138.4 A, b = 87.1 A, c = 73.6 A, and one protein molecule in the asymmetric unit.
An accurate three-dimensional structure is known for papain (1.65 A resolution) and actinidin (1.7 A). A detailed comparison of these two structures was performed to determine the effect of amino acid changes on the conformation. It appeared that, despite only 48% identity in their amino acid sequence, different crystallization conditions and different X-ray data collection techniques, their structures are surprisingly similar with a root-mean-square difference of 0.40 A between 76% of the main-chain atoms (differences less than 3 sigma). Insertions and deletions cause larger differences but they alter the conformation over a very limited range of two to three residues only. Conformations of identical side-chains are generally retained to the same extent as the main-chain conformation. If they do change, this is due to a modified local environment. Several examples are described. Spatial positions of hydrogen bonds are conserved to a greater extent than are the specific groups involved. The greatest structural similarity is found for the active site residues of papain and actinidin, for the internal water molecules and for the main-chain conformation of residues in alpha-helices and anti-parallel beta-sheet structure. This was reflected also in the similarity of the temperature factors. It suggests that the secondary structural elements form the skeleton of the molecule and that their interaction is the main factor in directing the fold of the polypeptide chain. Therefore, substitution of residues in the skeleton will, in general, have the most drastic effect on the conformation of the protein molecule. In papain and actinidin, some main-chain-side-chain hydrogen bonds are also strongly conserved and these may determine the folding of non-repetitive parts of the structure. Furthermore, we included primary structure information for three homologous thiol proteases: stem bromelain, and the cathepsins B and H. By combining the three-dimensional structural information for papain and actinidin with sequence homologies and identities, we conclude that the overall folding pattern of the polypeptide chain is grossly the same in all five proteases, and that they utilize the same catalytic mechanism.
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The structure of the blue copper protein, azurin, from Alcaligenes denitrificans has been determined from an electron density map at a nominal resolution of 3.0 A. Four isomorphous heavy-atom derivatives, prepared with KAu(CN)2, uranyl acetate, Hg(NH3)2Cl2 and KAu(CN)2 + uranyl acetate (a double derivative) were used to calculate phases by the method of isomorphous replacement. The overall figure of merit was 0.61. The two molecules in the asymmetric unit are related by an approximate 2-fold axis. Independent interpretations of the density were made for the two molecules, and the structures have since been partially refined. After 12 refinement cycles, using the Hendrickson-Konnert restrained least-squares program, the R factor is 0.318 for data to 2.5 A resolution and there are no major conformational differences between the two molecules. Refinement is continuing. Eight extended strands of the polypeptide chain form a beta-barrel structure whose topology is the same as that of plastocyanin and the alternative folding proposed for Pseudomonas aeruginosa azurin. As in the latter two proteins, the copper atom forms three short bonds, with His-46 N delta 1, His117 N delta 1 and Cys112 S gamma, and one longer bond, with Met121 S delta, these four ligands forming a very distorted tetrahedron. A possible additional interaction, between copper and the carbonyl oxygen of Gly45, cannot be discounted at the present stage of the analysis. A surface hydrophobic patch, around the edge of the imidazole ring of His117 appears the most likely electron transfer locus. The sequences of azurin and plastocyanin have been aligned and the homology between the two proteins is discussed.
The kinetic parameters kcat, Km and kcat/Km, have been determined for the actinidin-catalysed hydrolyses of N-substituted amino acid esters and amides and compared to the corresponding values for papain (EC 3.4.22.2). Substrates with aromatic N-substituents have lower kcat/Km values for actinidin (EC 3.4.22.14); the difference is much smaller for substrates with aliphatic substituents. The lower kcat/Km values for actinidin generally correspond to higher Km values suggesting that the strength of substrate binding differs between the two enzymes. This difference is explained in terms of the differences in the substrate binding sites found in X-ray crystallographic studies.
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Structure-function relationships for transferrins are discussed in the light of recent X-ray crystal structure determinations. A common folding pattern into two lobes, each comprising two domains is adopted; this allows the tight, but reversible binding of iron. Uptake and release of iron involve substantial domain movements which open and close the binding clefts. The iron binding sites are similar and the key role of the CO3(2-) anion bound with each Fe3+ can now be understood; structural differences near the iron binding sites suggest reasons for the different binding properties of serum transferrin and lactoferrin. The glycan moieties do not appear to affect the protein structure or metal binding properties; they are not clearly seen in the X-ray analyses but have been modelled. The accommodation of different metals and anions is illustrated by the crystal structures of Cu2+ and oxalate-substituted lactoferrins; Al3+ binding is of particular interest. New results on transferrin-receptor interactions with transferrin, and melanotransferrin and an invertebrate transferrin (both of which have defective C-terminal binding sites), emphasize possible functional differences between the two lobes. The availability of site-specific mutants of both transferrin and lactoferrin now offers the opportunity to probe the structural determinants of iron binding, iron release, and receptor binding.