Human CD81, a known receptor for hepatitis C virus envelope E2 glycoprotein, is a transmembrane protein belonging to the tetraspanin family. The crystal structure of human CD81 large extracellular domain is reported here at 1.6 A resolution. Each subunit within the homodimeric protein displays a mushroom-like structure, composed of five alpha-helices arranged in 'stalk' and 'head' subdomains. Residues known to be involved in virus binding can be mapped onto the head subdomain, providing a basis for the design of antiviral drugs and vaccines. Sequence analysis of 160 tetraspanins indicates that key structural features and the new protein fold observed in the CD81 large extracellular domain are conserved within the family. On these bases, it is proposed that tetraspanins may assemble at the cell surface into homo- and/or hetero-dimers through a conserved hydrophobic interface located in the stalk subdomain, while interacting with other liganding proteins, including hepatitis C virus E2, through the head subdomain. The topology of such interactions provides a rationale for the assembly of the so-called tetraspan-web.

The crystal structure at 1.8 A resolution of 8-HDF type photolyase from A. nidulans shows a backbone structure similar to that of MTHF type E. coli photolyase but reveals a completely different binding site for the light-harvesting cofactor.

Pasteurella multocida toxin (PMT), one of the virulence factors produced by the bacteria, exerts its toxicity by up-regulating various signaling cascades downstream of the heterotrimeric GTPases Gq and G12/13 in an unknown fashion. Here, we present the crystal structure of the C-terminal region (residues 575-1,285) of PMT, which carries an intracellularly active moiety. The overall structure of C-terminal region of PMT displays a Trojan horse-like shape, composed of three domains with a ''feet''-,''body''-, and ''head''-type arrangement, which were designated C1, C2, and C3 from the N to the C terminus, respectively. The C1 domain, showing marked similarity in steric structure to the N-terminal domain of Clostridium difficile toxin B, was found to lead the toxin molecule to the plasma membrane. The C3 domain possesses the Cys-HisAsp catalytic triad that is organized only when the Cys is released from a disulfide bond. The steric alignment of the triad corresponded well to that of papain or other enzymes carrying CysHis-Asp. PMT toxicities on target cells were completely abrogated when one of the amino acids constituting the triad was mutated. Our results indicate that PMT is an enzyme toxin carrying the cysteine protease-like catalytic triad dependent on the redox state and functions on the cytoplasmic face of the plasma membrane of target cells.crystallography ͉ cysteine protease ͉ membrane targeting

Clostridium perfringens enterotoxin (CPE) is a cause of food poisoning and is considered a pore-forming toxin, which damages target cells by disrupting the selective permeability of the plasma membrane. However, the pore-forming mechanism and the structural characteristics of the pores are not well documented. Here, we present the structure of CPE determined by x-ray crystallography at 2.0 Å . The overall structure of CPE displays an elongated shape, composed of three distinct domains, I, II, and III. Domain I corresponds to the region that was formerly referred to as C-CPE, which is responsible for binding to the specific receptor claudin. Domains II and III comprise a characteristic module, which resembles those of ␤-pore-forming toxins such as aerolysin, C. perfringens ⑀-toxin, and Laetiporus sulfureus hemolytic pore-forming lectin. The module is mainly made up of ␤-strands, two of which span its entire length. Domain II and domain III have three short ␤-strands each, by which they are distinguished. In addition, domain II has an ␣-helix lying on the ␤-strands. The sequence of amino acids composing the ␣-helix and preceding ␤-strand demonstrates an alternating pattern of hydrophobic residues that is characteristic of transmembrane domains forming ␤-barrel-made pores. These structural features imply that CPE is a ␤-pore-forming toxin. We also hypothesize that the transmembrane domain is inserted into the membrane upon the buckling of the two long ␤-strands spanning the module, a mechanism analogous to that of the cholesterol-dependent cytolysins. Clostridium perfringens enterotoxin (CPE),2 which damages intestinal epithelia, is a causative agent of food poisoning. The toxin consists of a single chain polypeptide of 319 amino acids. The C-terminal domain of CPE (C-CPE, residues 184 -319) recognizes and binds to certain members of the claudin family, components of tight junctions, as a receptor on target cells (1-4), and the N-terminal region is believed to be involved in forming physiological pores to disrupt the selective permeability of the plasma membrane, resulting in cell death (5-7). It was also reported that the physiological pores are composed of a large complex comprising CPE and cellular components such as claudins (2,8).The binding between C-CPE and claudins has been well characterized. The 16 -17 C-terminal amino acids of C-CPE were reportedly important for the interaction (9, 10), especially, Tyr 306 , Tyr 310 , Tyr 312, and Leu 315 (11-13). According to the crystal structure of C-CPE (14), these residues organize a cleft space that is considered to interact directly with claudins. Claudins are tetratransmembrane proteins. The region of claudins responsible for binding to CPE was located on the C-terminal side of the second extracellular loop and recently designated CPE-SR for CPE sensitivity-related region (15). The bottom of the cleft space of CPE is negatively charged, whereas the CPESRs of CPE-sensitive claudins are positively charged. Therefore, it was proposed that electrostatic attraction at...

Background: Botulinum HA is a component of the botulinum neurotoxin complex and dramatically increases the oral toxicity of the complex. Results: The crystal structure of botulinum HA reveals that 12 subcomponents of HA constitute a huge triskelion-shaped molecule. Conclusion:The complex is functionally and structurally separable into two parts. Significance: This is the first crystal structure of the whole botulinum HA complex.

Pasteurella multocida toxin (PMT) is a virulence factor responsible for the pathogenesis of some Pasteurellosis. PMT exerts its toxic effects through the activation of heterotrimeric GTPase (Gq, G12/13 and Gi)‐dependent pathways, by deamidating a glutamine residue in the α subunit of these GTPases. However, the enzymatic characteristics of PMT are yet to be analyzed in detail because the deamidation has only been observed in cell‐based assays. In the present study, we developed rat monoclonal antibodies, specifically recognizing the deamidated Gαq, to detect the actions of PMT by immunological techniques such as western blotting. Using the monoclonal antibodies, we found that the toxin deamidated Gαq only under reducing conditions. The C‐terminal region of PMT, C‐PMT, was more active than the full‐length PMT. The C3 domain possessing the enzyme core catalyzed the deamidation in vitro without any other domains. These results not only support previous observations on toxicity, but also provide insights into the enzymatic nature of PMT. In addition, we present several lines of evidence that Gα11, as well as Gαq, could be a substrate for PMT.

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Oxidative base damage leads to alteration of genomic information and is implicated as a cause of aging and carcinogenesis. To combat oxidative damage to DNA, cells contain several DNA glycosylases including OGG1, NTH1 and the Nei-like proteins, NEIL1 and NEIL2. A third Nei-like protein, NEIL3, is composed of an amino-terminal Nei-like domain and an unknown carboxy-terminal domain. In contrast to the other well-described DNA glycosylases, the DNA glycosylase activity and in vivo repair function of NEIL3 remains unclear. We show here that the structural modeling of the putative NEIL3 glycosylase domain (1-290) fits well to the known Escherichia coli Fpg crystal structure. In spite of the structural similarity, the recombinant NEIL3 and NEIL3(1-290) proteins do not cleave any of several test oligonucleotides containing a single modified base. Within the substrates, we detected AP lyase activity for single-stranded (ss) DNA but double-stranded (ds) DNA. The activity is abrogated completely in mutants with an aminoterminal deletion and at the zinc-finger motif. Surprisingly, NEIL3 partially rescues an E. coli nth nei mutant from hydrogen peroxide sensitivity. Taken together, repair of certain base damage including base loss in ssDNA may be mediated by NEIL3.