A Role for Weak Electrostatic Interactions in Peripheral Membrane Protein Binding

Abstract

Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) is a secreted virulence factor that binds specifically to phosphatidylcholine (PC) bilayers containing negatively charged phospholipids. BtPI-PLC carries a negative net charge and its interfacial binding site has no obvious cluster of basic residues. Continuum electrostatic calculations show that, as expected, nonspecific electrostatic interactions between BtPI-PLC and membranes vary as a function of the fraction of anionic lipids present in the bilayers. Yet they are strikingly weak, with a calculated ΔGel below 1 kcal/mol, largely due to a single lysine (K44). When K44 is mutated to alanine, the equilibrium dissociation constant for small unilamellar vesicles increases more than 50 times (∼2.4 kcal/mol), suggesting that interactions between K44 and lipids are not merely electrostatic. Comparisons of molecular-dynamics simulations performed using different lipid compositions reveal that the bilayer composition does not affect either hydrogen bonds or hydrophobic contacts between the protein interfacial binding site and bilayers. However, the occupancies of cation-π interactions between PC choline headgroups and protein tyrosines vary as a function of PC content. The overall contribution of basic residues to binding affinity is also context dependent and cannot be approximated by a rule-of-thumb value because these residues can contribute to both nonspecific electrostatic and short-range protein-lipid interactions. Additionally, statistics on the distribution of basic amino acids in a data set of membrane-binding domains reveal that weak electrostatics, as observed for BtPI-PLC, might be a less unusual mechanism for peripheral membrane binding than is generally thought.

Defining the Subcellular Distribution and Metabolic Channeling of Phosphatidylinositol

“…With these goals in mind, we examined the Bc PI-PLC crystal structure, as well as the existing in vitro binding studies, to find any exposed hydrophobic or charged residues on the membrane-oriented surface of the enzyme that were likely to contribute to interfacial binding. We identified two membrane-oriented tryptophan residues, namely Trp47 within helix B and Trp242 in the extended α7-βG loop (Figure 2a), facing which have already been shown to facilitate the association of Bacillus PI-PLCs with phospholipid vesicles (Feng et al, 2002; Guo et al, 2008; Khan et al, 2016). Aromatic or hydrophobic side chains, and tryptophan residues in particular, are known to reinforce the membrane attachment of peripheral proteins by directly penetrating into the hydrocarbon core of the bilayer (Yau et al, 1998; Lomize et al, 2007; Fuglebakk and Reuter, 2018).…”

The energetics of protein–lipid interactions as viewed by molecular simulations

“…Considerations of space preclude a more general survey (see e.g. [79][80][81][82][83] for this), so we will focus on one particular example, that of PH ( pleckstrin homology) domains. PH domains are a large and well-characterized family present in many membrane recognition proteins, which can bind to phosphatidylinositol-phosphates (mainly PIP 2 and/or PIP 3 ) in membranes in a specific fashion [84].…”

Regulation of Human Cytomegalovirus Secondary Envelopment by a C-Terminal Tetralysine Motif in pUL71

“…The function of positively charged amino acids is less clear. They are often part of nuclear localization signals (38,39) but also mediate membrane association of proteins by their interaction with negatively charged phospholipids (40)(41)(42)(43). To clarify whether one of these two motifs has a function in secondary envelopment, two additional recombinant viruses were generated in which either the dileucine motif (TB71mutL357-358A) or the tetralysine motif (TB71mutK348-351A) was mutated to alanine residues ( Fig.…”

“…By mutating the tetralysine motif, the positively charged polar lysine residues were exchanged with uncharged nonpolar alanine residues. There is good evidence that positively charged amino acids can directly interact with negatively charged components of the membrane, such as phospholipids (40)(41)(42)(43). Interestingly, several known lipid-binding domains, including PH and FYVE domains, are dominated by positively charged amino acids, supporting the idea that lysine and arginine interact with lipids (41).…”