Standard binding free energy and membrane desorption mechanism for a phospholipase C

Abstract

Peripheral membrane proteins (PMPs) bind temporarily to cellular membranes and play important roles in signalling, lipid metabolism and membrane trafficking. Obtaining accurate membrane-PMP affinities using experimental techniques is more challenging than for protein-ligand affinities in aqueous solution. At the theoretical level, calculation of standard protein-membrane binding free energy using molecular dynamics simulations remains a daunting challenge owing to the size of the biological objects at play, the slow lipid diffusion and the large variation in configurational entropy that accompanies the binding process. To overcome these challenges, we used a computational framework relying on a series of potential-of-mean-force (PMF) calculations including a set of geometrical restraints on collective variables. This methodology allowed us to determine the standard binding free energy of a PMP to a phospholipid bilayer using an all-atom force field. Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) was chosen due to its importance as a virulence factor and owing to the host of experimental affinity data available. We computed a standard binding free energy of -8.2±1.4 kcal/mol in reasonable agreement with the reported experimental values (-6.6±0.2 kcal/mol). In light of the 2.3-μs separation PMF calculation, we investigated the mechanism whereby BtPI-PLC disengages from interactions with the lipid bilayer during separation. We describe how a short amphipathic helix engages in transitory interactions to ease the passage of its hydrophobes through the interfacial region upon desorption from the bilayer.

Phosphatidylcholine Cation—Tyrosine π Complexes: Motifs for Membrane Binding by a Bacterial Phospholipase C

“…Recent work to calculate the absolute membrane binding free energy for Bt PI-PLC used a geometrical route and an atomistic force field to progressively detach the protein from the bilayer [ 26 ]. Along with a value for ΔG 0 bind (which agrees moderately well with the value from fluorescence correlation spectroscopy binding data for Bt PI-PLC binding to PC SUVs), the method provides atomic-level details that describe the membrane-bound protein and how interactions change during the desorption process.…”

“…So far, there are only a few other peripheral membrane proteins where PC-cation–aromatic Tyr/Trp/Phe complexes have been identified and shown to be important for membrane binding. These include a number of phospholipases in addition to Bt PI-PLC: the cytosolic phospholipase A 2 , whose C 2 domain forms a PC-cation–Tyr-π complex that is also stabilized by a Ca +2 interacting with the lipid phosphate group [ 33 ]; phospholipase A 2 Naja naja atra , where ΔΔG data are available for replacing aromatic amino acids by Ala [ 34 ], and the PC specificity was shown to arise from cation-π complexes by simulations [ 26 ]; and a spider phospholipase D that uses Tyr cages around the bound PC cation [ 35 ]. The PC cation-π formation in the PLD appears conserved in many of the other members in the same clade, emphasizing these are distinct complexes that are useful for ensuring PC specificity.…”

Dissecting peripheral protein-membrane interfaces

“…For example, the ratio between basic and hydrophobic amino acids varies between PMPs, from strong unspecific electrostatics to proteins with barely any basic cluster [21][22][23]. We have shown that lysine side chains might engage in hydrophobic contacts [23,24] and that the role of aromatic amino acids appears to be more elaborate than a mere hydrophobic anchor. The side chains of tyrosines and tryptophanes are instead able to take advantage of a wider palette of chemical interactions and insertion depths than first thought [25][26][27][28].…”

Dissecting peripheral protein-membrane interfaces

“…For example, the ratio between basic and hydrophobic amino acids varies between PMPs, from strong unspecific electrostatics to proteins with barely any basic cluster [ 20 – 22 ]. We have shown that lysine side chains might engage in hydrophobic contacts [ 22 , 23 ] and that the role of aromatic amino acids appears to be more elaborate than a mere hydrophobic anchor. The side chains of tyrosines and tryptophanes are instead able to take advantage of a wider palette of chemical interactions and insertion depths than first thought [ 24 – 27 ].…”