Structure-Function Analyses Reveal Arabidopsis Accelerated-Cell-Death11 (Acd11) is a Ceramide-1-Phosphate Transfer Protein that Forms a Gltp-Fold

Abstract

The accelerated-cell-death11 (acd11) mutant of Arabidopsis provides a genetic model for studying immune response activation and localized cell suicide that halts the spread of pathogen infection in plants. Here, we elucidate ACD11 structure/function. Using a Forster resonance energy transfer approach involving anthrylvinyl and perylenoyl lipid probes, we find that ACD11 exhibits selective intermembrane transfer of ceramide-1-phosphate (C1P) and phyto-C1P, metabolites of the cell death sphingolipids, ceramide and phytoceramide. Crystal structures establish C1P binding via a surface-localized, phosphate headgroup recognition center connected to an interior hydrophobic pocket that adaptively ensheaths lipid chains via a cleft-like gating mechanism. Point mutational mapping confirmed the functional involvement of specific binding-site residues that selectively target the C1P phosphate headgroup. C1P binding selectivity also is verified by changes in fluorescence intensity associated with ACD11 intrinsic tryptophan and by ESI-mass spectrometry. Phosphatidylserine at low mole fraction in donor membranes, but not other negatively-charged phospholipids, stimulates C1P intermembrane by ACD11. A π-helix (π-bulge) present near the lipid-binding cleft distinguishes apo-ACD11 from other GLTP-folds. The global two-layer, α-helically-dominated, ‘sandwich’ topology displaying C1P-selective binding identifies ACD11 as the plant prototype of a new GLTP-fold subfamily. [Support: NIGMS GM45928 & NCI CA121493, Danish Strategic Research Council 09-067148, Spanish Ministerio de Ciencia e Innovacion (MICINN BFU2010-17711), Russian Fdn. for Basic Research 012-04-00168; Abby Rockefeller Mauze Trust, Maloris Fnd., Hormel Fnd.]