The three-dimensional structure of acetylcholinesterase from Torpedo californica electric organ has been determined by x-ray analysis to 2.8 angstrom resolution. The form crystallized is the glycolipid-anchored homodimer that was purified subsequent to solubilization with a bacterial phosphatidylinositol-specific phospholipase C. The enzyme monomer is an alpha/beta protein that contains 537 amino acids. It consists of a 12-stranded mixed beta sheet surrounded by 14 alpha helices and bears a striking resemblance to several hydrolase structures including dienelactone hydrolase, serine carboxypeptidase-II, three neutral lipases, and haloalkane dehalogenase. The active site is unusual because it contains Glu, not Asp, in the Ser-His-acid catalytic triad and because the relation of the triad to the rest of the protein approximates a mirror image of that seen in the serine proteases. Furthermore, the active site lies near the bottom of a deep and narrow gorge that reaches halfway into the protein. Modeling of acetylcholine binding to the enzyme suggests that the quaternary ammonium ion is bound not to a negatively charged "anionic" site, but rather to some of the 14 aromatic residues that line the gorge.

We have identified a new protein fold--the alpha/beta hydrolase fold--that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta sheet, not barrel, of eight beta-sheets connected by alpha-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the alpha/beta hydrolase fold enzymes.

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Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins.

The alternative pathway of complement is important in innate immunity, attacking not only microbes but all unprotected biological surfaces through powerful amplification. It is unresolved how host and nonhost surfaces are distinguished at the molecular level, but key components are domains 19-20 of the complement regulator factor H (FH), which interact with host (i.e., nonactivator surface glycosaminoglycans or sialic acids) and the C3d part of C3b. Our structure of the FH19-20:C3d complex at 2.3-Å resolution shows that FH19-20 has two distinct binding sites, FH19 and FH20, for C3b. We show simultaneous binding of FH19 to C3b and FH20 to nonactivator surface glycosaminoglycans, and we show that both of these interactions are necessary for full binding of FH to C3b on nonactivator surfaces (i.e., for target discrimination). We also show that C3d could replace glycosaminoglycan binding to FH20, thus providing a feedback control for preventing excess C3b deposition and complement amplification. This explains the molecular basis of atypical hemolytic uremic syndrome, where mutations on the binding interfaces between FH19-20 and C3d or between FH20 and glycosaminoglycans lead to complement attack against host surfaces. structure and function | X-ray crystallography | hemolysis | kidney diseases | human mutations P reviously unencountered microbes invading a human body must be rapidly recognized and eliminated. This is the function of innate immunity, which includes the alternative pathway (AP) of complement. AP components can attack targets with hydroxyl or amine groups (i.e., all biological surfaces). This is a powerful defense mechanism, because there is rapid amplification leading to efficient opsonization or target lysis by the membrane attack complex (MAC). The AP attack is, therefore, also potentially dangerous for the host if one's cells and acellular structures are not protected.The AP activation is based on spontaneous hydrolysis of C3 in plasma leading to production of C3b, which then randomly attaches onto any surface hydroxyl or amine group through a reactive thioester located on the C3d part [i.e., thioester domain (TED)] of C3b. If these surface-attached C3b molecules are not quickly inactivated to iC3b and C3d, C3b deposition is rapidly amplified by a positive enzymatic feedback loop, leading to opsonophagocytosis and formation of the lytic membrane attack complex. On host surfaces, which are naturally nonactivators of the AP, efficient down-regulation of bound C3b occurs in three ways: factor I-mediated cleavage of C3b to inactive iC3b, acceleration of the decay of the preformed C3 convertases, or inhibition of factor B binding to C3b. Factor H (FH) is required for all these. It also down-regulates C3b deposition on noncellular surfaces, such as the heparan sulfate-rich glomerular basement membrane. FH is, thus, essential for restricting AP attack against host surfaces while allowing AP attack against foreign surfaces (i.e., for target discrimination) (1). A long-standing central question in complemen...

alpha/beta Hydrolase fold proteins are an important, diverse, widespread group of enzymes not yet fully exploited by structural biologists. We describe the current state of knowledge of this family, and suggest a smaller definition of the required core and some possible future avenues of exploration.

Our structure-based model of the PPase mechanism posits that the nucleophile is the hydroxide ion mentioned above. This aspect of the mechanism is formally analogous to the "two-metal ion' mechanism of alkaline phosphatase, exonucleases and polymerases. A third metal ion coordinates another water molecule that is probably the required general acid. Extensive Lewis acid coordination and hydrogen bonds provide charge shielding of the electrophile and lower the pKa of the leaving group. This "three-metal ion' mechanism is in detail different from that of other phosphoryl transfer enzymes, presumably reflecting how ancient the reaction is.

The crystal structure of the recombinant collagen-binding domain of Yersinia adhesin YadA from Yersinia enterocolitica serotype O:3 was solved at 1.55 Å resolution. The trimeric structure is composed of head and neck regions, and the collagen binding head region is a novel ninecoiled left-handed parallel b-roll. Before the b-roll, the polypeptide loops from one monomer to the rest, and after the b-roll the neck region does the same, making the transition from the globular head region to the narrower stalk domain. This creates an intrinsically stable 'lock nut' structure. The trimeric form of YadA is required for collagen binding, and mutagenesis of its surface residues allowed identification of a putative collagen-binding surface. Furthermore, a new structure-sequence motif for YadA b-roll was used to identify putative YadA-head-like domains in a variety of human and plant pathogens. Such domains may therefore be a common bacterial strategy for avoiding host response.