Clostridium perfringens alpha-toxin is the key virulence determinant in gas gangrene and has also been implicated in the pathogenesis of sudden death syndrome in young animals. The toxin is a 370-residue, zinc metalloenzyme that has phospholipase C activity, and can bind to membranes in the presence of calcium. The crystal structure of the enzyme reveals a two-domain protein. The N-terminal domain shows an anticipated structural similarity to Bacillus cereus phosphatidylcholine-specific phospholipase C (PC-PLC). The C-terminal domain shows a strong structural analogy to eukaryotic calcium-binding C2 domains. We believe this is the first example of such a domain in prokaryotes. This type of domain has been found to act as a phospholipid and/or calcium-binding domain in intracellular second messenger proteins and, interestingly, these pathways are perturbed in cells treated with alpha-toxin. Finally, a possible mechanism for alpha-toxin attack on membrane-packed phospholipid is described, which rationalizes its toxicity when compared to other, non-haemolytic, but homologous phospholipases C.

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The structure of HIV-1 ⌿-RNA has been elucidated by a concerted approach combining structural probes with mass spectrometric detection (MS3D), which is not affected by the size and crystallization properties of target biomolecules. Distance constraints from bifunctional cross-linkers provided the information required for assembling an all-atom model from the high-resolution coordinates of separate domains by triangulating their reciprocal placement in 3D space. The resulting structure revealed a compact cloverleaf morphology stabilized by a long-range tertiary interaction between the GNRA tetraloop of stemloop 4 (SL4) and the upper stem of stemloop 1 (SL1). The preservation of discrete stemloop structures ruled out the possibility that major rearrangements might produce a putative supersite with enhanced affinity for the nucleocapsid (NC) domain of the viral Gag polyprotein, which would drive genome recognition and packaging. The steric situation of single-stranded regions exposed on the cloverleaf structure offered a valid explanation for the stoichiometry exhibited by full-length ⌿-RNA in the presence of NC. The participation of SL4 in a putative GNRA loop-receptor interaction provided further indications of the plasticity of this region of genomic RNA, which can also anneal with upstream sequences to stabilize alternative conformations of the 5 untranslated region (5-UTR). Considering the ability to sustain specific NC binding, the multifaceted activities supported by the SL4 sequence suggest a mechanism by which Gag could actively participate in regulating the vital functions mediated by 5-UTR. Substantiated by the 3D structure of ⌿-RNA, the central role played by SL4 in specific RNA-RNA and protein-RNA interactions advances this domain as a primary target for possible therapeutic intervention.GNRA loop-receptor interaction ͉ high-resolution mass spectrometry ͉ HIV-1 Psi-RNA ͉ modeling ͉ structural probing T he multifaceted activities attributed to the packaging signal (⌿-RNA) (1, 2) of HIV-1 are mediated by discrete stemloop domains that exercise individual functions, but can also operate in concert to complete complex tasks [supporting information (SI) Fig. S1] (3-5). Stemloop 1 (SL1) is primarily responsible for genome dimerization (6, 7), but its mutations induce significant reductions of RNA encapsidation (8). Stemloop 2 (SL2) contains the major splice donor (SD) (9), but its high affinity for the nucleocapsid (NC) domain of the Gag polyprotein implies its participation in genome recognition and packaging (10, 11). Stemloop 3 (SL3) is sufficient by itself to induce the packaging of heterologous RNA into virus-like particles (12), but its deletion does not completely eliminate encapsidation that is still sustained with suboptimal efficiency by the remaining stemloops (13). Located downstream of the gag's starting codon, stemloop 4 (SL4) may be engaged in both coding and non-coding activities, as suggested by its possible involvement in long-range pairing interactions (14) and its ability to bind NC in vi...