The genes of N. pharaonis SRII and the carboxy terminal truncated transducer (1-114) were cloned into a pET27bmod expression vector 24 with a C-terminal £ 7 His tag, respectively. Proteins were expressed in Escherichia coli strain BL21 (DE3), and purified as described 25,26. After removal of imidazol by diethyl-aminoethyl chromatography, SRII-His and HtrII 114-His were mixed in a 1:1 ratio, followed by reconstitution into purple membrane (the bacteriorhodopsin containing membrane patches of H. salinarum) lipids 7 (protein to lipid ratio 1:35). After filtration, the reconstituted proteins were pelleted by centrifugation at 100,000g. For resolubilization, the samples were resuspended in a buffer containing 2% n-octyl-b-D-glucopyranoside and shaken for 16 h at 4 8C in the dark. The resolubilized complex was isolated by centrifugation at 100,000g. Crystallization, structure determination and refinement We added the solubilized complex in crystallization buffer (150 mM NaCl, 25 mM Na/KPi, pH 5.1, 0.8% n-octyl-b-D-glucopyranoside) to the lipidic phase, formed from monovaccenin (Nu-Chek Prep). Precipitant was 1 M salt Na/KPi, pH 5.6. Crystals were grown at 22 8C. X-ray diffraction data were collected at beamline ID14-1 of the European Synchrotron Radiation Facility (ESRF), Grenoble, France, using a Quantum ADSC Q4R CCD (charge-coupled device) detector. Data were integrated using MOSFILM 27 and SCALA 28. Molecular replacement using MOLREP 28 to phase a polyalanine model (from Protein Data Bank accession number 1JGJ (ref. 12)) gave a unique solution (R ¼ 0.568, correlation coefficient C ¼ 0.357) at 2.9 A ˚. After inserting side chains for SRII, the helices of HtrII were found (R ¼ 0.329, C ¼ 0.711). Simulated annealing, positional refinement and temperature factor refinement were performed in CNS 29 ; model rebuilding was carried out in O 30 (Table 1).

Retinal arrestin is the essential protein for the termination of the light response in vertebrate rod outer segments. It plays an important role in quenching the light-induced enzyme cascade by its ability to bind to phosphorylated light-activated rhodopsin (P-Rh*). Arrestins are found in various G-protein-coupled amplification cascades. Here we report on the three-dimensional structure of bovine arrestin (relative molecular mass, 45,300) at 3.3 A resolution. The crystal structure comprises two domains of antiparallel beta-sheets connected through a hinge region and one short alpha-helix on the back of the amino-terminal fold. The binding region for phosphorylated light-activated rhodopsin is located at the N-terminal domain, as indicated by the docking of the photoreceptor to the three-dimensional structure of arrestin. This agrees with the interpretation of binding studies on partially digested and mutated arrestin.

Cells steadily adapt their membrane glycerophospholipid (GPL) composition to changing environmental and developmental conditions. While the regulation of membrane homeostasis via GPL synthesis in bacteria has been studied in detail, the mechanisms underlying the controlled degradation of endogenous GPLs remain unknown. Thus far, the function of intracellular phospholipases A (PLAs) in GPL remodeling (Lands cycle) in bacteria is not clearly established. Here, we identified the first cytoplasmic membrane-bound phospholipase A1 (PlaF) from Pseudomonas aeruginosa, which might be involved in the Lands cycle. PlaF is an important virulence factor, as the P. aeruginosa ΔplaF mutant showed strongly attenuated virulence in Galleria mellonella and macrophages. We present a 2.0-Å-resolution crystal structure of PlaF, the first structure that reveals homodimerization of a single-pass transmembrane (TM) full-length protein. PlaF dimerization, mediated solely through the intermolecular interactions of TM and juxtamembrane regions, inhibits its activity. The dimerization site and the catalytic sites are linked by an intricate ligand-mediated interaction network, which might explain the product (fatty acid) feedback inhibition observed with the purified PlaF protein. We used molecular dynamics simulations and configurational free energy computations to suggest a model of PlaF activation through a coupled monomerization and tilting of the monomer in the membrane, which constrains the active site cavity into contact with the GPL substrates. Thus, these data show the importance of the PlaF mediated GPL remodeling pathway for virulence and could pave the way for the development of novel therapeutics targeting PlaF.

The Thermus aualcus DNA methyltransferase M'Taq I (EC 2.1.1.72) methylates N6 of adenine in the specific double-helical DNA sequence TCGA by transfer of -CH3 from the cofactor S-adenosyl-L-methionine. The x-ray crystal structure at 2.4-A resolution of this enzyme in complex with S-adenosylmethlonlne shows a/P folding of the polypeptide into two domains of about equal size. They are arranged in the form of a C with a wide deft suitable to accommodate the DNA substrate. The N-terminal domain Is dominated by a nine-stranded 3-sheet; it contains the two conserved segments typical for N-methyltranserases which form a pocket for cofactor binding. The C-terminal domain is formed by four small 1-sheets and a-helices. The three-dimensional folding of M'Taq I is similar to that of the cytosine-speciflc Hha I methyltransferase, where the large 1-sheet in the N-terminal domain contains all conserved segments and the enzymatically functional parts, and the smaller C-terminal domain is less structured.DNA-methyltransferases (MTases) are a family of enzymes that occur in nearly all living organisms. They catalyze the transfer of-CH3 from the cofactor S-adenosyl-L-methionine (AdoMet) to cytosine C5 (C-MTases) or cytosine N4 or adenine N6 (N-MTases) in di-to octanucleotide target sequences of double-stranded DNA (1). In bacteria, all three types of MTases are found and implicated in the protection of DNA from their own restriction endonucleases and in mismatch repair (2). In eukaryotes only C-MTases have been observed so far; they are involved in cell differentiation, genome imprinting, mutagenesis, and regulation of gene expression (3).The C-MTases are a homogeneous class of molecules with three-dimensional structures probably similar to the structure described recently for the M-Hha I enzyme from Haemophilus haemolyticus (4). This is because their amino acid sequences show sequential arrangement of 10 conserved segments (I to X) from the N to the C terminus (5); segments I (DXFXGXG, with X = any amino acid) and IV (FPCQ) are implicated in binding of AdoMet, and the cysteine in IV is involved in the transfer of -CH3. In contrast, the N-MTases show only two of the conserved segments (6). They correspond to segments I and IV in the C-MTases, namely I (DXFXGXG), which can degenerate so much that only one glycine is retained, and II (DPPY), where aspartate can be replaced by asparagine or seine, and tyrosine by phenylalanine. Because these two segments can occur in reversed order-i.e., one or the other N-terminal (7)-the N-MTases are a more heterogeneous class of molecules. When the amino acid sequences of only those N-MTases that recognize TNNA (N = any nucleotide) are compared, an additional segment III is found (8). It spans 38 amino acids, has no equivalent in C-MTases, and occurs sequentially-i.e., I, II, III. The mechanism of methyl transfer is different in C-and N-MTases. In the former the conserved cysteine SH in segment IV attacks C6 of cytosine to form a covalent intermediate with resonance-stabilized carbanionic ...