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Wolfram Saenger

Publications and source records attributed to Wolfram Saenger.

At least 19 recordsLinked to original sources
PubMed2006

Where water is oxidized to dioxygen: structure of the photosynthetic Mn4Ca cluster.

The oxidation of water to dioxygen is catalyzed within photosystem II (PSII) by a Mn(4)Ca cluster, the structure of which remains elusive. Polarized extended x-ray absorption fine structure (EXAFS) measurements on PSII single crystals constrain the Mn(4)Ca cluster geometry to a set of three similar high-resolution structures. Combining polarized EXAFS and x-ray diffraction data, the cluster was placed within PSII, taking into account the overall trend of the electron density of the metal site and the putative ligands. The structure of the cluster from the present study is unlike either the 3.0 or 3.5 angstrom-resolution x-ray structures or other previously proposed models.

Binding Sites↗
PubMed2006

Function of two beta-carotenes near the D1 and D2 proteins in photosystem II dimers.

The antenna proteins in photosystem II (PSII) not only promote energy transfer to the photosynthetic reaction center (RC) but provide also an efficient cation sink to re-reduce chlorophyll a if the electron transfer (ET) from the Mn-cluster is inhibited. Using the newest PSII dimer crystal structure (3.0 A resolution), in which 11 beta-carotene molecules (Car) and 14 lipids are visible in the PSII monomer, we calculated the redox potentials (Em) of one-electron oxidation for all Car (Em(Car)) by solving the Poisson-Boltzmann equation. In each PSII monomer, the D1 protein harbors a previously unlocated Car (CarD1) in van der Waals contact with the chlorin ring of ChlZ(D1). Each CarD1 in the PSII dimer complex is located in the interface between the D1 and CP47 subunits, together with another four Car of the other PSII monomer and several lipid molecules. The proximity of Car bridging between CarD1 and plastoquinone/Q(A) may imply a direct charge recombination of Car+Q(A)-. The calculated Em(CarD1) and Em(ChlZ(D1)) are, respectively, 83 and 126 mV higher than Em(CarD2) and Em(ChlZ(D2)), which could explain why CarD2+ and ChlZ(D2)+ are observed rather than the corresponding CarD1+ and ChlZ(D1)+.

Cyanobacteria↗
PubMed2006

Structures of human N-Acetylglucosamine kinase in two complexes with N-Acetylglucosamine and with ADP/glucose: insights into substrate specificity and regulation.

N-Acetylglucosamine (GlcNAc), a major component of complex carbohydrates, is synthesized de novo or salvaged from lysosomally degraded glycoconjugates and from nutritional sources. The salvage pathway requires that GlcNAc kinase converts GlcNAc to GlcNAc-6-phosphate, a component utilized in UDP-GlcNAc biosynthesis or energy metabolism. GlcNAc kinase belongs to the sugar kinase/Hsp70/actin superfamily that catalyze phosphoryl transfer from ATP to their respective substrates, and in most cases catalysis is associated with a large conformational change in which the N-terminal small and C-terminal large domains enclose the substrates. Here we report two crystal structures of homodimeric human GlcNAc kinase, one in complex with GlcNAc and the other in complex with ADP and glucose. The active site of GlcNAc kinase is located in a deep cleft between the two domains of the V-shaped monomer. The enzyme adopts a "closed" configuration in the GlcNAc-bound complex and GlcNAc interacts with residues of both domains. In addition, the N-acetyl methyl group contacts residues of the other monomer in the homodimer, a unique feature compared to other members of the sugar kinase/Hsp70/actin superfamily. This contrasts an "open" configuration in the ADP/glucose-bound structure, where glucose cannot form these interactions, explaining its low binding affinity for GlcNAc kinase. Our results support functional implications derived from apo crystal structures of GlcNAc kinases from Chromobacter violaceum and Porphyromonas gingivalis and show that Tyr205, which is phosphorylated in thrombin-activated platelets, lines the GlcNAc binding pocket. This suggests that phosphorylation of Tyr205 may modulate GlcNAc kinase activity and/or specificity.

Adenosine Diphosphate↗
PubMed2006

Saposin A mobilizes lipids from low cholesterol and high bis(monoacylglycerol)phosphate-containing membranes: patient variant Saposin A lacks lipid extraction capacity.

Saposin A (Sap-A) is one of five known sphingolipid activator proteins required for the lysosomal degradation of sphingolipids and for the loading of lipid antigens onto antigen-presenting molecules of the CD1 type. Sap-A assists in the degradation of galactosylceramide by galactosylceramide-beta-galactosidase in vivo, which takes place at the surface of intraendosomal/intralysosomal vesicles. Sap-A is believed to mediate the interaction between the enzyme and its membrane-bound substrate. Its dysfunction causes a variant form of Krabbe disease. In the present study we prepared glycosylated Sap-A free of other Saps, taking advantage of the Pichia pastoris expression system. Using liposomes and surface plasmon resonance spectroscopy, we tested the binding and lipid mobilization capacity of Sap-A under different conditions. Along the endocytic pathway, the pH value decreases, and the lipid composition of intraendosomal and intralysosomal membranes changes drastically. In the inner membranes the cholesterol concentration decreases, and that of the anionic phospholipid bis(monoacylglycero)phosphate increases. Here, we show that Sap-A is able to bind to liposomes and to mobilize lipids out of them at acidic pH values below pH 4.7. Low cholesterol levels and increasing concentrations of bis(monoacylglycero)phosphate favor lipid extraction significantly. Galactosylceramide as a bilayer component is not essential for lipid mobilization by Sap-A, which requires intact disulfide bridges for activity. We also show for the first time that glycosylation of Sap-A is essential for its lipid extraction activity. Variant Sap-A proteins, which cause storage of galactosylceramide in humans (Krabbe disease, Spiegel, R., Bach, G., Sury, V., Mengistu, G., Meidan, B., Shalev, S., Shneor, Y., Mandel, H., and Zeigler, M. (2005) Mol. Genet. Metab. 84, 160-166) and in mutant mice (Matsuda, J., Vanier, M. T., Saito, Y., Tohyama, J., and Suzuki, K. (2001) Hum. Mol. Genet. 10, 1191-1199) are deficient in lipid extraction capacity.

Cholesterol↗
PubMed2006

How photosynthetic reaction centers control oxidation power in chlorophyll pairs P680, P700, and P870.

At the heart of photosynthetic reaction centers (RCs) are pairs of chlorophyll a (Chla), P700 in photosystem I (PSI) and P680 in photosystem II (PSII) of cyanobacteria, algae, or plants, and a pair of bacteriochlorophyll a (BChla), P870 in purple bacterial RCs (PbRCs). These pairs differ greatly in their redox potentials for one-electron oxidation, E(m). For P680, E(m) is 1,100-1,200 mV, but for P700 and P870, E(m) is only 500 mV. Calculations with the linearized Poisson-Boltzmann equation reproduce these measured E(m) differences successfully. Analyzing the origin for these differences, we found as major factors in PSII the unique Mn(4)Ca cluster (relative to PSI and PbRC), the position of P680 close to the luminal edge of transmembrane alpha-helix d (relative to PSI), local variations in the cd loop (relative to PbRC), and the intrinsically higher E(m) of Chla compared with BChla (relative to PbRC).

Bacteriochlorophylls↗
PubMed2006

Structures of omega repressors bound to direct and inverted DNA repeats explain modulation of transcription.

Repressor omega regulates transcription of genes required for copy number control, accurate segregation and stable maintenance of inc18 plasmids hosted by Gram-positive bacteria. omega belongs to homodimeric ribbon-helix-helix (RHH2) repressors typified by a central, antiparallel beta-sheet for DNA major groove binding. Homodimeric omega2 binds cooperatively to promotors with 7 to 10 consecutive non-palindromic DNA heptad repeats (5'-(A)/(T)ATCAC(A)/(T)-3', symbolized by -->) in palindromic inverted, converging (--><--) or diverging (<---->) orientation and also, unique to omega2 and contrasting other RHH2 repressors, to non-palindromic direct (-->-->) repeats. Here we investigate with crystal structures how omega2 binds specifically to heptads in minimal operators with (-->-->) and (--><--) repeats. Since the pseudo-2-fold axis relating the monomers in omega(2) passes the central C-G base pair of each heptad with approximately 0.3 A downstream offset, the separation between the pseudo-2-fold axes is exactly 7 bp in (-->-->), approximately 0.6 A shorter in (--><--) but would be approximately 0.6 A longer in (<---->). These variations grade interactions between adjacent omega2 and explain modulations in cooperative binding affinity of omega2 to operators with different heptad orientations.

Bacterial Proteins↗
PubMed2006

Energetics of a possible proton exit pathway for water oxidation in photosystem II.

The crystal structure of photosystem II (PSII) at 3.0-A resolution suggests that titratable residues on the lumenal side of D1/D2 and PsbO form a polar channel, which might serve as a proton exit pathway associated with water oxidation on the Mn-cluster. With full account of protein environment, we calculated the pK(a) of these residues by solving the linearized Poisson-Boltzmann equation. Along the prospective proton channel, the calculated pK(a) of titratable residues (namely via D1-Asp61, D1-Glu65, D2-Glu312, D2-Lys317 D1-Asp59, D1-Arg64, PsbO-Arg152, and PsbO-Asp224) monotonically increase from the Mn-cluster to the lumenal bulk side. We suggest that these residues form the exit pathway guiding protons, which are released at the Mn-cluster as a product of water oxidation, in an exergonic process out of PSII. Upon the S2 to S3 transition, CP43-Arg357 showed a dramatic deprotonation of ca. one H(+), suggesting that this residue is coupled to the redox states of the Mn-cluster and the tyrosine Y(Z). The calculated pK(a) values of 4.2-4.4 for D2-Glu312 and those of approximately 8-10.9 for D1-Asp59 and D1-Arg64 are indicative of the experimentally determined pK(a) values for inhibition of S-state transitions. Upon removal of the atomic coordinates of PsbO, the pK(a) of these residues are dramatically affected, indicating a significant role of PsbO in tuning the pK(a) of those residues in the proton exit pathway.

Binding Sites↗
PubMed2006

Crystallization and preliminary characterization of three different crystal forms of human saposin C heterologously expressed in Pichia pastoris.

The amphiphilic saposin proteins (A, B, C and D) act at the lipid-water interface in lysosomes, mediating the hydrolysis of membrane building blocks by water-soluble exohydrolases. Human saposin C activates glucocerebrosidase and beta-galactosylceramidase. The protein has been expressed in Pichia pastoris, purified and crystallized in three different crystal forms, diffracting to a maximum resolution of 2.5 A. Hexagonal crystals grew from 2-propanol-containing solution and contain a single molecule in the asymmetric unit according to the Matthews coefficient. Orthorhombic and tetragonal crystals were both obtained with pentaerythritol ethoxylate and are predicted to contain two molecules in the asymmetric unit. Attempts to determine the respective crystal structures by molecular replacement using either the known NMR structure of human saposin C or a related crystal structure as search models have so far failed. The failure of the molecular-replacement method is attributed to conformational changes of the protein, which are known to be required for its biological activity. Crystal structures of human saposin C therefore might be the key to mapping out the conformational trajectory of saposin-like proteins.

Crystallization↗
PubMed2006

The enzyme-binding region of human GM2-activator protein.

The GM2-activator protein (GM2AP) is an essential cofactor for the lysosomal degradation of ganglioside GM2 by beta-hexosaminidase A (HexA). It mediates the interaction between the water-soluble exohydrolase and its membrane-embedded glycolipid substrate at the lipid-water interface. Functional deficiencies in this protein result in a fatal neurological storage disorder, the AB variant of GM2 gangliosidosis. In order to elucidate this cofactor's mode of action and identify the surface region of GM2AP responsible for binding to HexA, we designed several variant forms of this protein and evaluated the consequences of these mutations for lipid- and enzyme-binding properties using a variety of biophysical and functional studies. The point mutants D113K, M117V and E123K showed a drastically decreased capacity to stimulate HexA-catalysed GM2 degradation. However, surface plasmon resonance (SPR) spectroscopy showed that the binding of these variants to immobilized lipid bilayers and their ability to solubilize lipids from anionic vesicles were the same as for the wild-type protein. In addition, a fluorescence resonance energy transfer (FRET)-based assay system showed that these variants had the same capacity as wild-type GM2AP for intervesicular lipid transfer from donor to acceptor liposomes. The concentration-dependent effect of these variants on hydrolysis of the synthetic substrate 4-methylumbelliferyl-2-acetamido-2-deoxy-6-sulfo-beta-D-glucopyranoside (MUGS) indicated a weakened association with the enzyme's alpha subunit. This identifies the protein region affected by these mutations, the single short alpha helix of GM2AP, as the major determinant for the interaction with the enzyme. These results further confirm that the function of GM2AP is not restricted to a biological detergent that simply disrupts the membrane structure or lifts the substrate out of the lipid plane. In contrast, our data argue in favour of the critical importance of distinct activator-hexosaminidase interactions for GM2 degradation, and corroborate the view that the activator/lipid complex represents the true substrate for the degrading enzyme.

Animals↗
PubMed2005

Towards complete cofactor arrangement in the 3.0 A resolution structure of photosystem II.

Oxygenic photosynthesis in plants, algae and cyanobacteria is initiated at photosystem II, a homodimeric multisubunit protein-cofactor complex embedded in the thylakoid membrane. Photosystem II captures sunlight and powers the unique photo-induced oxidation of water to atmospheric oxygen. Crystallographic investigations of cyanobacterial photosystem II have provided several medium-resolution structures (3.8 to 3.2 A) that explain the general arrangement of the protein matrix and cofactors, but do not give a full picture of the complex. Here we describe the most complete cyanobacterial photosystem II structure obtained so far, showing locations of and interactions between 20 protein subunits and 77 cofactors per monomer. Assignment of 11 beta-carotenes yields insights into electron and energy transfer and photo-protection mechanisms in the reaction centre and antenna subunits. The high number of 14 integrally bound lipids reflects the structural and functional importance of these molecules for flexibility within and assembly of photosystem II. A lipophilic pathway is proposed for the diffusion of secondary plastoquinone that transfers redox equivalents from photosystem II to the photosynthetic chain. The structure provides information about the Mn4Ca cluster, where oxidation of water takes place. Our study uncovers near-atomic details necessary to understand the processes that convert light to chemical energy.

Crystallography, X-Ray↗
PubMed2005

X-ray diffraction analysis of crystals from the human major histocompatibility antigen HLA-B*2706 in complex with a viral peptide and with a self-peptide.

The human leukocyte antigen (HLA) alleles HLA-B*2704 and HLA-B*2706 show an ethnically restricted distribution and are differentially associated with ankylosing spondylitis, with HLA-B*2706 lacking association with this autoimmune disease. However, the products of the two alleles differ by only two amino acids, at heavy-chain residues 114 (His in HLA-B*2704; Asp in HLA-B*2706) and 116 (Asp in HLA-B*2704; Tyr in HLA-B*2706). Both residues could be involved in contacting amino acids of a bound peptide, suggesting that peptides presented by these subtypes play a role in disease pathogenesis. Two HLA-B*2706-peptide complexes were crystallized using the hanging-drop vapour-diffusion method with PEG as precipitant. Data sets were collected to resolutions of 2.70 A (viral peptide pLMP2, RRRWRRLTV; space group P2(1)2(1)2(1)) and 1.83 A (self-peptide pVIPR, RRKWRRWHL; space group P2(1)). Using HLA-B*2705 complexed with the pGR peptide (RRRWHRWRL) as a search model, unambiguous molecular-replacement solutions were found for both HLA-B*2706 complexes.

Crystallization↗
PubMed2005

Conformational dimorphism of self-peptides and molecular mimicry in a disease-associated HLA-B27 subtype.

An interesting property of certain peptides presented by major histocompatibility complex (MHC) molecules is their acquisition of a dual binding mode within the peptide binding groove. Using x-ray crystallography at 1.4 A resolution, we show here that the glucagon receptor-derived self-peptide pGR ((412)RRRWHRWRL(420)) is presented by the disease-associated human MHC class I subtype HLA-B*2705 in a dual conformation as well, with the middle of the peptide bent toward the floor of the peptide binding groove of the molecule in both binding modes. The conformations of pGR are compared here with those of another self-peptide (pVIPR, RRKWRRWHL) that is also displayed in two binding modes by HLA-B*2705 antigens and with that of the viral peptide pLMP2 (RRRWRRLTV). Conserved structural features suggest that the N-terminal halves of the peptides are crucial in allowing cytotoxic T lymphocyte (CTL) cross-reactivity. In addition, an analysis of T cell receptors (TCRs) from pGR- or pVIPR-directed, HLA-B27-restricted CTL clones demonstrates that TCR from distinct clones but with comparable reactivity may share CDR3alpha but not CDR3beta regions. Therefore, the cross-reactivity of these CTLs depends on TCR-CDR3alpha, is modulated by TCR-CDR3beta sequences, and is ultimately a consequence of the conformational dimorphism that characterizes binding of the self-peptides to HLA-B*2705. These results lend support to the concept that conformational dimorphisms of MHC class I-bound peptides might be connected with the occurrence of self-reactive CTL.

Antigens↗
PubMed2005

Preliminary X-ray diffraction analysis of crystals from the recombinantly expressed human major histocompatibility antigen HLA-B*2704 in complex with a viral peptide and with a self-peptide.

The product of the human leukocyte antigen (HLA) gene HLA-B*2704 differs from that of the prototypical subtype HLA-B*2705 by three amino acids at heavy-chain residues 77 (Ser instead of Asp), 152 (Glu instead of Val) and 211 (Gly instead of Ala). In contrast to the ubiquitous HLA-B*2705 subtype, HLA-B*2704 occurs only in orientals. Both subtypes are strongly associated with spondyloarthropathies and the peptides presented by these subtypes are suspected to play a role in disease pathogenesis. HLA-B*2704 was crystallized in complex with a viral peptide and with a self-peptide using the hanging-drop vapour-diffusion method with PEG as a precipitant. Both crystals belong to space group P2(1)2(1)2(1). Data sets were collected to 1.60 A (complex with the self-peptide pVIPR) or to 1.90 A (complex with the viral peptide pLMP2) resolution using synchrotron radiation. With HLA-B*2705 complexed with pVIPR as a search model, unambiguous molecular-replacement solutions were found for the complexes of HLA-B*2704 with both peptides.

Crystallization↗
PubMed2005

Conjugated bile acid hydrolase is a tetrameric N-terminal thiol hydrolase with specific recognition of its cholyl but not of its tauryl product.

Bacterial bile salt hydrolases catalyze the degradation of conjugated bile acids in the mammalian gut. The crystal structures of conjugated bile acid hydrolase (CBAH) from Clostridium perfringens as apoenzyme and in complex with taurodeoxycholate that was hydrolyzed to the reaction products taurine and deoxycholate are described here at 2.1 and 1.7 A resolution, respectively. The crystal structures reveal close relationship between CBAH and penicillin V acylase from Bacillus sphaericus. This similarity together with the N-terminal cysteine classifies CBAH as a member of the N-terminal nucleophile (Ntn) hydrolase superfamily. Both crystal structures show an identical homotetrameric organization with dihedral (D(2) or 222) point group symmetry. The structure analysis of C. perfringens CBAH identifies critical residues in catalysis, substrate recognition, and tetramer formation which may serve in further biochemical characterization of bile acid hydrolases.

Amidohydrolases↗
PubMed2005

Thermostability and Ca2+ binding properties of wild type and heterologously expressed PsbO protein from cyanobacterial photosystem II.

Oxygenic photosynthesis takes place in the thylakoid membrane of cyanobacteria, algae, and higher plants. Initially light is absorbed by an oligomeric pigment-protein complex designated as photosystem II (PSII), which catalyzes light-induced water cleavage under release of molecular oxygen for the biosphere on our planet. The membrane-extrinsic manganese stabilizing protein (PsbO) is associated on the lumenal side of the thylakoids close to the redox-active (Mn)(4)Ca cluster at the catalytically active site of PSII. Recombinant PsbO from the thermophilic cyanobacterium Thermosynechococcus elongatus was expressed in Escherichia coli and spectroscopically characterized. The secondary structure of recombinant PsbO (recPsbO) was analyzed in the absence and presence of Ca(2+) using Fourier transform infrared spectroscopy (FTIR) and circular dichroism spectropolarimetry (CD). No significant structural changes could be observed when the PSII subunit was titrated with Ca(2+) in vitro. These findings are compared with data for spinach PsbO. Our results are discussed in the light of the recent 3D-structural analysis of the oxygen-evolving PSII and structural/thermodynamic differences between the two homologous proteins from thermophilic cyanobacteria and plants.

Bacterial Proteins↗
PubMed2005

Size determination of cyanobacterial and higher plant photosystem II by gel permeation chromatography, light scattering, and ultracentrifugation.

The oxygen-evolving photosystem II core complexes (PSIIcc) from the thermophilic cyanobacterium Thermosynechococcus elongatus (PSIIccTe) and the higher plant Spinacia oleracea (PSIIccSo) have been isolated from the thylakoid membrane by solubilization with n-dodecyl-beta-d-maltoside, purified and characterized by gel permeation chromatography (GPC), dynamic light scattering (DLS), and analytical ultracentrifugation (AUC). DLS suggests that PSIIcc from both organisms exists as a monomer in dilute solution and aggregates with increasing protein concentration. In contrast to DLS, GPC and AUC showed that PSIIcc of both organisms occur as monomers and dimers, and it became clear from our studies that calibration of GPC columns with soluble proteins leads to wrong estimates of the molecular masses of membrane proteins. At a PSIIcc protein concentration of 0.2 mg/mL, molar masses, M, of 756 +/- 18 kDa and 710 +/- 15 kDa for dimeric PSIIccTe and PSIIccSo, respectively, were determined by analytical ultracentrifugation. At very low protein concentrations, at or below 0.05 mg/mL, the dimeric form of PSIIccTe partially dissociates (20-30%) to form monomers. On the basis of these studies 3-dimensional crystals of PSIIccTe were obtained that contain dimers in the asymmetric unit [Zouni, A. et al. (2001) Nature 409, 739-743]. Using synchrotron radiation the crystals diffract to a resolution of 3.8 A, which has been improved recently to 3.2 A [Biesiadka, J., et al. (2004) Phys. Chem. Chem. Phys. 6, 4733-4736].

Bacterial Proteins↗