NAD(H)-mediated tetramerization controls the activity of Legionella pneumophila phospholipase PlaB

The virulence factor and phospholipase PlaB promotes lung colonization, tissue destruction, and intracellular replication of Legionella pneumophila, the causative agent of Legionnaires disease. It is exposed at the bacterial surface and shows an extraordinary activation mechanism by tetramer deoligomerization. To unravel the molecular basis for enzyme activation and localization, we determined the crystal structure of PlaB in its tetrameric form. We found that the tetramer is a dimer of identical dimers, and a monomer consists of an N-terminal phospholipase /{beta}-hydrolase domain augmented by two non-canonical two-stranded {beta}-sheets, {beta}6/{beta}7 and {beta}9/{beta}10. The C- terminal domain reveals a novel fold displaying a bilobed {beta}-sandwich with a hook structure that is required for dimer formation and complementation of the phospholipase domain in the neighboring monomer. Unexpectedly, we observed eight NAD(H) molecules at the dimer/dimer interface, suggesting that these molecules stabilize the tetramer and hence lead to enzyme inactivation. Indeed, addition of NAD(H) increased the fraction of the tetrameric form and concomitantly reduced activity. {beta}9/{beta}10 mutants revealed a decrease in the tetrameric fraction, altered activity profiles, and mislocalization. Protein variants lacking the hook or strands {beta}6/{beta}7 were unaffected in terms of localization but lost their activity, and lid mutants changed substrate specificity. Together, these data reveal structural elements and an unprecedented NAD(H)- mediated tetramerization mechanism required for spatial and enzymatic control of a phospholipase virulence factor. The regulatory process identified is ideally suited to fine tune PlaB in a way that protects L. pneumophila from self-inflicted lysis while ensuring its activity at the pathogen-host interface.