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A diverse superfamily of phospholipases consisting of the type VI lipase effectors Tle1-Tle5 secreted by the bacterial type VI secretion system (T6SS) have recently been identified as antibacterial effectors that hydrolyze membrane phospholipids. These effectors show no significant homology to known lipases, and their mechanism of membrane targeting and hydrolysis of phospholipids remains unknown. Here, the crystal structure of Tle1 (∼96.5 kDa) from Pseudomonas aeruginosa refined to 2.0 Å resolution is reported, representing the first structure of this superfamily. Its overall structure can be divided into two distinct parts, the phospholipase catalytic module and the putative membrane-anchoring module; this arrangement has not previously been observed in known lipase structures. The phospholipase catalytic module has a canonical α/β-hydrolase fold and mutation of any residue in the Ser-Asp-His catalytic triad abolishes its toxicity. The putative membrane-anchoring module adopts an open conformation composed of three amphipathic domains, and its partial folds are similar to those of several periplasmic or membrane proteins. A cell-toxicity assay revealed that the putative membrane-anchoring module is critical to Tle1 antibacterial activity. A molecular-dynamics (MD) simulation system in which the putative membrane-anchoring module embedded into a bilayer was stable over 50 ns. These structure-function studies provide insight into the hydrolysis and membrane-targeting process of the unique phospholipase Tle1. [ABSTRACT FROM AUTHOR]

The death receptors Fas, p75NTR and DR6 are key components of extrinsically activated apoptosis. Characterization of how they interact with the adaptors is crucial in order to unravel the signalling mechanisms. However, the exact conformation that their intracellular death domain adopts upon binding downstream partners remains unclear. One model suggests that it adopts a typical compact fold, whilst a second model proposed an open conformation. Calmodulin (CaM), a major calcium sensor, has previously been reported to be one of the Fas adaptors that modulate apoptosis. This work reports that CaM also binds directly to the death domains of p75NTR and DR6, indicating that it serves as a common modulator of the death receptors. Two crystal structures of CaM in complexes with the corresponding binding regions of Fas and p75NTR are also reported. Interestingly, the precise CaM-binding sites were mapped to different regions: helix 1 in Fas and helix 5 in p75NTR and DR6. A novel 1-11 motif for CaM binding was observed in p75NTR. Modelling the complexes of CaM with full-length receptors reveals that the opening of the death domains would be essential in order to expose their binding sites for CaM. These results may facilitate understanding of the diverse functional repertoire of death receptors and CaM and provide further insights necessary for the design of potential therapeutic peptide agents. [ABSTRACT FROM AUTHOR]