DsbB is an E. coli membrane protein that oxidizes DsbA, the primary protein disulfide donor present in the periplasm. To understand how disulfide bonds are generated and introduced into secreted proteins, we determined the crystal structure of DsbB in a complex with DsbA and endogenous ubiquinone at 3.7 Å resolution. The first structure of DsbB revealed that DsbB contains the four-helix bundle scaffold in the transmembrane region and one short membrane-parallel α-helix in the long periplasmic loop. Strikingly, the disulfide-generating reaction center composed of Cys41, Cys44, Arg48 and ubiquinone is located near the N-terminus of the transmembrane helix 2, where oxidizing equivalents of ubiquinone are converted to a protein disulfide bond de novo. Whereas DsbB in the resting state contains a Cys104-Cys130 disulfide, Cys104 in the ternary complex is engaged in the intermolecular disulfide bond and captured by the hydrophobic groove of DsbA, resulting in its separation from Cys130. This DsbA-induced conformational change in DsbB seems to prevent the backward resolution of the complex and thereby promote the physiological electron flow from DsbA to DsbB. Recently, I examined functional roles of the membrane-parallel α-helix with strong amphiphilicity by systematic mutation analyses. Introduction of charged or helix-breaking residues into this region not only disrupted the peripheral membrane-association of this helix but also impaired DsbA oxidation activity of DsbB. On the basis of structural and biochemical data so far obtained, I propose the "cysteine relocation mechanism", by which DsbB oxidizes the extremely oxidizing (reduction-prone) dithiol oxidase, DsbA, efficiently.

We have characterized a putative Ca 2؉ -ATPase from the pathogenic bacterium Listeria monocytogenes with the locus tag lmo0841.

Two high resolution crystal structures of isatin hydrolase (IH) from the Baltic seabed bacteria Labrenzia aggregata are presented. The crystals structure capture both the apo and the product state. This hydrolase present a new fold and the first metal-dependent hydrolase with this fold to be functionally characterized [1]. The Isatin hydrolase catalyze the reaction that convert isatin to isatinate and belongs to a novel family of metalloenzymes that include bacterial kynurenine formamidase (KynB) also recently published, however hoste a binclear zink site in the active site[2] as compared to a single manganese in IH. The product state, mimicked by thioisatinate, has captured an additional water molecule that bridges the thioisatinate to a water channel and that could act as a proton wire and thus allows the proton to be released during the hydrolysis reaction only when the product is formed. The functional proton wire is therefore locked by thioisatinate and represents a unique catalytic feature trapped and visualized. Biochemical evidence for the proton wire is also presented as single point mutation from S225C enhances the Vmax of the enzyme. Ser-225 is the only side chain residue that is included in the proton wire. The molecular basis for thioisatinate recognition allows stronger and more confident identification of orthologous genes encoding isatin hydrolases within the prokaryotic kingdom. The isatin hydrolase orthologues found in human gut bacteria raise the question as to whether the indole-3-acetic acid degradation pathway is present in human gut flora.

Toll-like Receptors (TLRs) are central to vertebrate innate immune responses. To facilitate soluble expression and crystallization of human TLRs with bound ligands, we have developed a novel technique that we term the Hybrid LRR Technique. The hagfish VLR proteins were chosen as the fusion partners and connected to human TLRs at the conserved LxxLxLxxN regions. The hybrid LRR technique neither interrupts function of TLR nor causes substantial structural changes. TLR4 and MD-2 form a heterodimer that recognizes LPS from Gram negative bacteria. TLR2 in association with TLR1 or TLR6 responses to microbial lipoproteins and lipopeptides. The crystal structures reveal that TLR1, 2 and 4 are atypical members of the LRR family and are composed of N-terminal, central and C-terminal domains. The beta sheet of the central domain shows unusually small radii and large twist angles. MD-2 binds to the concave surface of the N-terminal and central domains of TLR4. The interaction with Eritoran, a candidate antisepsis drug, is mediated by a hydrophobic internal pocket in MD-2. Binding of the tri-acylated lipopeptide, Pam3CSK4, induced the formation of an m shaped heterodimer of the TLR1 and TLR2 ectodomains whereas binding of the di-acylated lipopeptide, Pam2CSK4 did not. The three lipid chains of Pam3CSK4 mediate the heterodimerization of the receptor; the two ester-bound lipid chains are inserted into a pocket in TLR2, while the amide-bound lipid chain is inserted into a hydrophobic channel in TLR1. An extensive hydrogen bonding network, as well as hydrophobic interactions, between TLR1 and TLR2 further stabilize the heterodimer. We propose that formation of the TLR dimer brings the intracellular TIR domains close to each other to promote dimerization and initiate signaling.Keywords: protein structure, TLR, LRR MS.50.4 Acta Cryst. (2008). A64, C90Crystal structure of the sodium pump at 3.5 Å The Na + ,K + -ATPase, the sodium-potassium pump, was first described in 1957 by Jens C. Skou (1) -a discovery for which he was awarded the Nobel prize in Chemistry in 1997. The Na + ,K + -ATPase belongs to the P-type ATPase family, and via formation and break-down of phosphoenzyme intermediates it derives the energy from ATP hydrolysis to pump Na + out of the cell and K + into the cell, thereby energizing the plasma membrane with steep electrochemical gradients for these key cations. The Na + ,K + -ATPase is a heterotrimeric complex composed of an α, β and γ chain that all contain transmembrane segments. A complete native dataset was obtained at 3.5 Å resolution on the X06SA beam line at the Swiss Light Source (SLS). The brilliant light source present at SLS was necessary to obtain useful data from these very weakly diffracting crystals. A low resolution molecular replacement solution allowed us to identify two heavy-atom derivatives by difference-Fourier analysis forming the basis for MIRAS phasing at 7 Å resolution. The crystal form has 75% solvent and contains two-fold NCS. Careful density modification with NCS and inter-crystal averag...

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