Quantifying Transient Interactions between Bacillus Phosphatidylinositol-Specific Phospholipase-C and Phosphatidylcholine-Rich Vesicles

Abstract

Bacillus thuringiensis secretes the virulence factor phosphatidylinositol-specific phospholipase C (BtPI-PLC), which specifically binds to phosphatidylcholine (PC) and cleaves GPI-anchored proteins off eukaryotic plasma membranes. To elucidate how BtPI-PLC searches for GPI-anchored proteins on the membrane surface, we measured residence times of single fluorescently labeled proteins on PC-rich small unilamellar vesicles (SUVs). BtPI-PLC interactions with the SUV surface are transient with a lifetime of 379 ± 49 ms. These data also suggest that BtPI-PLC does not directly sense curvature, but rather prefers to bind to the numerous lipid packing defects in SUVs. Despite this preference for defects, all-atom molecular dynamics simulations of BtPI-PLC interacting with PC-rich bilayers show that the protein is shallowly anchored with the deepest insertions ∼18 Å above the bilayer center. Membrane partitioning is mediated, on average, by 41 hydrophobic, 8 hydrogen-bonding, and 2 cation−π (between PC choline headgroups and Tyr residues) transient interactions with phospholipids. These results lead to a quantitative model for BtPI-PLC interactions with cell membranes where protein binding is mediated by lipid packing defects, possibly near GPI-anchored proteins, and the protein diffuses on the membrane for ∼100–380 ms, during which time it may cleave ∼10 GPI-anchored proteins before dissociating. This combination of short two-dimensional scoots followed by three-dimensional hops may be an efficient search strategy on two-dimensional surfaces with obstacles.

Defining the Subcellular Distribution and Metabolic Channeling of Phosphatidylinositol

“…These data highlight the fact that membrane recognition by the Bc PI-PLC requires a combination of interfacial as well as active site binding. Consequently, at present, it cannot be ruled out that, in addition to the presence of PtdIns, other membrane features, such as DAG content and local membrane packing defects (Lehto and Sharom, 2002; Ahyayauch et al, 2015) or the relative phosphatidylcholine abundance (Zhang et al, 2004; Pu et al, 2009; Yang et al, 2015), may also play a role in the association of the Bc PI-PLC H82A probe with membranes. It is important to emphasize that our conclusions regarding the membrane distribution of PtdIns are based on a combination of approaches and not solely on Bc PI-PLC H82A localization; even though this probe appears to faithfully map the cellular PtdIns landscape.…”

Regulation of bifunctional proteins in cells: Lessons from the phospholipase Cβ/G protein pathway

“…Binding of PLCβ to model membranes is strong and fairly nonspecific with partition coefficients ranging from 10 to 100 μM . This range of affinities suggests a transient membrane association in the range of 0.1 to 1 s (see References ). Studies using purified proteins and model membranes suggest that PLCβ initially binds to membranes where it diffuses and hops along the membrane until it encounters Gαq .…”

Atomic-level description of protein–lipid interactions using an accelerated membrane model

“…Furthermore, due to the transient nature of the interaction between peripheral proteins and membrane lipids, crystallizing the membrane-bound complexes of peripheral proteins for X-ray analyses is exceedingly difficult. Other techniques, such as SAXS [16–18], EPR [19–21], NMR [22–24] including high–resolution field–cycling NMR [25–28], FRET [29, 30], fluorescence correlation spectroscopy [31–33], x–ray reflectivity [34, 35], neutron reflectometry [36, 37], and mutagenesis studies can bridge the gap, and provide low-resolution information on protein-lipid interactions such as determination of the binding face of the protein as it interacts with a membrane interface. However, without the guidance of a structural model of the protein-membrane complex, even when structures of constituent proteins are resolved, hypothesis-driven experimental investigations into the biological mechanisms are limited by the uncertainties inherent in missing the contribution of the membrane.…”