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E N Baker

Publications and source records attributed to E N Baker.

At least 19 recordsLinked to original sources
PubMed1992

Preliminary crystallographic studies of the amino terminal half of human lactoferrin in its iron-saturated and iron-free forms.

The amino terminal half of human lactoferrin (LfN) produced from transfected baby hamster kidney cells has been crystallized in its iron-saturated and iron-free forms. The crystals of glycosylated LfN and deglycosylated LfN are monoclinic, space group C2, with cell dimensions a = 133.0 A, b = 58.3 A, c = 58.3 A, alpha = 90.0 degrees, beta = 114.7 degrees, gamma = 90.0 degrees, and one molecule per asymmetric unit. Crystals of apo LfN have also been prepared using deglycosylated protein. These crystals are tetragonal, space group P4(1)2(1)2 (or P4(3)2(1)2), with cell dimensions of a = b = 58.4 A and c = 217.2 A and one molecule per asymmetric unit. Both the iron-saturated and the iron-free crystals are suitable for high resolution X-ray analysis.

Apoproteins↗
PubMed1992

Studies of the N-terminal half of human lactoferrin produced from the cloned cDNA demonstrate that interlobe interactions modulate iron release.

The factors influencing iron binding and release by lactoferrin have been addressed by comparison of the native full length molecule (Lf) with the N-terminal half of human lactoferrin (LfN) produced from the cloned cDNA expressed in baby hamster kidney (BHK) cells. The coding sequences for LfN were inserted into the expression vector pNUT between the metallothionein promoter and the human growth hormone transcription termination sequences. Transformed BHK cells were grown in roller bottles where concentrations of LfN as high as 35 mg/liter were obtained. The pure protein, produced by the transformed BHK cells, was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protein blotting and immunodetection, N-terminal sequence analysis, UV-visible spectroscopy, electron spin resonance spectroscopy, and measurements of metal binding and release. By these criteria LfN was found to be correctly processed, glycosylated, and able to bind iron reversibly. Both UV-visible and electron spin resonance spectra of the half molecule were very similar to those of native lactoferrin and the full length lactoferrin produced in BHK cells, but there were marked differences in the pH at which iron release occurred. Iron release from LfN occurs in the pH range 6.0-4.0, compared with 4.0-2.5 for native lactoferrin and 6.2-4.0 for transferrin. These results suggest that the more facile release of iron from LfN compared with native lactoferrin results from the absence of stabilizing contacts between the N- and C-terminal halves and that the characteristic difference in pH stability between lactoferrins and transferrins is due primarily to differences in these interactions.

Amino Acid Sequence↗
PubMed1992

Anion binding by human lactoferrin: results from crystallographic and physicochemical studies.

The anion-binding properties of lactoferrin (Lf), with Fe3+ or Cu2+ as the associated metal ion, have been investigated by physicochemical and crystallographic techniques. These highlight differences between the two sites and in the anion-binding behavior when different metals are bound. Carbonate, oxalate, and hybrid carbonate-oxalate complexes have been prepared and their characteristic electronic and EPR spectra recorded. Oxalate can displace carbonate from either one or both anion sites of Cu2(CO3)2Lf, depending on the oxalate concentration, but no such displacement occurs for Fe2(CO3)2Lf. Addition of oxalate and the appropriate metal ion to apoLf under carbonate-free conditions gives dioxalate complexes with both Fe3+ and Cu2+, except when traces of EDTA remain associated with the protein, when hybrid complexes M2(CO3)(C2O4)Lf can result. The anion sites in the crystal structures of Fe2(CO3)2Lf, Cu2-(CO3)2Lf, and Cu2(CO3)(C2O4)Lf, refined at 2.2, 2.1, and 2.2 A, respectively, have been compared. In every case, the anion is hydrogen bonded to the N-terminus of helix 5, an associated arginine side chain, and a nearby threonine side chain. The carbonate ion binds in bidentate fashion to the metal, except in the N-lobe site of dicupric lactoferrin, where it is monodentate; the difference arises from slight movement of the metal ion. The hybrid complex shows that the oxalate ion binds preferentially in the C-lobe site, in 1,2-bidentate mode, but with the displacement of several nearby side chains. These observations lead to a generalized model for synergistic anion binding by transferrins.

Binding, Competitive↗
PubMed1992

Metal substitution in transferrins: the crystal structure of human copper-lactoferrin at 2.1-A resolution.

The structural consequences of binding a metal other than iron to a transferrin have been examined by crystallographic analysis of human copper-lactoferrin, Cu2Lf. X-ray diffraction data were collected from crystals of Cu2Lf, using a diffractometer, to 2.6-A resolution, and oscillation photography on a synchrotron source, to 2.1-A resolution. The structure was refined crystallographically, by restrained least-squares methods, starting with a model based on the isomorphous diferric structure from which the ligands, metal ions, anions, and solvent molecules had been deleted. The final model, comprising 5321 protein atoms (691 residues), 2 Cu2+ ions, 2 (bi)carbonate ions, and 308 solvent molecules has good stereochemistry (rms deviation of bond lengths from standard values of 0.018 A) and gives a crystallographic R value of 0.196 for 43,525 reflections in the range 7.5-2.1-A resolution. The copper coordination is different in the two binding sites. In the N-terminal site, the geometry is square pyramidal, with equatorial bonds to Asp 60, Tyr 192, His 253, and a monodentate anion and a longer apical bond to Tyr 92. In the C-terminal site, the geometry is distorted octahedral, with bonds to Asp 395, Tyr 435, Tyr 528, and His 597 and an asymmetrically bidentate anion. The protein structure is the same as for the diferric protein, Fe2Lf, demonstrating that the closure of the protein domains over the metal is the same in each case irrespective of whether Fe3+ or Cu2+ is bound and that copper could be transported and delivered to cells equally well as iron. The differences in metal coordination are achieved by small movements of the metal ion and anion within each binding site, which do not affect the protein structure.

Binding Sites↗
PubMed1992

Human melanotransferrin (p97) has only one functional iron-binding site.

The iron-binding properties of melanotransferrin, the tumour-associated antigen also known as p97, have been investigated by UV/visible and fluorescence spectroscopy, amino acid sequence comparison, and modelling. These show that, in contrast to other transferrins, melanotransferrin binds only one Fe3+ ion per molecule. The binding properties of its N-terminal site are similar to other transferrins, but its C-terminal site does not bind iron at all. The differences can be related to specific amino acid changes in the C-terminal site.

Antigens, Neoplasm↗
PubMed1991

Molecular replacement solution of the structure of apolactoferrin, a protein displaying large-scale conformational change.

The crystal structure of an orthorhombic form of human apolactoferrin (ApoLf) has been determined from 2.8 A diffractometer data by molecular replacement methods. A variety of search models derived from the diferric lactoferrin structure (Fe2Lf) were used to obtain a consistent solution to the rotation function. An R-factor search gave the correct translational solution and the model was refined by rigid-body least-squares refinement (program CORELS). Only three of the four domains were located correctly by this procedure, however; the fourth was finally placed correctly by rotating it manually onto three strands of electron density which were recognized as part of its central beta-sheet. The final model, refined by restrained least-squares methods to an R factor of 0.214 for data in the resolution range 10.0 to 2.8 A, shows a large domain movement in the N-terminal half of the molecule (a 54 degree rotation of domain N2) and smaller domain movements elsewhere, when compared with Fe2Lf. A feature of the crystal structure is that although the ApoLf and Fe2Lf unit cells appear very similar, their crystal packing and molecular structures are quite different.

Apoproteins↗
PubMed1991

Preliminary crystallographic studies of copper(II)- and oxalate-substituted human lactoferrin.

As part of a comparative study on the binding of different metals and anions by human lactoferrin, we have prepared and crystallized: (1) dicupric lactoferrin with Cu2+ and carbonate in each site (Cu2Lf); and (2) a lactoferrin complex with Cu2+ and carbonate in one site, and Cu2+ and oxalate in the other (Cu2oxLf). Crystals of Cu2Lf are orthorhombic: a = 155.9, b = 97.0, c = 56.0 A, space-group P2(1)2(1)2(1); those of Cu2oxLf are also orthorhombici a = 155.9, b = 97.1, c = 56.2 A, space-group P2(1)2(1)2(1). Both are isomorphous with diferric human lactoferrin, Fe2Lf. Diffractometer data to 2.6 A and 2.5 A have been collected for Cu2Lf and Cu2oxLf, respectively. Difference maps show that the main effect of substitution of Cu2+ for Fe3+ is a small shift (0.5 to 1.0 A) in the metal position in each site. For Cu2oxLf the oxalate ion is found to be accommodated in the C-lobe, bound to copper in a bidentate mode, causing only small local changes, in the positions of adjacent Arg and Tyr side-chains.

Binding Sites↗
PubMed1991

Structure, function and flexibility of human lactoferrin.

X-ray structure analyses of four different forms of human lactoferrin (diferric, dicupric, an oxalate-substituted dicupric, and apo-lactoferrin), and of bovine diferric lactoferrin, have revealed various ways in which the protein structure adapts to different structural and functional states. Comparison of diferric and dicupric lactoferrins has shown that different metals can, through slight variations in the metal position, have different stereochemistries and anion coordination without any significant change in the protein structure. Substitution of oxalate for carbonate, as seen in the structure of a hybrid dicupric complex with oxalate in one site and carbonate in the other, shows that larger anions can be accommodated by small side-chain movements in the binding site. The multidomain nature of lactoferrin also allows rigid body movements. Comparison of human and bovine lactoferrins, and of these with rabbit serum transferrin, shows that the relative orientations of the two lobes in each molecule can vary; these variations may contribute to differences in their binding properties. The structure of apo-lactoferrin demonstrates the importance of large-scale domain movements for metal binding and release and suggests that in solution an equilibrium exists between open and closed forms, with the open form being the active binding species. These structural forms are shown to be similar to those seen for bacterial periplasmic binding proteins, and lead to a common model for the various steps in the binding process.

Animals↗
PubMed1990

Apolactoferrin structure demonstrates ligand-induced conformational change in transferrins.

Proteins of the transferrin family, which contains serum transferrin and lactoferrin, control iron levels in higher animals through their very tight (Kapp approximately 10(20)) but reversible binding of iron. These bilobate molecules have two binding sites, one per lobe, each housing one Fe3+ and the synergistic CO3(2-) ion. Crystallographic studies of human lactoferrin and rabbit serum transferrin in their iron-bound forms have characterized their binding sites and protein structure. Physical studies show that a substantial conformational change accompanies iron binding and release. We have addressed this phenomenon through crystal structure analysis of human apolactoferrin at 2.8 A resolution. In this structure the N-lobe binding cleft is wide open, following a domain rotation of 53 degrees, mediated by the pivoting of two helices and flexing of two interdomain polypeptide strands. Remarkably, the C-lobe cleft is closed, but unliganded. These observations have implications for transferrin function and for binding proteins in general.

Animals↗
PubMed1989

Structure of human lactoferrin: crystallographic structure analysis and refinement at 2.8 A resolution.

The structure of human lactoferrin has been refined crystallographically at 2.8 A (1 A = 0.1 nm) resolution using restrained least squares methods. The starting model was derived from a 3.2 A map phased by multiple isomorphous replacement with solvent flattening. Rebuilding during refinement made extensive use of these experimental phases, in combination with phases calculated from the partial model. The present model, which includes 681 of the 691 amino acid residues, two Fe3+, and two CO3(2-), gives an R factor of 0.206 for 17,266 observed reflections between 10 and 2.8 A resolution, with a root-mean-square deviation from standard bond lengths of 0.03 A. As a result of the refinement, two single-residue insertions and one 13-residue deletion have been made in the amino acid sequence, and details of the secondary structure and tertiary interactions have been clarified. The two lobes of the molecule, representing the N-terminal and C-terminal halves, have very similar folding, with a root-mean-square deviation, after superposition, of 1.32 A for 285 out of 330 C alpha atoms; the only major differences being in surface loops. Each lobe is subdivided into two dissimilar alpha/beta domains, one based on a six-stranded mixed beta-sheet, the other on a five-stranded mixed beta-sheet, with the iron site in the interdomain cleft. The two iron sites appear identical at the present resolution. Each iron atom is coordinated to four protein ligands, 2 Tyr, 1 Asp, 1 His, and the specific Co3(2-), which appears to bind to iron in a bidentate mode. The anion occupies a pocket between the iron and two positively charged groups on the protein, an arginine side-chain and the N terminus of helix 5, and may serve to neutralize this positive charge prior to iron binding. A large internal cavity, beyond the Arg side-chain, may account for the binding of larger anions as substitutes for CO3(2-). Residues on the other side of the iron site, near the interdomain crossover strands could provide secondary anion binding sites, and may explain the greater acid-stability of iron binding by lactoferrin, compared with serum transferrin. Interdomain and interlobe interactions, the roles of charged side-chains, heavy-atom binding sites, and the construction of the metal site in relation to the binding of different metals are also discussed.

Amino Acid Sequence↗
PubMed1989

Preliminary crystallographic studies on human apo-lactoferrin in its native and deglycosylated forms.

Human apo-lactoferrin in both native and deglycosylated forms has been purified, and crystals obtained by dialysis against low ionic strength buffer solutions. The crystals of native apo-lactoferrin are orthorhombic, space group P2(1)2(1)2(1) with cell dimensions a = 222.0 A, b = 115.6 A, c = 77.8 A and have two protein molecules per asymmetric unit. Two crystal forms of deglycosylated apo-lactoferrin have been obtained. One is orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 152.1 A, b = 94.6 A, c = 55.8 A. The second is tetragonal, space group I4, with cell dimensions a = b = 189.4 A, c = 55.1 A. Both of the latter have only one molecule per asymmetric unit, and are suitable for high-resolution X-ray structure analysis.

Apoproteins↗
PubMed1988

Structure of azurin from Alcaligenes denitrificans refinement at 1.8 A resolution and comparison of the two crystallographically independent molecules.

The structure of the blue copper protein azurin, from Alcaligenes denitrificans, has been refined crystallographically by restrained least-squares methods. The final crystallographic R value for 21,980 observed reflections to 1.8 A (1 A = 0.1 nm) resolution is 0.157. The asymmetric unit of the crystal contains two independent azurin molecules, the model for which comprises 1973 protein atoms, together with three SO2-4 ions, and 281 water molecules. Comparison of the two molecules shows very high correspondence. For 125 out of 129 residues (excluding only the chain termini, residues 1 to 2 and 128 to 129) the root-mean-square (r.m.s.) deviation in main-chain atom positions is 0.27 A. For other structural parameters r.m.s. deviations are also low; torsion angles 6.5 degrees, hydrogen bond lengths 0.12 A, bonds to copper 0.04 A and bond angles at the copper 3.9 degrees. The only significant differences are at the chain termini and in several loops. Some of these can be attributed to crystal packing effects, others to genuine structural microheterogeneity. Refinement has confirmed that the copper co-ordination is best described as distorted trigonal planar, with strong in-plane bonds to His46 N delta 1, His117 N delta 1 and Cys112 S gamma, and much weaker axial interactions with Met121 S delta and Gly45 C = O. Two N-H...S hydrogen bonds characterize Cys112 S gamma as a thiolate (S-) sulphur and may influence the visible absorption maximum. Atoms in and around the copper site have very low mobility, whereas the most mobile regions of the molecule are the chain termini and some of the connecting loops between secondary structure elements, especially those at the "southern" end, remote from the copper site. Main-chain to side-chain hydrogen bonds supply important stabilizing interactions at the "northern" end. Surface features include the hydrophobic patch around His117, probably important for electron transfer, the SO2-4 site at His83, and the general absence of ion pairs, despite the presence of many charged amino acid residues. The 281 water molecules include 182 that occur as approximately twofold-related pairs. There are no internal water molecules. The water sites common to both azurin molecules include those in surface pockets and some in intermolecular contact regions. They are characterized by relatively low thermal parameters and numerous protein contacts.

Alcaligenes↗
PubMed1988

The structure of 2Zn pig insulin crystals at 1.5 A resolution.

The paper describes the arrangement of the atoms within rhombohedral crystals of 2Zn pig insulin as seen in electron density maps calculated from X-ray data extending to 1.5 A (1 A = 10(-10) m = 10(-1) nm) at room temperature and refined to R = 0.153. The unit cell contains 2 zinc ions, 6 insulin molecules and about 3 x 283 water molecules. The atoms in the protein molecules appear well defined, 7 of the 102 side chains in the asymmetric unit have been assigned alternative disordered positions. The electron density over the water molecules has been interpreted in terms of 349 sites, 217 weighted 1.0, 126 weighted 0.5, 5 at 0.33 and 1 at 0.25 giving ca. 282 molecules. The positions and contacts of all the residues belonging to the two A and B chains of the asymmetric unit are shown first and then details of their arrangement in the two insulin molecules, 1 and 2, which are different. The formation from these molecules of a compact dimer and the further aggregation of three dimers to form a hexamer around two zinc ions, follows. It appears that in the packing of the hexamers in the crystal there are conflicting influences; too-close contacts between histidine B5 residues in neighbouring hexamers are probably responsible for movements of atoms at the beginning of the A chain of one of the two molecules of the dimer that initiate movements in other parts, particularly near the end of the B chain. At every stage of the building of the protein structure, residues to chains of definite conformation, molecules, dimers, hexamers and crystals, we can trace the effect of the packing of like groups to like, aliphatic groups together, aromatic groups together, hydrogen-bonded structures, positive and negative ions. Between the protein molecules, the water is distributed in cavities and channels that are continuous throughout the crystals. More than half the water molecules appear directly hydrogen bonded to protein atoms. These are generally in contact with other water molecules in chains and rings of increasing disorder, corresponding with their movement through the crystals. Within the established crystal structure we survey next the distribution of hydrogen bonds within the protein molecules and between water and protein and water and water; all but eight of the active atoms in the protein form at least one hydrogen bond.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Spectrochemical studies on the blue copper protein azurin from Alcaligenes denitrificans.

Spectroscopic and electrochemical studies, incorporating electronic spectra, electron paramagnetic resonance (EPR) spectra, resonance Raman (RR) spectra, and measurements of the redox potential, have been carried out on the blue copper protein azurin, from Alcaligenes denitrificans. These data are correlated with the refined crystal structure of this azurin and with corresponding data for other blue copper proteins. The electronic spectrum, characterized by an intense (epsilon = 5100 M-1 cm-1) charge-transfer band at 619 nm, the EPR spectral parameters (g perpendicular = 2.059, g parallel of = 2.255, A parallel of = 60 X 10(-4) cm-1), and the resonance Raman spectrum are similar to those obtained from other azurins and from plastocyanins. Both the electronic spectrum and the EPR spectrum are unchanged over the pH range 4-10.5, but major changes occur above pH 12 and below pH 3.5. A small reversible change occurs at pH approximately 11.4. In the RR spectrum the Cu-S stretching mode is shown to contribute to all of the five principal RR peaks. Deuterium substitution produces shifts in at least seven of the peaks; these shifts may be attributable, at least in part, to the NH...S hydrogen bond to the copper-ligated Cys-112. Measurements of the redox potential, using spectroelectrochemical methods, over the temperature range 4.8-40.0 degrees C, give values for delta H0' and delta S0' of -55.6 kJ mol-1 and -97.0 J K-1 mol-1, respectively. The redox potential of A. denitrificans azurin at pH 7.0, Eo', is 276 mV. These data are interpreted in terms of a copper site, in azurin, comprising three strong bonds, in an approximately trigonal plane, from Cys-112, His-46, and His-117 and much longer axial approaches from Met-121 and the peptide carbonyl oxygen of Gly-45. Spectral differences within the azurin family and between azurin and plastocyanin are attributed to differences in the strengths of these axial interactions. Likewise, the distinctly lower Eo values for azurins, as compared with plastocyanins, are related to the more copper(II)-like site in azurin [with a weaker Cu-S(Met) interaction and a Cu-O interaction not found in plastocyanin]. On the other hand, the relative constancy of the EPR parameters between azurin and plastocyanin suggests they are not strongly influenced by weakly interacting axial groups.

Alcaligenes↗
PubMed1987

Structure of human lactoferrin at 3.2-A resolution.

The three-dimensional structure of human milk lactoferrin, a member of the transferrin family, has been determined crystallographically at 3.2-A resolution. The molecule has two-fold internal homology. The N- and C-terminal halves form two separate globular lobes, connected by a short alpha-helix, and carry one iron-binding site each. Each lobe has the same folding, based on two domains of similar supersecondary structure, with the iron site at the domain interface. Each iron atom is coordinated by four protein ligands: two tyrosines, one histidine, and one aspartate. A probable CO3(2-) (or HCO3-) ion is suggested by the electron density, bound to iron and adjacent to an arginine side chain and a helix N terminus. The protein folding and location of the binding sites show marked similarities with those of other binding proteins, notably the sulfate-binding protein from Salmonella typhimurium.

Binding Sites↗
PubMed1986

Preliminary crystallographic studies on bovine lactoferrin.

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.

Animals↗
PubMed1985

Thiol proteases. Comparative studies based on the high-resolution structures of papain and actinidin, and on amino acid sequence information for cathepsins B and H, and stem bromelain.

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.

Amino Acid Sequence↗