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Abstract: Here we present the crystal structure of the Methanococcus jannaschii RelE–RelB (RelBE) toxin–antitoxin (TA) protein complex determined by the MIRAS (multiple isomorphous replacement with anomalous signal) method. The genes encoding this TA system are located in the chromosome of this archaeon and involved in stress response. RelE acts as an endoribonuclease that cleaves mRNA on the ribosome, and we compare the RelBE complex to the known structures of other TA systems belonging to this group and to endoribonucleases. M. jannaschii RelBE forms a heterotetramer with the antitoxin in the centre of the complex, a configuration that differs vastly from the heterotetramer structure of the previously published RelBE from another archaeon, Pyrococcus horikoshii. The long N-terminal α-helix of the tightly bound M. jannaschii antitoxin RelB covers the presumed active site of the toxin RelE that is formed by a central β-sheet, a loop on one side and a C-terminal α-helix on the other side. The active site of the M. jannaschii toxin RelE harbours positive charges that are thought to neutralize the negative charges of the substrate mRNA, including Arg62 that was changed to Ser62 by the Escherichia coli expression system, thereby leading to inactive toxin RelE. Comparative studies suggest that Asp43 and His79 are also involved in the activity of the toxin. [Copyright &y& Elsevier]

Abstract: The catabolite control protein A (CcpA) from Bacillus megaterium is a member of the bacterial repressor protein family GalR–LacI. CcpA functions as master transcriptional regulator of carbon catabolite repression/regulation in firmicutes. Here we present the crystal structure of full-length apo CcpA at 2.5 Å resolution from B. megaterium. The structure reveals the location of the helix–turn–helix domain as well as the hinge region, which were not visible due to their high flexibility in earlier crystallographic studies on CcpA molecules. The structure of the apo CcpA homodimer in the present form is in contrast to other reported structures for CcpA. [Copyright &y& Elsevier]

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, Em. For P680, Em is 1,100–1,200 mV, but for P700 and P870, Em is only 500 mV. Calculations with the linearized Poisson-Boltzmann equation reproduce these measured Em differences successfully. Analyzing the origin for these differences, we found as major factors in PSII the unique Mn4Ca cluster (relative to PSI and PbRC), the position of P680 close to the luminal edge of transmembrane α-helix d (relative to PSI), local variations in the cd loop (relative to PbRC), and the intrinsically higher Em of Chla compared with BChla (relative to PbRC). [ABSTRACT FROM AUTHOR]

The crystal structure of photosystem H (PSII) at 3.0-Å 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 pKa of these residues by solving the linearized Poisson-Boltzmann equation. Along the prospective proton channel, the calculated pKa of titratable residues (namely via D1-Asp61, D1-G1u65, 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 P5II. 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 Yz. The calculated pKa values of 4.2-4.4 for D2-Glu312 and those of ∼8-10.9 for Dl-Asp59 and Dl-Arg64 are indicative of the experimentally determined PKa values for inhibition of S-state transitions. Upon removal of the atomic coordinates of PsbO, the pKa of these residues are dramatically affected, indicating a significant role of PsbO in tuning the pKa of those residues in the proton exit pathway. [ABSTRACT FROM AUTHOR]

The recently determined crystal structures of photosystems I and II at 2.5 A˚ and 3.8 A˚ resolution, respectively, have improved the structural basis for understanding the processes of light trapping, exciton transfer and electron transfer occurring in the primary steps of oxygenic photosynthesis. Understanding the assembly of the 12 protein subunits and 128 cofactors in photosystem I allows us to study the possible functions of the individual players in this protein–cofactor complex. [Copyright &y& Elsevier]

Presents information on a study which identified the chemical structure of homodimeric tetracycline repressor which regulates resistance to the antibiotic tetracycline at the transcriptional level. Materials and methods; Results and discussion.

Presents an x-ray crystal structure analysis of cyclodextrins or cycloamyloses. Structural features of cyclodextrins molecules; Types of crystal structures.

Six different crystal forms of the long neurotoxin α-cobratoxin isolated from the venom of Naja naja siamensis have been obtained and the conditions for their crystallisation are described. Heavy atom derivatives for two of these forms have been prepared and have proven suitable for high-resolution X-ray studies and, with one of them, a 0.28-nm resoution structure has been determined. All but one of the crystal forms were grown between pH 2 and pH 3. Two novel suggestions are made which may be applicable to other protein crystallisation studies. One involves cocrystallisation with mercurous iodide and the other describes a two-step microdialysis procedure. first against salt solution, followed by analysis against poly(ethylene glycol). [ABSTRACT FROM AUTHOR]

Analyzes the neutron diffraction data to understand the role of C-H(bullets)O hydrogen bonds in the coordination of water molecules. Distances and frequency of C-H(bullets)O bonds; Acceptor and donor functions of the water molecule; Comparison with the hydrogen bonds of CH, OH and NH; Geometric characterization of the different hydrogen bonds.

Discusses the crystal structure of complex gamma-cyclodextrin-methanol-n-hydrate with focus on its unusual crystal packing mode. Stacking of the gamma-cyclodextrin molecules in columns which have internal channels; Conditions that prevent characterization of the solvent and guest arrangements.

An overview is given on the structural characteristics of α-. β-, and γ-cyclodextrins (CDs). These cyclic oligosaccharides consisting of six, seven and eight glucoses form inclusion complexes with guest molecules small enough to fit into their central cavities and serve as good model systems for non-covalent bonding. Depending on the size and the ionic or molecular nature of the guests, the complexes crystallize in cage- or channel-type structures, X-ray and neutron diffraction have been used to study intermolecular interactions; they provide insight into O-H···O and C-H···O hydrogen bonds stabilizing the macrocyclic conformations and the inclusion of the guest molecules. Throughout the crystal lattices of the CD hydrates, which abound with O-H groups, cooperative networks are formed by O-H···O hydrogen bonds; in the β-CD macrocycle, dynamic disorder of the flip-flop type is observed, O(½H)···(½H)-O, between O(2)-H and O(3)-H groups of adjacent glucoses. [ABSTRACT FROM AUTHOR]

The tRNA minor constituent 4-thiouridine and four of its derivatives with identical or similar nucleobases (arabino-4-thouridine, 1 -methyl-4-thiouracil, 3'-O-acetyl-4-thiothyinidine and 2,4- dithiouridine) have been crystallized from water and examined by single-crystal X-ray diffraction methods. From the packing patterns and hydrogen-bonding schemes found in the crystal structures the following observations were made. 1. The forces which determine the crystal structures are mainly stacking interactions due to hydrophobic and polarization forces The hydrogen-bonding forces play only a minor or no role 2. The hydrogen-bond formation is determined by geometrical factors imposed on the system through packing patterns. 3. Oxygen atoms, due to their greater electro-negativity, are better proton acceptors than sulfur atoms. However, when certain stacking patterns must be followed, all predictions concerning hydrogen-bonding schemes become meaningless. 4 In cocrystals of complementary base pairs, however, the hydrogen bonding schemes are determining the crystal structures. [ABSTRACT FROM AUTHOR]

The molecular structure of the uridine analog 6,7-dimethyllumazine-N-1-β-D.ribofuranoside has been determined from 4114 three-dimensional X-ray data. The substance crystallized in the monoclinic space group P21 with two crystallographically independent molecules per asymmetric unit. The intensity data were measured diffractometrically, and the positions of the 46 nonhydrogen atoms were located by direct methods. The atomic parameters were refined by fullmatrix least-squares methods to a discrepancy index of 5.9%. Both molecules are in the unusual syn conformation with C(2')-endo sugar puckering and a gauche, gauche conformation about the C(')-C(5') bond; thus an intramolecular O(5')-H · O(2) hydrogen bond can be formed. The molecules are packed such that the "hydrophobic" pteridine heterocycles are stacked in endless ribbons which are separated by regions containing the sugar residues. The lumazine substituents at O(2), 0(4) and N(3)-H but not N(5) and N(8) are involved in hydrogen bonding. A discussion of the possible biochemical consequences are given. [ABSTRACT FROM AUTHOR]

We present a model for quasielastic neutron scattering (QENS) by an aqueous solution of compact and inflexible molecules. This model accounts for time-dependent spatial pair correlations between the atoms of the same as well as of distinct molecules and includes all coherent and incoherent neutron scattering contributions. The extension of the static theory of the excluded volume effect [A. K. Soper, J. Phys.: Condens. Matter 9, 2399 (1997)] to the time-dependent (dynamic) case allows us to obtain simplified model expressions for QENS spectra in the low Q region in the uniform fluid approximation. The resulting expressions describe the quasielastic small-angle neutron scattering (QESANS) spectra of D2O solutions of native and methylated cyclodextrins well, yielding in particular translational and rotational diffusion coefficients of these compounds in aqueous solution. Finally, we discuss the full potential of the QESANS analysis (that is, beyond the uniform fluid approximation), in particular, the information on solute-solvent interactions (e.g., hydration shell properties) that such an analysis can provide, in principle. [ABSTRACT FROM AUTHOR]

Abstract: Clathrin-mediated endocytosis mediates the internalization of signaling and nutrient receptors, ion channels and regulates the endocytic recycling of pre- and postsynaptic membrane proteins. During early stages endocytic adaptors recognize sorting signals within this diverse array of cargo proteins destined for internalization. Cargo sequestration is mechanistically coupled to membrane deformation, a process involving BAR domain proteins, resulting in the generation of endocytic intermediates that finally undergo dynamin-mediated fission. Here we summarize recent insights gathered from a combination of structural, biochemical, and cell biological studies that have revealed a remarkable complexity of the machinery for endocytic sorting and membrane deformation. [Copyright &y& Elsevier]

The existence of cytotoxic T cells (CTL) cross-reacting with the human major histocompatibiity antigens HLA-B14 and HLA-B27 suggests that their alloreactivity could be due to presentation of shared peptides in similar binding modes by these molecules. We therefore determined the crystal structures of the subtypes HLA-B*1402, HLA-B*2705, and HLA-B*2709 in complex with a proven seif-ligand, pCatA (peptide with the sequence IRAAPPPLF derived from cathepsin A (residues 2-10)), and of HLA-B* 1402 in complex with a viral peptide, pLMP2 (RRRWRRLTV, derived from latent membrane protein 2 (residues 236-244) of Epstein-Barr virus). Despite the exchange of 18 residues within the binding grooves of HLA-B*1402 and HLA-B*2705 or HLA-B*2709, the pCatA peptide is presented in nearly identical conformations. However, pLMP2 is displayed by HLA-B*1402 in a conformation distinct from those previously found in the two HLA-B27 subtypes. In addition, the complexes of HLA-B*1402 with the two peptides reveal a nonstandard, tetragonal mode of the peptide N terminus anchoring in the binding groove because of the exchange of the common Tyr171 by His-171 of the HLA-B*1402 heavy chain. This exchange appears also responsible for reduced stability of HLA-B14.-peptide complexes in vivo and slow assembly in vitro. The studies with the pCatA peptide uncover that CTL cross-reactive between HLA-B14 and HLA-B27 might primarily recognize the common structural features of the bound peptide, thus neglecting amino acid replacements within the rim of the binding grooves. In contrast, structural alterations between the three complexes with the pLMP2 peptide indicate how heavy chain polymorphisms can influence peptide display and prevent CTL cross-reactivity between HLA-B14 and HLA-B27 antigens. [ABSTRACT FROM AUTHOR]

Abstract: Photosystem II (PSII) is a homodimeric protein–cofactor complex embedded in the thylakoid membrane that catalyses light-driven charge separation accompanied by the oxidation of water during oxygenic photosynthesis. Biochemical analysis of the lipid content of PSII indicates a number of integral lipids, their composition being similar to the average lipid composition of the thylakoid membrane. The crystal structure of PSII at 3.0 Å resolution allowed for the first time the assignment of 14 integral lipids within the protein scaffold, all of them being located at the interface of different protein subunits. The reaction centre subunits D1 and D2 are encircled by a belt of 11 lipids providing a flexible environment for the exchange of D1. Three lipids are located in the dimerization interface and mediate interactions between the PSII monomers. Several lipids are located close to the binding pocket of the mobile plastoquinone QB, forming part of a postulated diffusion pathway for plastoquinone. Furthermore two lipids were found, each ligating one antenna chlorophyll a. A detailed analysis of lipid–protein and lipid–cofactor interactions allows to derive some general principles of lipid binding pockets in PSII and to suggest possible functional properties of the various identified lipid molecules. [Copyright &y& Elsevier]

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 Å) 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 β-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. [ABSTRACT FROM AUTHOR]

Selected HLA-B27 subtypes are associated with spondyloarthropathies, but the underlying mechanism is not understood. To explain this association in molecular terms, a comparison of peptide-dependent dynamic and structural properties of the differentially disease-associated subtypes HLA-B*2705 and HLA-B*2709 was cartied out. These molecules differ only by a single amino acid at the floor of the peptide binding groove. The thermostabilities of a series of HLA-B27 molecules complexed with nonameric and decameric peptides were determined and revealed substantial differences depending on the subtype as well as the residues at the termini of the peptides. In addition we present the crystal structure of the B*2709 subtype complexed with a decameric peptide. This structure provides an explanation for the preference of HLA-B27 for a peptide with an N-terminal arginine as secondary anchor and the lack of preference for tyrosine as peptide C terminus in B*2709. The data show that differences in thermodynamic properties between peptide-complexed HLA-B27 subtypes are correlated with a variety of structural properties. [ABSTRACT FROM AUTHOR]

The crystal structure at 2.5 A˚ resolution of the membrane-intrinsic, homotrimeric photosystem I (PSI) isolated from the thermophilic cyanobacterium Synechococcus elongatus shows that each monomer is composed of 12 protein subunits of which nine are embedded in the membrane and feature a total of 34 transmembrane α-helices (TMH). Hence, PSI provides an ideal case to study “conventional” and Cα–H⋯O hydrogen bonds between TMH engaged in intra- and intersubunit interactions. Of the total of 75 Cα–H⋯O hydrogen bonds between TMHs, 72 are intrasubunit and only three are intersubunit. The two largest subunits PsaA and PsaB are each folded into 11 TMHs showing 29 and 24 intrasubunit Cα–H⋯O hydrogen bonds, respectively, that are not distributed randomly but many of them flank chlorophyll a (Chl a) co-ordinating amino acids, suggesting stabilisation of these structural segments. As major constituent of the trimerisation domain, subunit PsaL is located next to the 3-fold axis relating the three monomers of PSI. PsaL features a unique number of 19 intrasubunit Cα–H⋯O hydrogen bonds that connect two of its three TMHs but there are no intersubunit Cα–H⋯O hydrogen bonds between the three PsaL. Of the three intersubunit Cα–H⋯O hydrogen bonds, two are formed between PsaA and PsaB and one between PsaB and PsaM. The large number of 75 Cα–H⋯O hydrogen bonds contrasts the 49 conventional hydrogen bonds, indicating that the former and van der Waals contacts determine association and orientation of TMHs in PSI. [Copyright &y& Elsevier]

The binding of six herbicides of the triazine family to the photosynthetic reaction center of Rhodopseudomonas viridis was investigated with energy-minimization techniques, in order to correlate experimental with calculated data. The inhibitors were modeled in the active site according to the Xray structure analysis of the complex formed between the triazine terbutryn (2-ethylamino-4-tbutylamino-6-methylthio-s-triazine) and the reaction center of R. viridis [Michel, H., Epp, O. & Deisenhofer, J. (1986) EMBO J. 5, 2445-2451]. 40 different energy minimizations were carried out with varying cutoff radii, partial charges on inhibitor atoms and dielectric constants, i.e. 10 different combinations of these were tested. The impact of these parameters on the calculated binding and interaction energy was either examined for all protein/triazine complexes or, in the case of the dielectric constant, a smaller sample was used. The calculated energies are dominated by van der Waals interactions, which change by up to 20% when extending the cutoff radius from 0.8 nm to 1.5 nm. The use of uniform or distance-dependent dielectric constant or partial charges on the inhibitor atoms does not severely influence the resulting structures, but shows a great impact on the calculated energies. In the two groups of triazines, each containing three inhibitors with methoxy or methylthio substituents, correlations of biological and calculated data were found quite often, but only once with all six triazines. The energy-minimized structures were compared and analysed. A third hydrogen bond, not seen in the X-ray analysis of the reaction center/terbutryn complex, was found between the t-butylamino moiety of terbutryn (and equivalent moieties in the other triazines) and the carbonyl oxygen of TyrL222. [ABSTRACT FROM AUTHOR]

The crystal and molecular structure of proteinase K was determined by X-ray diffraction data to 0.15-nm resolution. The enzyme belongs to the subtilisin family with an active-site catalytic triad Asp39-His69-Ser224 but is a representative of a subgroup with a free Cys73 close to and 'below' the active His69. Besides this Cys72, proteinase K has two disulfide bonds, Cys34-Cys123 and Cys178-Cys249, which contribute to the stability of the tertiary structure consisting of an extended central parallel β-sheet decorated by six α-helices, three short antiparallel β-sheets, 18 β-turns and involving several internal, structurally important water molecules. Proteinase K exhibits two Ca2+-binding sites, one very strong and the other weak, which were the sites of the heavy atoms (Pb2+, Sm3+) used to solve the crystal structure. The weak binding site is liganded to the N and C termini, Thr16 and Asp260, and is only incompletely coordinated by oxygen ligands. The strong binding site is coordinated in the form of a pentagonal bipyramid with the side chain carboxylate of Asp200 and the C = O of Pro175 as apex, and C = O of Val177 and four water molecules in the equatorial plane. Upon removal of this Ca2+, proteinase K loses activity which is interpreted in terms of a local structural deformation involving the substrate-recognition site (Ser132-Gly136), probably associated with a cis→trans isomerization of cis Pro171. Several water molecules are located in the active site. One, W335, is positioned in the 'oxyanion hole' and is displaced by the C = O of the scissile peptide bond of the substrate, as indicated by crystallographic studies with peptide chloromethane inhibitors. Based on these experiments, a reaction mechanism is proposed where the peptide substrate forms a three-stranded antiparallel pleated sheet with the recognition site of proteinase K consisting of Ser132-Leu133-Gly134 on one side and Gly100-Ser101 on the other, followed by expulsion of the oxyanion hole water W335 and hydrolytic cleavage by the Asp39-His69-Serr224 triad. These latter residues display low thermal motion corresponding to well-defined geometry and are hardly accessible to solvent molecules, whereas the recognition-site amino acids are more flexible and partially exposed to solvent. [ABSTRACT FROM AUTHOR]

The fungal proteinase K (EC 3.4.21.14) is a very potent unusually stable member of the subtilisin family. Its X-ray structure determined at 0.15-nm resolution shows two bound Ca2+ ions. Ca1 is in near-ideal pentagonal bipyramidal configuration with Asp200 carboxylate and Pro175 peptide C = O in an apical, and Val177 peptide C = O and four water molecules in an equatorial position, whereas Ca2 displays incomplete octahedral coordination with the carboxylate of Asp260, the peptide C=O of Val16 and two water molecules. Scatchard analysis of the titration of Ca2+-free proteinase K with Ca2+ yields a single dissociation constant (7.6 ± 2.5) × 10-5 M associated with the tightly bound Ca1 whereas Ca2 is so weakly bound that it cannot be titrated. If proteinase K is depleted of Ca2+ by treatment with EDTA, followed by gel filtration, its enzymatic activity drops within 6 h to 20% of its original value, without autolysis. Addition of excess Ca2+ immediately raises the residual activity to 28%, but full activity is not achieved. Removal of Ca2+ triggers a conformational change of the substrate recognition site because there is a direct connection, via secondary structure hydrogen bonds, between the Ca1 binding site and the substrate-recognition site. This is indicated further by circular dichroism and fluorescencespectroscopic data, and by reversed-phase FPLC, carried out in the presence and absence of Ca2+, but the overall structure of the enzyme is not affected. Depletion of Ca2+ also influences binding of Longer peptide inhibitors of the chloromethane type, it increases the tale of autolysis after about 48 h, it reduces the thermal stability (measured by activity tests from 65 °C to 46 °C), and it enhances the deactivation by 8 M urea which inactivates to only 65%, whereas sodium dodecyl sulfate totally inactivates at a concentration of 12.5%. [ABSTRACT FROM AUTHOR]

Presents a study which employed hen egg white lysozyme to study the aggregation and nucleation events induced by electrolytes such as NaCI. How the process was monitored; Details on the experiments conducted; Methodology used in this study; Results of this study.

The tetrameric glucose dehydrogenase from Bacillus megaterium M1286 belongs to the 'short' family of dehydrogenases with 262 amino acids per subunit (Mr ≈ 30 000), and does not require Zn2+ for enzymatic action. It was crystallized as complex with its coenzyme NAD from a 1-2% protein solution by the batch method using ammonium sulfate as precipitant at pH 6.5. Crystals appeared within two days as clusters of large plates with maximum dimensions of 2 mm, which diffract X-rays to a resolution of at least 0.2 nm. The space group is orthorhombic P212121, unit-cell dimensions are a = 15.03 nm, b = 10.42 nm and c = 6.74 nm. Assuming one molecule (≈ 120 kDa) per asymmetric unit the VM value is 0.0022 nm³/Da and the solvent content of the crystals is 45% based on a partial specific volume for the protein of 0.723 ml/g. The crystallization was further improved by using the microdialysis technique where instead of clusters, single crystals appeared within 7 days. [ABSTRACT FROM AUTHOR]

Hen egg-white avidin was crystallized at pH 6 from solutions containing 8-9% (w/v) poly(ethyleneglycol) 6000 as precipitant. X-ray investigation showed that the crystalsare orthorhombic with cell constants a = 7.47 nm, b = 8.00 nm, c = 4.31 nm and belong to space group P212121. They contain half of a tetrameric molecule in the asymmetric unit. [ABSTRACT FROM AUTHOR]

Sialic acids are essential components of membrane glycoconjugates. They are responsible for the interaction, structure, and functionality of all deuterostome cells and have major functions in cellular processes in health and diseases. The key enzyme of the biosynthesis of sialic acid is the bifunctional UDP-N-acetylglucosamine- 2-epimerase/N-acetylmannosamine kinase that transforms UDP-N-acetylglucosamine to N-acetylmannosamine (ManNAc) followed by its phosphorylation to ManNAc 6-phosphate and has a direct impact on the sialylation of cell surface components. Here, we present the crystal structures of the human N-acetylmannosamine kinase (MNK) domain of UDP-N-acetylglucosamine- 2-epimerase/N-acetylmannosamine kinase in complexes with ManNAc at 1.64 Å resolution, MNK·ManNAc·ADP (1.82 Å) and MNK·ManNAc 6-phosphate·ADP (2.10 Å). Our findings offer detailed insights in the active center of MNK and serve as a structural basis to design inhibitors. We synthesized a novel inhibitor, 6-O-acetyl-ManNAc, which is more potent than those previously tested. Specific inhibitors of sialic acid biosynthesis may serve to further study biological functions of sialic acid. [ABSTRACT FROM AUTHOR]

Abstract: Although most autoimmune diseases are connected to major histocompatibility complex (MHC) class II alleles, a small number of these disorders exhibit a variable degree of association with selected MHC class I genes, like certain human HLA-A and HLA-B alleles. The basis for these associations, however, has so far remained elusive. An understanding might be obtained by comparing functional, biochemical, and biophysical properties of alleles that are minimally distinct from each other, but are nevertheless differentially associated to a given disease, like the HLA-B*27:05 and HLA-B*27:09 antigens, which differ only by a single amino acid residue (Asp116His) that is deeply buried within the binding groove. We have employed a number of approaches, including X-ray crystallography and isotope-edited infrared spectroscopy, to investigate biophysical characteristics of the two HLA-B27 subtypes complexed with up to ten different peptides. Our findings demonstrate that the binding of these peptides as well as the conformational flexibility of the subtypes is greatly influenced by interactions of the C-terminal peptide residue. In particular, a basic C-terminal peptide residue is favoured by the disease-associated subtype HLA-B*27:05, but not by HLA-B*27:09. This property appears also as the only common denominator of distinct HLA class I alleles, among them HLA-B*27:05, HLA-A*03:01 or HLA-A*11:01, that are associated with diseases suspected to have an autoimmune etiology. We postulate here that the products of these alleles, due to their unusual ability to bind with high affinity to a particular peptide set during positive T cell selection in the thymus, are involved in shaping an abnormal T cell repertoire which predisposes to the acquisition of autoimmune diseases. [Copyright &y& Elsevier]

Abstract: This paper briefly reviews contemporary protein crystallography and focuses on six receptor proteins of membrane-intrinsic ATP binding cassette (ABC) transporters. Three of these receptors are specific for carbohydrates and three for amino acids. The receptor GacH of the transporter GacFGH from Streptomyces glaucescens is specific for acarbose and its homologs, and MalE of Salmonella typhimurium is specific for maltose but also forms a complex with acarbose, and the third receptor is the highly specific d-galactose receptor AcbH of the transporter AcbFGH from Actinoplanes sp. Concerning the receptors for amino acids, ArtJ belongs to the ArtJ-(MP)2 transporter of Geobacillus stearotermophilus and recognizes and binds to positively charged arginine, lysine, and histidine with different sizes of side chains, contrasting the receptors Ngo0372 and Ngo2014 from Neisseria gonorrhaeae that are highly specific for cystine and cysteine, respectively. The differences in the rather unspecific receptors GacH, MalE and ArtJ are compared with the highly specific receptors AcbH, Ngo0372 and Ngo2014. [Copyright &y& Elsevier]

Abstract: ATP-binding cassette (ABC) transport systems mediate the translocation of solutes across biological membranes at the expense of ATP. They share a common modular architecture comprising two pore-forming transmembrane domains and two nucleotide binding domains. In prokaryotes, ABC transporters are involved in the uptake of a large variety of chemicals, including nutrients, osmoprotectants and signal molecules. In pathogenic bacteria, some ABC importers are virulence factors. Canonical ABC import systems require an additional component, a substrate-specific receptor or binding protein for function. Interaction of the liganded receptor with extracytoplasmic loop regions of the transmembrane domains initiate the transport cycle. In this review we summarize the current knowledge on receptor–transporter interplay provided by crystal structures as well as by biochemical and biophysical means. In particular, we focus on the maltose/maltodextrin transporter of enterobacteria and the transporters for positively charged amino acids from the thermophile Geobacillus stearothermophilus and Salmonella enterica serovar Typhimurium. [Copyright &y& Elsevier]

Abstract: Dipeptidyl peptidase IV (DPPIV or CD26) is a multifunctional membrane glycoprotein. As an exopeptidase it regulates the activity of a series of biologically important peptides. Through its interaction with specific proteins and peptides, DPPIV is also involved in a wide range of biologically relevant processes such as cell adhesion, T cell activation and apoptosis. In this paper, we review our recent studies on the interactions of DPPIV with adenosine deaminase (ADA) and the transcription transactivator of the human immunodeficiency virus type-1 (HIV-1 Tat) as revealed by three-dimensional structure reconstructed by single particle analysis of cryo-electron microscopy (EM) and crystal structures of the human DPPIV-bovine ADA complex as well as the crystal structures of DPPIV in complex with HIV-1 Tat-derived nonapeptides. These results contribute importantly to the clarification of the molecular mechanisms of this multifunctional protein. The biological relevance of these interactions is discussed. [Copyright &y& Elsevier]

Abstract: ATP-binding cassette (ABC) transporters are integral membrane proteins that carry a variety of substrates across biological membranes at the expense of ATP. The here considered prokaryotic canonical importers consist of three entities: an extracellular solute receptor, two membrane-intrinsic proteins forming a translocation pathway, and two cytoplasmic ATP-binding subunits. The ngo0372-74 and ngo2011-14 gene clusters from the human pathogen Neisseria gonorrhoeae were predicted by sequence homology as ABC transporters for the uptake of cystine and cysteine, respectively, and chosen for structural characterization. The structure of the receptor component Ngo0372 was obtained in a ligand-free “open” conformation and in a “closed” conformation when co-crystallized with l-cystine. Our data provide the first structural information of an l-cystine ABC transporter. Dissociation constants of 21 and 33 nM for l-cystine and l-selenocystine, respectively, were determined by isothermal titration calorimetry. In contrast, l-cystathionine and l-djenkolic acid are weak binders, while no binding was detectable for S-methyl-l-cysteine. Mutational analysis of two residues from the binding pocket, Trp97 and Tyr59, revealed that the latter is crucial for l-cystine binding. The structure of the Ngo2014 receptor was obtained in closed conformation in complex with co-purified l-cysteine. The protein binds l-cysteine with a K d of 26 nM. Comparison of the structures of both receptors and analysis of the ligand binding sites shed light on the mode of ligand recognition and provides insight into the tight binding of both substrates. Moreover, since l-cystine limitation leads to reduction in virulence of N. gonorrhoeae, Ngo0372 might be suited as target for an antimicrobial vaccine. [Copyright &y& Elsevier]

Summary: Clathrin-mediated endocytosis (CME) regulates many cell physiological processes such as the internalization of growth factors and receptors, entry of pathogens, and synaptic transmission. Within the endocytic network, clathrin functions as a central organizing platform for coated pit assembly and dissociation via its terminal domain (TD). We report the design and synthesis of two compounds named pitstops that selectively block endocytic ligand association with the clathrin TD as confirmed by X-ray crystallography. Pitstop-induced inhibition of clathrin TD function acutely interferes with receptor-mediated endocytosis, entry of HIV, and synaptic vesicle recycling. Endocytosis inhibition is caused by a dramatic increase in the lifetimes of clathrin coat components, including FCHo, clathrin, and dynamin, suggesting that the clathrin TD regulates coated pit dynamics. Pitstops provide new tools to address clathrin function in cell physiology with potential applications as inhibitors of virus and pathogen entry and as modulators of cell signaling. [ABSTRACT FROM AUTHOR]

Herbicides that target photosystem II (PSII) compete with the native electron acceptor plastoquinone for binding at the QB site in the D1 subunit and thus block the electron transfer from QA to QB. Here, we present the first crystal structure of PSII with a bound herbicide at a resolution of 3.2 Å. The crystallized PSII core complexes were isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus. The used herbicide terbutryn is found to bind via at least two hydrogen bonds to the QB site similar to photosynthetic reaction centers in anoxygenic purple bacteria. Herbicide binding to PSII is also discussed regarding the influence on the redox potential of QA, which is known to affect photoinhibition. We further identified a second and novel chloride position close to the water-oxidizing complex and in the vicinity of the chloride ion reported earlier (Guskov, A., Kern, J., Gabdulkhakov, A., Broser, M., Zouni, A., and Saenger, W. (2009) Nat. Struct. Mol. Biol. 16, 334-342). This discovery is discussed in the context of proton transfer to the lumen. [ABSTRACT FROM AUTHOR]

Abstract: The manganese-stabilizing protein PsbO is associated with the luminal side of thylakoids close to the redox-active Mn4Ca cluster at the catalytically active site of photosystem II (PSII). PsbO is believed to increase the efficiency of oxygen evolution and to stabilize the Mn4Ca cluster against photoinhibition. Using small-angle X-ray scattering, we investigated the low-resolution structure of wild-type spinach PsbO and that of chimeric spinach PsbO fused with maltose-binding protein. Small-angle X-ray scattering data revealed that both proteins are monomeric in solution, and that plant and cyanobacterial PsbO have similar structures. We show a highly efficient expression system that allows recombinant production of the active wild type and the chimeric PsbO from spinach and cyanobacteria, with yields compatible with biophysical and structural studies. The binding of spinach PsbO fused with maltose-binding protein to PSII depleted of extrinsic subunits (PSII-ΔpsbO,P,Q) was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The reconstituted PSII was shown to have similar oxygen evolution rates as obtained with wild-type spinach PsbO. [Copyright &y& Elsevier]

Abstract: Solute receptors (binding proteins) are indispensable components of canonical ATP-binding cassette importers in prokaryotes. Here, we report on the characterization and crystal structures in the closed and open conformations of AcbH, the solute receptor of the putative carbohydrate transporter AcbFG which is encoded in the acarbose (acarviosyl-1,4-maltose) biosynthetic gene cluster from Actinoplanes sp. SE50/110. Binding assays identified AcbH as a high-affinity monosaccharide-binding protein with a dissociation constant (K d) for β-d-galactopyranose of 9.8±1.0 nM. Neither galactose-containing di- and trisaccharides, such as lactose and raffinose, nor monosaccharides including d-galacturonic acid, l-arabinose, d-xylose and l-rhamnose competed with [1 4C]galactose for binding to AcbH. Moreover, AcbH does not bind d-glucose, which is a common property of all but one d-galactose-binding proteins characterized to date. Strikingly, determination of the X-ray structure revealed that AcbH is structurally homologous to maltose-binding proteins rather than to glucose-binding proteins. Two helices are inserted in the substrate-binding pocket, which reduces the cavity size and allows the exclusive binding of monosaccharides, specifically β-d-galactopyranose, in the 4C1 conformation. Site-directed mutagenesis of three residues from the binding pocket (Arg82, Asp361 and Arg362) that interact with the axially oriented O4-H hydroxyl of the bound galactopyranose and subsequent functional analysis indicated that these residues are crucial for galactose binding. To our knowledge, this is the first report of the tertiary structure of a solute receptor with exclusive affinity for β-d-galactopyranose. The putative role of a galactose import system in the context of acarbose metabolism in Actinoplanes sp. is discussed. [Copyright &y& Elsevier]

Lactococcus lactis IL1403 is a lactic acid bacterium that is used widely for food fermentation. Copper homeostasis in this organism chiefly involves copper secretion by the CopA copper ATPase. This enzyme is under the control of the CopR transcriptional regulator. CopR not only controls its own expression and that of CopA, but also that of an additional three operons and two monocistronic genes. One of the genes under the control of CopR, yahD, encodes an α/β-hydrolase. YahD expression was induced by copper and cadmium, but not by other metals or oxidative or nitrosative stress. The three-dimensional structure of YahD was determined by X-ray crystallography to a resolution of 1.88 Å. The protein was found to adopt an α/β-hydrolase fold with the characteristic Ser-His-Asp catalytic triad. Functional testing of YahD for a wide range of substrates for esterases, lipases, epoxide hydrolases, phospholipases, amidases and proteases was, however, unsuccessful. A copper-inducible serine hydrolase has not been described previously and YahD appears to be a new functional member of this enzyme family. [ABSTRACT FROM AUTHOR]

The membrane-embedded photosystem II core complex (PSIIcc) uses light energy to oxidize water in photosynthesis. Information about the spatial structure of PSIIcc obtained from x-ray crystallography was so far derived from homodimeric PSIIcc of thermophilic cyanobacteria. Here, we report the first crystallization and structural analysis of the monomeric form PSIIcc with high oxygen evolution capacity, isolated from Thermosynechococcus elongatus. The crystals belong to the space group C2221 contain one monomer per asymmetric unit, and diffract to a resolution of 3.6 Å. The x-ray diffraction pattern of the PSllcc-monomer crystals exhibit less anisotropy (dependence of resolution on crystal orientation) compared with crystals of dimeric PSllcc, and the packing of the molecules within the unit cell is different. In the monomer, 19 protein subunits, 35 chlorophylls, two pheophytins, the non-heme iron, the primary plastoquinone QA, two heme groups, 11 β-carotenes, 22 lipids, seven detergent molecules, and the Mn4Ca cluster of the water oxidizing complex could be assigned analogous to the dimer. Based on the new structural information, the roles of lipids and protein subunits in dimer formation of PSIIcc are discussed. Due to the lack of noncrystallographic symmetry and the orientation of the membrane normal of PSIIcc perpendicular (~87°) to the crystallographic b-axis, further information about the structure of the Mn4Ca cluster is expected to become available from orientation-dependent spectroscopy on this new crystal form. [ABSTRACT FROM AUTHOR]

Members of the Bin/amphiphysin/Rvs (BAR) domain protein super-family are involved in membrane remodeling in various cellular pathways ranging from endocytic vesicleand T-tubule formation to cell migration and neuromorphogenesis. Membrane curvature induction and stabilization are encoded within the BAR or Fer-CIP4 homology-BAR (F-BAR) domains, a-helical coiled coils that dimerize into membrane-binding modules. BAR/F-BAR domain proteins often contain an SH3 domain, which recruits binding partners such as the oligomeric membrane-fissioning GTPase dynamin. How precisely BAR/F-BAR domain-mediated membrane deformation is regulated at the cellular level is unknown. Here we present the crystal structures of full-length syndapin 1 and its F-BAR domain. Our data show that syndapin 1 F-BAR-mediated membrane deformation is subject to autoinhibition by its SH3 domain. Release from the clamped conformation is driven by association of syndapin 1 SH3 with the proline-rich domain of dynamin 1, thereby unlocking its potent membrane-bending activity. We hypothesize that this mechanism might be commonly used to regulate BAR/F-BAR domain-induced membrane deformation and to potentially couple this process to dynamin-mediated fission. Our data thus suggest a structure-based model for SH3-mediated regulation of BAR/F-BAR domain function. [ABSTRACT FROM AUTHOR]

Abstract: GacH is the solute binding protein (receptor) of the putative oligosaccharide ATP-binding cassette transporter GacFG, encoded in the acarbose biosynthetic gene cluster (gac) from Streptomyces glaucescens GLA.O. In the context of the proposed function of acarbose (acarviosyl-1,4-maltose) as a ‘carbophor,’ the transporter, in complex with a yet to be identified ATPase subunit, is supposed to mediate the uptake of longer acarbose homologs and acarbose for recycling purposes. Binding assays using isothermal titration calorimetry identified GacH as a maltose/maltodextrin-binding protein with a low affinity for acarbose but with considerable binding activity for its homolog, component 5C (acarviosyl-1,4-maltose-1,4-glucose-1,1-glucose). In contrast, the maltose-binding protein of Salmonella typhimurium (MalE) displays high-affinity acarbose binding. We determined the crystal structures of GacH in complex with acarbose, component 5C, and maltotetraose, as well as in unliganded form. As found for other solute receptors, the polypeptide chain of GacH is folded into two distinct domains (lobes) connected by a hinge, with the interface between the lobes forming the substrate-binding pocket. GacH does not specifically bind the acarviosyl group, but displays specificity for binding of the maltose moiety in the inner part of its binding pocket. The crystal structure of acarbose-loaded MalE showed that two glucose units of acarbose are bound at the same region and position as maltose. A comparative analysis revealed that in GacH, acarbose is buried deeper into the binding pocket than in MalE by exactly one glucose ring shift, resulting in a total of 18 hydrogen-bond interactions versus 21 hydrogen-bond interactions for MalEacarbose. Since the substrate specificity of ATP-binding cassette import systems is determined by the cognate binding protein, our results provide the first biochemical and structural evidence for the proposed role of GacHFG in acarbose metabolism. [Copyright &y& Elsevier]

Clathrin-mediated synaptic vesicle (SV) recycling involves the spatiotemporally controlled assembly of clathrin coat components at phosphatidylinositiol (4, 5)-bisphosphate [Pl(4,5)P2]-enriched membrane sites within the periactive zone. Such spatiotemporal control is needed to coordinate SV cargo sorting with clathrin/AP2 recruitment and to restrain membrane fission and synaptojanin-mediated uncoating until membrane deformation and clathnn coat assembly are completed. The molecular events underlying these control mechanisms are unknown. Here we show that the endocytic SH3 domaincontaining accessory protein intersectin 1 scaffolds the endocytic process by directly associating with the clathrin adaptor AP2. Acute perturbation of the intersectin 1-AP2 interaction in lamprey synapses in situ inhibits the onset of SV recycling. Structurally, complex formation can be attributed to the direct association of hydrophobic peptides within the intersectin 1 SH3A-B linker region with the "side sites" of the AP2α and β-appendage domains. AP2 appendage association of the SH3A-B linker region inhibits binding of the inositol phosphatase synaptojanin 1 to intersectin 1. These data identify the intersectin-AP2 complex as an important regulator of clathrinmediated SV recycling in synapses. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential determinant in clathrin-mediated endocytosis (CME). In mammals three type I phosphatidylinositol-4-phosphate 5-kinase (PIPK) enzymes are expressed, with the Iγ-p90 isoform being highly expressed in the brain where it regulates synaptic vesicle (SV) exo-/endocytosis at nerve terminals. How precisely PI(4,5)P2 metabolism is controlled spatially and temporally is still uncertain, but recent data indicate that direct interactions between type I PIPK and components of the endocytic machinery, in particular the AP-2 adaptor complex, are involved. Here we demonstrated that PIPKIγ-p90 associates with both the μ and β2 subunits of AP-2 via multiple sites. Crystallographic data show that a peptide derived from the splice insert of the human PIPKIγ-p90 tail binds to a cognate recognition site on the sandwich subdomain of the β2 appendage. Partly overlapping aromatic and hydrophobic residues within the same peptide also can engage the C-terminal sorting signal binding domain of AP-2μ, thereby potentially competing with the sorting of conventional YXXØ motif-containing cargo. Biochemical and structure-based mutagenesis analysis revealed that association of the tail domain of PIPKIγ-p90 with AP-2 involves both of these sites. Accordingly the ability of overexpressed PIPKIγ tail to impair endocytosis of SVs in primary neurons largely depends on its association with AP-2β and AP-2μ. Our data also suggest that interactions between AP-2 and the tail domain of PIPKIγ-p90 may serve to regulate complex formation and enzymatic activity. We postulate a model according to which multiple interactions between PIPKIγ-p90 and AP-2 lead to spatiotemporally controlled PI(4,5)P2 synthesis during clathrin-mediated SV endocytosis. [ABSTRACT FROM AUTHOR]

Abstract: The magnesium atom of chlorophylls (Chls) is always five- or six-coordinated within chlorophyll–protein complexes which are the main light-harvesting systems of plants, algae and most photosynthetic bacteria. Due to the presence of stereocenters and the axial ligation of magnesium the two faces of Chls are diastereotopic. It has been previously recognized that the α-configuration having the magnesium ligand on the opposite face of the 17-propionic acid moiety is more frequently encountered and is more stable than the more seldom β-configuration that has the magnesium ligand on the same face [T.S. Balaban, P. Fromme, A.R. Holzwarth, N. Krauβ, V.I. Prokhorenko, Relevance of the diastereotopic ligation of magnesium atoms in chlorophylls in Photosystem I, Biochim. Biophys. Acta (Bioenergetics), 1556 (2002) 197–207; T. Oba, H. Tamiaki, Which side of the π-macrocycle plane of (bacterio)chlorophylls is favored for binding of the fifth ligand? Photosynth. Res. 74 (2002) 1–10]. In photosystem I only 14 Chls out of a total of 96 are in a β-configuration and these occupy preferential positions around the reaction center. We have now analyzed the α/β dichotomy in the homodimeric photosystem II based on the 2.9 Å resolution crystal structure [A. Guskov, J. Kern, A. Gabdulkhakov, M. Broser, A. Zouni, W. Saenger, Cyanobacterial photosystem II at 2.9 Å resolution: role of quinones, lipids, channels and chloride, Nature Struct. Mol. Biol. 16 (2009) 334–342] and find that out of 35 Chls in each monomer only 9 are definitively in the β-configuration, while 4 are uncertain. Ab initio calculations using the approximate coupled-cluster singles-and-doubles model CC2 [O. Christiansen, H. Koch, P. Jørgensen, The second-order approximate coupled cluster singles and doubles model CC2, Chem. Phys. Lett. 243 (1995) 409–418] now correctly predict the absorption spectra of Chls a and b and conclusively show for histidine, which is the most frequent axial ligand of magnesium in chlorophyll–protein complexes, that only slight differences (<4 nm) are encountered between the α- and β-configurations. Significant red shifts (up to 50 nm) can, however, be encountered in excitonically coupled β–β-Chl dimers. Surprisingly, in both photosystems I and II very similar “special” β–β dimers are encountered at practically the same distances from P700 and P680, respectively. In purple bacteria LH2, the B850 ring is composed exclusively of such tightly coupled β-bacteriochlorophylls a. A statistical analysis of the close contacts with the protein matrix (<5 Å) shows significant differences between the α- and β-configurations and the subunit providing the axial magnesium ligand. The present study allows us to conclude that the excitation energy transfer in light-harvesting systems, from a peripheral antenna towards the reaction center, may follow preferential pathways due to structural reasons involving β-ligated Chls. [Copyright &y& Elsevier]

Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD) of H. pylori DnaB (HpDnaB) helicase at 2.2 Å resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria. [ABSTRACT FROM AUTHOR]

Summary: Using the 2.9 Å resolution structure of the membrane-intrinsic protein-cofactor complex photosystem II (PSII) from the cyanobacterium Thermosynechococcus elongatus, we calculated and characterized nine possible substrate/product channels leading to/away from the Mn4Ca cluster, where water is oxidized to dioxygen, protons, and electrons. Five narrow channels could function in proton transport, assuming that no large structural changes are associated with water oxidation. Four wider channels could serve to supply water to or remove oxygen from the Mn4Ca cluster. One of them might be regulated by conformational changes of Lys134 in subunit PsbU. Data analyses of Kr derivatized crystals and complexes with dimethyl sulfoxide (DMSO) confirm the accessibility of the proposed dioxygen channels to other molecules. Results from Xe derivatization suggest that the lipid clusters within PSII could serve as a drain for oxygen because of their predominant hydrophobic character and mediate dioxygen release from the lumen. [Copyright &y& Elsevier]

For the initiation of DNA replication, dsDNA is unwound by helicases. Primases then recognize specific sequences on the template DNA strands and synthesize complementary oligonucleotide primers that are elongated by DNA polymerases in leading- and lagging-strand mode. The bacterial plasmid RSF1010 provides a model for the initiation of DNA replication, because it encodes the smallest known primase RepB' (35.9 kDa). features only 1 single-stranded primase initiation site on each strand (ssiA and ssiB, each 40 nt long with 5'- and 3,'-terminal 6 and 13 single-stranded nucleotides. respectively, and nucleotides 7-27 forming a hairpin), and is replicated exclusively in leading strand mode. We present the crystal structure of full-length dumbbell-shaped RepB' consisting of an N-terminal catalytic domain separated by a long α-helix and tether from the C-terminal helix-bundle domain and the structure of the catalytic domain in a specific complex with the 6 5'-terminal single-stranded nucleotides and the C7-G27 base pair of ssiA, its single-stranded 3'-terminus being deleted. The catalytic domains of RepB' and the archaeal/eukaryotic family of Pri-type primases share a common fold with conserved catalytic amino acids, but RepB' lacks the zinc-binding motif typical of the Pri-type primases. According to complementation studies the catalytic domain shows primase activity only in the presence of the helix-bundle domain. Primases that are highly homologous to RepB' are encoded by broad-host-range IncQ and lncQ-like plasmids that share primase initiation sites ssiA and ssiB and high sequence identity with RSF1010. [ABSTRACT FROM AUTHOR]

Photosystem II (PSII) is a large homodimeric protein–cofactor complex located in the photosynthetic thylakoid membrane that acts as light-driven water:plastoquinone oxidoreductase. The crystal structure of PSII from Thermosynechococcus elongatus at 2.9-Å resolution allowed the unambiguous assignment of all 20 protein subunits and complete modeling of all 35 chlorophyll a molecules and 12 carotenoid molecules, 25 integral lipids and 1 chloride ion per monomer. The presence of a third plastoquinone QC and a second plastoquinone-transfer channel, which were not observed before, suggests mechanisms for plastoquinol-plastoquinone exchange, and we calculated other possible water or dioxygen and proton channels. Putative oxygen positions obtained from a Xenon derivative indicate a role for lipids in oxygen diffusion to the cytoplasmic side of PSII. The chloride position suggests a role in proton-transfer reactions because it is bound through a putative water molecule to the Mn4Ca cluster at a distance of 6.5 Å and is close to two possible proton channels. [ABSTRACT FROM AUTHOR]

Inflammatory processes are accompanied by the posttranslational modification of certain arginine residues within proteins to yield citrulline, although it is largely unknown how this modification influences antigen presentation. We employed crystallographic and functional studies to investigate whether the exchange of arginine to citrulline affects the display of a peptide by two human major histocompatibility antigen class I subtypes, HLA-B*2705 and HLA-B*2709. Both differ only in residue 116 within the peptide binding groove despite their differential association with ankylosing spondylitis, an inflammatory rheumatic disorder. The crystal structures described here show that a modified self-peptide, pVIPR-U5 (RRKWURWHL; U - citrulline), is presented by the two HLA-B27 molecules in distinct conformations. These binding modes differ not only drastically from each other but also from the conformations exhibited by the non-citrullinated peptide in a given subtype. The differential reactivity of HLA-B27-restricted cytotoxic T cells with modified or unmodified pVIPR supports the structural findings and shows that the presentation of citrullinated peptides has the potential to influence immune responses. [ABSTRACT FROM AUTHOR]