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Abstract: Cholesterol-dependent cytolysins (CDCs) are a large family of bacterial toxins that exhibit a dependence on the presence of membrane cholesterol in forming large pores in cell membranes. Significant changes in the three-dimensional structure of these toxins are necessary to convert the soluble monomeric protein into a membrane pore. We have determined the crystal structure of the archetypical member of the CDC family, streptolysin O (SLO), a virulence factor from Streptococcus pyogenes. The overall fold is similar to previously reported CDC structures, although the C-terminal domain is in a different orientation with respect to the rest of the molecule. Surprisingly, a signature stretch of CDC sequence called the undecapeptide motif, a key region involved in membrane recognition, adopts a very different structure in SLO to that of the well-characterized CDC perfringolysin O (PFO), although the sequences in this region are identical. An analysis reveals that, in PFO, there are complementary interactions between the motif and the rest of domain 4 that are lost in SLO. Molecular dynamics simulations suggest that the loss of a salt bridge in SLO and a cation–pi interaction are determining factors in the extended conformation of the motif, which in turn appears to result in a greater flexibility of the neighboring L1 loop that houses a cholesterol-sensing motif. These differences may explain the differing abilities of SLO and PFO to efficiently penetrate target cell membranes in the first step of toxin insertion into the membrane. [Copyright &y& Elsevier]

Abstract: GSTD1 is one of several insect glutathione S-transferases capable of metabolizing the insecticide DDT. Here we use crystallography and NMR to elucidate the binding of DDT and glutathione to GSTD1. The crystal structure of Drosophila melanogaster GSTD1 has been determined to 1.1 Å resolution, which reveals that the enzyme adopts the canonical GST fold but with a partially occluded active site caused by the packing of a C-terminal helix against one wall of the binding site for substrates. This helix would need to unwind or be displaced to enable catalysis. When the C-terminal helix is removed from the model of the crystal structure, DDT can be computationally docked into the active site in an orientation favoring catalysis. Two-dimensional 1H,15N heteronuclear single-quantum coherence NMR experiments of GSTD1 indicate that conformational changes occur upon glutathione and DDT binding and the residues that broaden upon DDT binding support the predicted binding site. We also show that the ancestral GSTD1 is likely to have possessed DDT dehydrochlorinase activity because both GSTD1 from D. melanogaster and its sibling species, Drosophila simulans, have this activity. [Copyright &y& Elsevier]

Abstract: The commonly used anti-cancer drug chlorambucil is the primary treatment for patients with chronic lymphocytic leukaemia. Chlorambucil has been shown to be detoxified by human glutathione transferase Pi (GST P1-1), an enzyme that is often found over-expressed in cancer tissues. The allelic variants of GST P1-1 are associated with differing susceptibilities to leukaemia and differ markedly in their efficiency in catalysing glutathione (GSH) conjugation reactions. Here, we perform detailed kinetic studies of the allelic variants with the aid of three representative co-substrates. We show that the differing catalytic properties of the variants are highly substrate-dependent. We show also that all variants exhibit the same temperature stability in the range 10 °C to 45 °C. We have determined the crystal structures of GST P1-1 in complex with chlorambucil and its GSH conjugate for two of these allelic variants that have different residues at positions 104 and 113. Chlorambucil is found to bind in a non-productive mode to the substrate-binding site (H-site) in the absence of GSH. This result suggests that under certain stress conditions where GSH levels are low, GST P1-1 can inactivate the drug by sequestering it from the surrounding medium. However, in the presence of GSH, chlorambucil binds in the H-site in a productive mode and undergoes a conjugation reaction with GSH present in the crystal. The crystal structure of the GSH–chlorambucil complex bound to the *C variant is identical with the *A variant ruling out the hypothesis that primary structure differences between the variants cause structural changes at the active site. Finally, we show that chlorambucil is a very poor inhibitor of the enzyme in contrast to ethacrynic acid, which binds to the enzyme in a similar fashion but can act as both substrate and inhibitor. [Copyright &y& Elsevier]

Abstract: Alzheimer''s disease (AD) is the most common form of dementia. Amyloid-β (Aβ) peptide, generated by proteolytic cleavage of the amyloid precursor protein, is central to AD pathogenesis. Most pharmaceutical activity in AD research has focused on Aβ, its generation and clearance from the brain. In particular, there is much interest in immunotherapy approaches with a number of anti-Aβ antibodies in clinical trials. We have developed a monoclonal antibody, called WO2, which recognises the Aβ peptide. To this end, we have determined the three-dimensional structure, to near atomic resolution, of both the antibody and the complex with its antigen, the Aβ peptide. The structures reveal the molecular basis for WO2 recognition and binding of Aβ. The Aβ peptide adopts an extended, coil-like conformation across its major immunodominant B-cell epitope between residues 2 and 8. We have also studied the antibody-bound Aβ peptide in the presence of metals known to affect its aggregation state and show that WO2 inhibits these interactions. Thus, antibodies that target the N-terminal region of Aβ, such as WO2, hold promise for therapeutic development. [Copyright &y& Elsevier]

Abstract: Chloride intracellular channel (CLIC) proteins possess the remarkable property of being able to convert from a water-soluble state to a membrane channel state. We determined the three-dimensional structure of human CLIC2 in its water-soluble form by X-ray crystallography at 1.8-Å resolution from two crystal forms. In contrast to the previously characterized CLIC1 protein, which forms a possibly functionally important disulfide-induced dimer under oxidizing conditions, we show that CLIC2 possesses an intramolecular disulfide and that the protein remains monomeric irrespective of redox conditions. Site-directed mutagenesis studies show that removal of the intramolecular disulfide or introduction of cysteine residues in CLIC2, equivalent to those that form the intramolecular disulfide in CLIC1, does not cause dimer formation under oxidizing conditions. We also show that CLIC2 forms pH-dependent chloride channels in vitro with higher channel activity at low pH levels and that the channels are subject to redox regulation. In both crystal forms, we observed an extended loop region from the C-terminal domain, called the foot loop, inserting itself into an interdomain crevice of a neighboring molecule. The equivalent region in the structurally related glutathione transferase superfamily corresponds to the active site. This so-called foot-in-mouth interaction suggests that CLIC2 might recognize other proteins such as the ryanodine receptor through a similar interaction. [Copyright &y& Elsevier]

Abstract: Cholesterol-dependent cytolysins (CDCs), a large family of bacterial toxins, are secreted as water-soluble monomers and yet are capable of generating oligomeric pores in membranes. Previous work has demonstrated that large scale structural rearrangements occur during this transition but the detailed mechanism by which these changes take place remains a puzzle. Despite evidence of structural and functional couplings between domains 3 and 4, the crystal structure of the CDC, perfringolysin O (PFO), shows the two domains do not make direct contact. Here, we present crystal structures of PFO that demonstrate movements of domain 4 are sufficient to trigger conformational changes that are transmitted through the molecule to the distant domain 3. These coupled movements result in a loss of many contacts between domain 3 and rest of the molecule that would eventually lead to the exposure of transmembrane regions in preparation for membrane insertion. The structures reveal a detailed molecular pathway that may be the basis for the allosteric transition that occurs on initial membrane binding leading to the exposure of membrane-spanning regions in a domain distant from the initial site of interaction. [Copyright &y& Elsevier]

Abstract: Alzheimer''s disease (AD) is the major cause of dementia. Amyloid β peptide (Aβ), generated by proteolytic cleavage of the amyloid precursor protein (APP), is central to AD pathogenesis. APP can function as a metalloprotein and modulate copper (Cu) transport, presumably via its extracellular Cu-binding domain (CuBD). Cu binding to the CuBD reduces Aβ levels, suggesting that a Cu mimetic may have therapeutic potential. We describe here the atomic structures of apo CuBD from three crystal forms and found they have identical Cu-binding sites despite the different crystal lattices. The structure of Cu2+-bound CuBD reveals that the metal ligands are His147, His151, Tyr168 and two water molecules, which are arranged in a square pyramidal geometry. The site resembles a Type 2 non-blue Cu center and is supported by electron paramagnetic resonance and extended X-ray absorption fine structure studies. A previous study suggested that Met170 might be a ligand but we suggest that this residue plays a critical role as an electron donor in CuBDs ability to reduce Cu ions. The structure of Cu+-bound CuBD is almost identical to the Cu2+-bound structure except for the loss of one of the water ligands. The geometry of the site is unfavorable for Cu+, thus providing a mechanism by which CuBD could readily transfer Cu ions to other proteins. [Copyright &y& Elsevier]

Proteolytic cleavage of the amyloid precursor protein (APP) by β and γ-secretases gives rise to the β-amyloid peptide, considered to be a causal factor in Alzheimer''s disease. Conversely, the soluble extracellular domain of APP (sAPPα), released upon its cleavage by α-secretase, plays a number of important physiological functions. Several APP fragments have been structurally characterized at atomic resolution, but the structures of intact APP and of full-length sAPPα have not been determined. Here, ab initio reconstruction of molecular models from high-resolution solution X-ray scattering (SAXS) data for the two main isoforms of sAPPα (sAPPα695 and sAPPα770) provided models of sufficiently high resolution to identify distinct structural domains of APP. The fragments for which structures are known at atomic resolution were fitted within the solution models of full-length sAPPα, allowing localization of important functional sites (i.e. glycosylation, protease inhibitory and heparin-binding sites). Furthermore, combined results from SAXS, analytical ultracentrifugation (AUC) and size-exclusion chromatography (SEC) analysis indicate that both sAPPα isoforms are monomeric in solution. On the other hand, SEC, bis-ANS fluorescence, AUC and SAXS measurements showed that sAPPα forms a 2:1 complex with heparin. A conformational model for the sAPPα:heparin complex was also derived from the SAXS data. Possible implications of such complex formation for the physiological dimerization of APP and biological signaling are discussed in terms of the structural models proposed. [Copyright &y& Elsevier]

We have sought the structural basis for the differing substrate specificities of human glutathione transferase P1-1 (class Pi) and human glutathione transferase A1-1 (class Alpha) by adding an extra helix (helix 9), found in the electrophilic substrate-binding site (H-site) of the human class Alpha enzyme, at the C terminus of the human class Pi enzyme. This class Pi-chimera (CODA) was expressed in Escherichia coli, purified and characterized by kinetic and crystallographic approaches. The presence of the newly engineered tail in the H-site of the human Pi enzyme alters its catalytic properties towards those exhibited by the human Alpha enzyme, as assessed using cumene hydroperoxide (diagnostic for class Alpha enzymes) and ethacrynic acid (diagnostic for class Pi) as co-substrates. There is a change of substrate selectivity in the latter case, as the kcat/KmEA value decreases about 70-fold, compared to that of class Pi. With 1-chloro-2,4-dinitrobenzene as co-substrate there is a loss of catalytic activity to about 2% with respect to that of the Pi enzyme. Crystallographic and kinetic studies of the class Pi-chimera provide important clues to explain these altered catalytic properties. The new helix forms many complimentary interactions with the rest of the protein and re-models the original electrophilic substrate-binding site towards one that is more enclosed, albeit flexible. Of particular note are the interactions between Glu205 of the new tail and the catalytic residues, Tyr7 and Tyr108, and the thiol moiety of glutathione (GSH). These interactions may provide an explanation of the more than one unit increase in the pKa value of the GSH thiolate and affect both the turnover number and GSH binding, using 1-chloro-2,4-dinitrobenzene as co-substrate. The data presented are consistent with the engineered tail adopting a highly mobile or disordered state in the apo form of the enzyme. [Copyright &y& Elsevier]