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E N Baker

Publications and source records attributed to E N Baker.

At least 19 recordsLinked to original sources
PubMed2006

Purification, crystallization and preliminary X-ray analysis of Mycobacterium tuberculosisfolylpolyglutamate synthase (MtbFPGS).

The gene encoding Mycobacterium tuberculosis FPGS (MtbFPGS; Rv2447c) has been cloned and the protein (51 kDa) expressed in Escherichia coli. The purified protein was crystallized either by the batch method in the presence of adenosine diphosphate (ADP) and CoCl2 or by vapour diffusion in the presence of ADP, dihydrofolate and CaCl2. X-ray diffraction data to approximately 2.0 and 2.6 A resolution were collected at the Stanford Synchrotron Radiation Laboratory (SSRL) for crystals grown under the respective conditions. Both crystals belong to the cubic space group P2(1)3, with a unit-cell parameter of 112.6 and 111.8 A, respectively. Structure determination is proceeding.

Bacterial Proteins↗
PubMed2005

Molecular structure, binding properties and dynamics of lactoferrin.

Lactoferrin (Lf), a prominent protein in milk, many other secretory fluids and white blood cells, is a monomeric, 80-kDa glycoprotein, with a single polypeptide chain of about 690 amino acid residues. Amino acid sequence relationships place it in the wider transferrin family. Crystallographic analyses of human Lf, and of the Lfs from cow, horse, buffalo and camel, reveal a highly conserved three-dimensional structure, but with differences in detail between species. The molecule is folded into homologous N- and C-terminal lobes, each comprising two domains that enclose a conserved iron binding site. Iron binding and release is accompanied by domain movements that close or open the sites, and is influenced by cooperative interactions between the lobes. Patches of high positive charge on the surface contribute to other binding properties, but the attached glycan chains appear to have little impact on structure and function.

Animals↗
PubMed2003

The TB structural genomics consortium: a resource for Mycobacterium tuberculosis biology.

The TB Structural Genomics Consortium is an organization devoted to encouraging, coordinating, and facilitating the determination and analysis of structures of proteins from Mycobacterium tuberculosis. The Consortium members hope to work together with other M. tuberculosis researchers to identify M. tuberculosis proteins for which structural information could provide important biological information, to analyze and interpret structures of M. tuberculosis proteins, and to work collaboratively to test ideas about M. tuberculosis protein function that are suggested by structure or related to structural information. This review describes the TB Structural Genomics Consortium and some of the proteins for which the Consortium is in the progress of determining three-dimensional structures.

Amino Acid Sequence↗
PubMed2003

Human milk lactoferrin is a serine protease that cleaves Haemophilus surface proteins at arginine-rich sites.

Lactoferrin is a member of the lactotransferrin family of non-haem, iron-binding glycoproteins and is found at high concentrations in all human secretions, where it plays a major role in mucosal defence. In recent work, we observed that lactoferrin has proteolytic activity and attenuates the pathogenic potential of Haemophilus influenzae by cleaving and removing two putative colonization factors, namely the IgA1 protease protein and the Hap adhesin. Experiments with protease inhibitors further suggested that lactoferrin may belong to a serine protease family. In the present study we explored the mechanism of lactoferrin protease activity and discovered that mutation of either Ser259 or Lys73 results in a dramatic decrease in proteolysis. Examination of the crystal structure revealed that these two residues are located in the N-terminal lobe of the protein, adjacent to a 12-15 A cleft that separates the N-lobe and the C-lobe and that can readily accommodate large polypeptide substrates. In additional work, we found that lactoferrin cleaves IgA1 protease at an arginine-rich region defined by amino acids 1379-1386 (RRSRRSVR) and digests Hap at an arginine-rich sequence between amino acids 1016 and 1023 (VRSRRAAR). Based on our results, we conclude that lactoferrin is a serine protease capable of cleaving arginine-rich sequences. We speculate that Ser259 and Lys73 form a catalytic dyad, reminiscent of a number of bacterial serine proteases. In addition, we speculate that lactoferrin may cleave arginine-rich sequences in a variety of microbial virulence proteins, contributing to its long-recognized antimicrobial properties.

Amino Acid Sequence↗
PubMed2001

The role of beta chains in the control of the hemoglobin oxygen binding function: chimeric human/mouse proteins, structure, and function.

By using transgenic methodologies, we have produced a number of mouse/human chimeric hemoglobins containing adult mouse and human embryonic globin chains. A detailed analysis of the oxygen binding properties of these proteins identifies the dominant role played by the specific beta-type globin chains in the control of the oxygen binding characteristics. Further analysis traces the origins of these effects to alterations in the properties of the T states of these proteins. The human zeta/mouse beta chimeric protein has been crystallized, and its structure has been determined by X-ray diffraction to a resolution of 2.1 A with R (R(free)) values of 21.6% (24.9%). Close examination of the structure indicates that the subunit interfaces contain contacts which, although different from those present in either the parent human or the parent mouse proteins, retain the overall stabilizing interactions seen in other R state hemoglobins.

Adult↗
PubMed2001

Crystal structures of the precursor form of glucose-fructose oxidoreductase from Zymomonas mobilis and its complexes with bound ligands.

The NADP(H)-dependent enzyme glucose-fructose oxidoreductase (GFOR) is a classic example of a redox protein that is translocated across a membrane in fully folded form. GFOR is synthesized in the cytoplasm with a 52-residue signal peptide, giving a precursor form, preGFOR, that is fully active and has its cofactor tightly bound. A twin-arginine motif in the signal peptide directs it to a Sec-independent pathway by which it is translocated, in fully folded form, into the periplasm where it functions to produce sorbitol for osmoprotection. We have determined the crystal structures of four different forms of preGFOR, (i) oxidized preGFOR, with succinate bound in the active site, (ii) oxidized preGFOR with glycerol bound, (iii) reduced preGFOR in 0.3 M glucose, and (iv) reduced preGFOR in 1.5 M sorbitol, at resolutions of 2.2, 2.05, 2.5, and 2.6 A, respectively. In all four crystal structures, the signal peptide is disordered, implying a flexibility that may be important for its interaction with the translocation apparatus; a factor contributing to this disorder may be the high positive charge of the protein surface in the region where the signal peptide emerges. This may disfavor a stable association between the signal peptide and the rest of the protein. The crystal structures show that the mature enzyme portion of preGFOR is identical to native GFOR, in structure and cofactor binding, explaining the enzymatic activity of the precursor form. In the glycerol complex, preGFOR(gll), a bound glycerol molecule models the binding of the glucose substrate, with its O1 atom hydrogen bonded to the essential acid/base catalyst, Tyr269, and C1 only 3 A from C4 of the nicotinamide. In the glucose-soaked structure, preGFOR(glu), we identify a conformational change of the nearby Lys181 that probably results from the oxidation of glucose to gluconolactone, and functions to prevent rebinding of glucose prior to the binding of fructose. In this conformational change, the Lys181 side chain moves closer to the nicotinamide ring, stabilized by its increased negative charge.

Catalysis↗
PubMed2001

Structure of HisF, a histidine biosynthetic protein from Pyrobaculum aerophilum.

HisF (imidazole glycerol phosphate synthase) is an important branch-point enzyme in the histidine biosynthetic pathway of microorganisms. Because of its potential relevance for structure-based drug design, the crystal structure of HisF from the hyperthermophilic archaeon Pyrobaculum aerophilum has been determined. The structure was determined by molecular replacement and refined at 2.0 A resolution to a crystallographic R factor of 20.6% and a free R of 22.7%. The structure adopts a classic (beta/alpha)(8) barrel fold and has networks of surface salt bridges that may contribute to thermostability. The active site is marked out by the presence of two bound phosphate ions and two glycerol molecules that delineate a long groove at one end of the (beta/alpha)(8) barrel. The two phosphate ions, 17 A apart, are bound to sequence-conserved structural motifs that seem likely to provide much of the specificity for the two phosphate groups of the HisF substrate. The two glycerol molecules bind in the vicinity of other sequence-conserved residues that are likely to be involved in binding and/or catalysis. Comparisons with the homologous HisF from Thermatoga maritima reveal a displaced loop that may serve as a lid over the active site.

Amino Acid Sequence↗
PubMed2001

Ligand variation in the transferrin family: the crystal structure of the H249Q mutant of the human transferrin N-lobe as a model for iron binding in insect transferrins.

Proteins of the transferrin (Tf) family play a central role in iron homeostasis in vertebrates. In vertebrate Tfs, the four iron-binding ligands, 1 Asp, 2 Tyr, and 1 His, are invariant in both lobes of these bilobal proteins. In contrast, there are striking variations in the Tfs that have been characterized from insect species; in three of them, sequence changes in the C-lobe binding site render it nonfunctional, and in all of them the His ligand in the N-lobe site is changed to Gln. Surprisingly, mutagenesis of the histidine ligand, His249, to glutamine in the N-lobe half-molecule of human Tf (hTf/2N) shows that iron binding is destabilized and suggests that Gln249 does not bind to iron. We have determined the crystal structure of the H249Q mutant of hTf/2N and refined it at 1.85 A resolution (R = 0.221, R(free) = 0.246). The structure reveals that Gln249 does coordinate to iron, albeit with a lengthened Fe-Oepsilon1 bond of 2.34 A. In every other respect, the protein structure is unchanged from wild-type. Examination of insect Tf sequences shows that the K206.K296 dilysine pair, which aids iron release from the N-lobes of vertebrate Tfs, is not present in the insect proteins. We conclude that substitution of Gln for His does destabilize iron binding, but in the insect Tfs this is compensated by the loss of the dilysine interaction. The combination of a His ligand with the dilysine pair in vertebrate Tfs may have been a later evolutionary development that gives more sophisticated pH-mediated control of iron release from the N-lobe of transferrins.

Amino Acid Sequence↗
PubMed2001

The structure of truncated recombinant human bile salt-stimulated lipase reveals bile salt-independent conformational flexibility at the active-site loop and provides insights into heparin binding.

Human bile salt-stimulated lipase (BSSL), which is secreted from the pancreas into the digestive tract and from the lactating mammary gland into human milk, is important for the effective absorption of dietary lipids. The dependence of BSSL on bile acids for activity with water-insoluble substrates differentiates it from other lipases. We have determined the crystal structure of a truncated variant of human BSSL (residues 1-5.8) and refined it at 2.60 A resolution, to an R-factor of 0.238 and R(free) of 0.275. This variant lacks the C-terminal alpha-helix and tandem C-terminal repeat region of native BSSL, but retains full catalytic activity. A short loop (residues 115-126) capable of occluding the active-site (the active site loop) is highly mobile and exists in two conformations, the most predominant of which leaves the active-site open for interactions with substrate. The bile salt analogue 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonic acid (CHAPS) was present in the crystallisation medium, but was not observed bound to the enzyme. However, the structure reveals a sulfonate group from the buffer piperizine ethane sulfonic acid (PIPES), making interactions with Arg63 and His115. His115 is part of the active-site loop, indicating that the loop could participate in the binding of a sulphate group from either the glycosaminoglycan heparin (known to bind BSSL) or a bile acid such as deoxycholate. Opening of the 115-126 active-site loop may be cooperatively linked to a sulphate anion binding at this site. The helix bundle domain of BSSL (residues 319-398) exhibits weak electron density and high temperature factors, indicating considerable structural mobility. This domain contains an unusual Asp:Glu pair buried in a hydrophobic pocket between helices alpha(H) and alpha(K) that may be functionally important. We have also solved the structure of full-length glycosylated human BSSL at 4.1 A resolution, using the refined coordinates of the truncated molecule as a search model. This structure reveals the position of the C-terminal helix, missing in the truncated variant, and also shows the active-site loop to be in a closed conformation.

Animals↗
PubMed2001

LISA: an intranet-based flexible database for protein crystallography project management.

The increase in the number of projects carried out in protein crystallography laboratories has emphasized the need for effective management of project information and data. To meet this need, a flexible web-accessible database for protein crystallography project management (LISA) has been developed using the open-source software MySQL and PHP4. The database contains information about all aspects of structure-determination projects, including primer and plasmid sequences, protein expression, purification and crystallization results, structure coordinate files and resultant publications. The database web pages include links to relevant servers and contain, in addition, tools for processing stored information. The software package is freely available.

Crystallography↗
PubMed2001

Folate-binding triggers the activation of folylpolyglutamate synthetase.

Folic acid is an essential vitamin for normal cell growth, primarily through its central role in one-carbon metabolism. Folate analogs (antifolates) are targeted at the same reactions and are widely used as therapeutic drugs for cancer and bacterial infections. Effective retention of folates in cells and the efficacy of antifolate drugs both depend upon the addition of a polyglutamate tail to the folate or antifolate molecule by the enzyme folylpolyglutamate synthetase (FPGS). The reaction mechanism involves the ATP-dependent activation of the free carboxylate group on the folate molecule to give an acyl phosphate intermediate, followed by attack by the incoming L-glutamate substrate. FPGS shares a number of structural and mechanistic details with the bacterial cell wall ligases MurD, MurE and MurF, and these enzymes, along with FPGS, form a subfamily of the ADP-forming amide bond ligase family. High-resolution crystallographic analyses of binary and ternary complexes of Lactobacillus casei FPGS reveal that binding of the first substrate (ATP) is not sufficient to generate an active enzyme. However, binding of folate as the second substrate triggers a large conformational change that activates FPGS and allows the enzyme to adopt a form that is then able to bind the third substrate, L-glutamate, and effect the addition of a polyglutamate tail to the folate.

Adenosine Triphosphate↗
PubMed2001

Crystal structure of methylmalonyl-coenzyme A epimerase from P. shermanii: a novel enzymatic function on an ancient metal binding scaffold.

BACKGROUND: Methylmalonyl-CoA epimerase (MMCE) is an essential enzyme in the breakdown of odd-numbered fatty acids and of the amino acids valine, isoleucine, and methionine. Present in many bacteria and in animals, it catalyzes the conversion of (2R)-methylmalonyl-CoA to (2S)-methylmalonyl-CoA, the substrate for the B12-dependent enzyme, methylmalonyl-CoA mutase. Defects in this pathway can result in severe acidosis and cause damage to the central nervous system in humans. RESULTS: The crystal structure of MMCE from Propionibacterium shermanii has been determined at 2.0 A resolution. The MMCE monomer is folded into two tandem betaalphabetabetabeta modules that pack edge-to-edge to generate an 8-stranded beta sheet. Two monomers then pack back-to-back to create a tightly associated dimer. In each monomer, the beta sheet curves around to create a deep cleft, in the floor of which His12, Gln65, His91, and Glu141 provide a binding site for a divalent metal ion, as shown by the binding of Co2+. Modeling 2-methylmalonate into the active site identifies two glutamate residues as the likely essential bases for the epimerization reaction. CONCLUSIONS: The betaalphabetabetabeta modules of MMCE correspond with those found in several other proteins, including bleomycin resistance protein, glyoxalase I, and a family of extradiol dioxygenases. Differences in connectivity are consistent with the evolution of these very different proteins from a common precursor by mechanisms of gene duplication and domain swapping. The metal binding residues also align precisely, and striking structural similarities between MMCE and glyoxalase I suggest common mechanisms in their respective epimerization and isomerization reactions.

Binding Sites↗
PubMed2001

Immunological and biochemical characterization of streptococcal pyrogenic exotoxins I and J (SPE-I and SPE-J) from Streptococcus pyogenes.

Recently, we described the identification of novel streptococcal superantigens (SAgs) by mining the Streptococcus pyogenes M1 genome database at Oklahoma University. Here, we report the cloning, expression, and functional analysis of streptococcal pyrogenic exotoxin (SPE)-J and another novel SAg (SPE-I). SPE-I is most closely related to SPE-H and staphylococcal enterotoxin I, whereas SPE-J is most closely related to SPE-C. Recombinant forms of SPE-I and SPE-J were mitogenic for PBL, both reaching half maximum responses at 0.1 pg/ml. Evidence from binding studies and cell aggregation assays using a human B-lymphoblastoid cell line (LG-2) suggests that both toxins exclusively bind to the polymorphic MHC class II beta-chain in a zinc-dependent mode but not to the generic MHC class II alpha-chain. The results from analysis by light scattering indicate that SPE-J exists as a dimer in solution above concentrations of 4.0 mg/ml. Moreover, SPE-J induced a rapid homotypic aggregation of LG-2 cells, suggesting that this toxin might cross-link MHC class II molecules on the cell surface by building tetramers of the type HLA-DRbeta-SPE-J-SPE-J-HLA-DRbeta. SPE-I preferably stimulates T cells bearing the Vbeta18.1 TCR, which is not targeted by any other known SAG: SPE-J almost exclusively stimulates Vbeta2.1 T cells, a Vbeta that is targeted by several other streptococcal SAgs, suggesting a specific role for this T cell subpopulation in immune defense. Despite a primary sequence diversity of 51%, SPE-J is functionally indistinguishable from SPE-C and might play a role in streptococcal disease, which has previously been addressed to SPE-C.

Amino Acid Sequence↗
PubMed2001

Subunit dissociation and reassociation leads to preferential crystallization of haemoglobin Bart's (gamma4) from solutions of human embryonic haemoglobin Portland (zeta2gamma2) at low pH.

A variety of human haemoglobins (Hbs) are produced at different stages of human development, including three embryonic Hbs, foetal Hb and adult Hb. All are heterotetramers. During crystallization experiments on human embryonic Hb Portland (zeta(2)gamma(2)), it was discovered by crystallographic and biochemical analysis that the homotetramer Hb Bart's (gamma(4)) preferentially crystallizes from zeta(2)gamma(2) solutions below pH 5. This results from dissociation of Hb Portland into gamma(2) dimers and zeta monomers and has interesting implications for subunit interactions and tetramer stability in Hbs. It also makes possible a full crystallographic analysis of Hb Bart's, which is of considerable medical significance because of its presence in the red blood cells of millions of people worldwide who suffer from alpha-thalassaemia.

Chromatography, High Pressure Liquid↗
PubMed2001

Expression, crystallization and preliminary characterization of methylmalonyl coenzyme A epimerase from Propionibacterium shermanii.

Methylmalonyl-CoA epimerase (MMCE) is an enzyme that interconverts the R and S epimers of methylmalonyl-CoA in the pathway that links propionyl-CoA with succinyl-CoA. This is used for both biosynthetic and degradative processes, including the breakdown of odd-numbered fatty acids and some amino acids. The enzyme has been expressed in Escherichia coli both as the native enzyme and as its selenomethionine (SeMet) derivative. Crystals of both forms have been obtained by vapour diffusion using monomethylether PEG 2000 as precipitant. The native MMCE crystals are orthorhombic, with unit-cell parameters a = 56.0, b = 114.0, c = 156.0 A, and the SeMet-MMCE crystals are monoclinic, with unit-cell parameters a = 43.6, b = 78.6, c = 89.4 A, beta = 92.0 degrees; both diffract to better than 2.8 A resolution.

Crystallization↗
PubMed2001

Removing a hydrogen bond in the dimer interface of Escherichia coli manganese superoxide dismutase alters structure and reactivity.

Among manganese superoxide dismutases, residues His30 and Tyr174 are highly conserved, forming part of the substrate access funnel in the active site. These two residues are structurally linked by a strong hydrogen bond between His30 NE2 from one subunit and Tyr174 OH from the other subunit of the dimer, forming an important element that bridges the dimer interface. Mutation of either His30 or Tyr174 in Escherichia coli MnSOD reduces the superoxide dismutase activity to 30--40% of that of the wt enzyme, which is surprising, since Y174 is quite remote from the active site metal center. The 2.2 A resolution X-ray structure of H30A-MnSOD shows that removing the Tyr174-->His30 hydrogen bond from the acceptor side results in a significant displacement of the main-chain segment containing the Y174 residue, with local rearrangement of the protein. The 1.35 A resolution structure of Y174F-MnSOD shows that disruption of the same hydrogen bond from the donor side has much greater consequences, with reorientation of F174 having a domino effect on the neighboring residues, resulting in a major rearrangement of the dimer interface and flipping of the His30 ring. Spectroscopic studies on H30A, H30N, and Y174F mutants show that (like the previously characterized Y34F mutant of E. coli MnSOD) all lack the high pH transition of the wt enzyme. This observation supports assignment of the pH sensitivity of MnSOD to coordination of hydroxide ion at high pH rather than to ionization of the phenolic group of Y34. Thus, mutations near the active site, as in the Y34F mutant, as well as at remote positions, as in Y174F, similarly affect the metal reactivity and alter the effective pK(a) for hydroxide ion binding. These results imply that hydrogen bonding of the H30 imidazole N--H group plays a key role in substrate binding and catalysis.

Amino Acid Substitution↗
PubMed2001

Crystal structure of the NADP(H)-dependent ketose reductase from Bemisia argentifolii at 2.3 A resolution.

Polyhydric alcohols are widely found in nature and can be accumulated to high concentrations as a protection against a variety of environmental stresses. It is only recently, however, that these molecules have been shown to be active in protection against heat stress, specifically in the use of sorbitol by the silverleaf whitefly, Bemisia argentifolii. We have determined the structure of the enzyme responsible for production of sorbitol in Bemisia argentifolii, NADP(H)-dependent ketose reductase (BaKR), to 2.3 A resolution. The structure was solved by multiwavelength anomalous diffraction (MAD) using the anomalous scattering from two zinc atoms bound in the structure, and was refined to an R factor of 21.9 % (R(free)=25.1 %). BaKR belongs to the medium-chain dehydrogenase family and its structure is the first for the sorbitol dehydrogenase branch of this family. The enzyme is tetrameric, with the monomer having a very similar fold to the alcohol dehydrogenases (ADHs). Although the structure determined is for the apo form, a phosphate ion in the active site marks the likely position for the adenyl phosphate of NADP(H). The catalytic zinc ion is tetrahedrally coordinated to Cys41, His66, Glu67 and a water molecule, in a modification of the zinc site usually found in ADHs. This modified zinc site seems likely to be a conserved feature of the sorbitol dehydrogenase sub-family. Comparisons with other members of the ADH family have also enabled us to model a ternary complex of the enzyme, and suggest how structural differences may influence coenzyme binding and substrate specificity in the reduction of fructose to sorbitol.

Alcohol Oxidoreductases↗