Defining Specific Lipid Binding Sites for a Peripheral Membrane Protein in Situ Using Subtesla Field-cycling NMR

Abstract

Despite the profound physiological consequences associated with peripheral membrane protein localization, only a rudimentary understanding of the interactions of proteins with membrane surfaces exists because these questions are inaccessible by commonly used structural techniques. Here, we combine high resolution field-cycling 31 P NMR relaxation methods with spinlabeled proteins to delineate specific interactions of a bacterial phospholipase C with phospholipid vesicles. Unexpectedly, discrete binding sites for both a substrate analogue and a different phospholipid (phosphatidylcholine) known to activate the enzyme are observed. The lifetimes for the occupation of these sites (when the protein is anchored transiently to the membrane) are >1-2 s (but <1 ms), which represents the first estimate of an off-rate for a lipid dissociating from a specific site on the protein and returning to the bilayer. Furthermore, analyses of the spin-label induced NMR relaxation corroborates the presence of a discrete tyrosine-rich phosphatidylcholine binding site whose location is consistent with that suggested by modeling studies. The methodology illustrated here may be extended to a wide range of peripheral membrane proteins.Transient interactions of peripheral membrane proteins with bilayers play critical roles in signal transduction cascades. Many of these proteins are proposed to have binding interactions with multiple distinct types of phospholipids (now usually characterized by bulk protein binding with different lipid components and mutagenesis to see whether suspected regions on the protein are correlated with higher affinity binding of the protein). Confirming whether there is a discrete site, whether a lipid binds, and with what affinity, when the protein interacts with a multicomponent bilayer is difficult at best. Nonspecific interactions with the bilayer also can occur. Conformation of the bound macromolecule also is hard to deduce, particularly if the affinity of the protein for the interface is of only moderate affinity (a trait desirable in many peripheral proteins if they are to bind and dissociate from the membrane repeatedly). These proteins often have flexible loops or domains that are likely to be affected by membrane binding. Methods that can address whether and where specific phospholipid sites exist on a peripheral protein when anchored to a bilayer would aid in understanding how its function may be modulated.An interesting case is the secreted phosphatidylinositol-specific phospholipase C (PI-PLC) 2 from Bacillus thuringiensis. This small (35 kDa) enzyme is a good model for the catalytic domain of the human PI-PLC. It specifically catalyzes the cleavage of PI to form diacylglycerol and inositol 1-phosphate. Crystal structures of this protein show a monomeric ␣␤-barrel (1). Anionic phospholipids such as phosphatidylmethanol (PMe) are competitive inhibitors and can therefore serve as substrate analogues (2). PI-PLC is activated specifically by phosphatidylcholine (PC) present in the interface (2-4); the PC ...

The Cation-π Box Is a Specific Phosphatidylcholine Membrane Targeting Motif

“…This position is comparable with D205C in B. thuringiensis PI-PLC, where a spin label had the largest effect on 31 P relaxation (22). The much larger dipole of the unpaired electron can relax 31 P fast-exchanging into and out of a discrete binding site and back into the bilayer.…”

“…Although B. thuringiensis PI-PLC has a high affinity for PC vesicles (8), the similar PI-PLC from S. aureus has virtually no affinity for PC vesicles. High resolution field-cycling NMR experiments on B. thuringiensis identified a discrete binding site for PC that is consistent with Tyr residues forming a cation-box or sandwich (22). However, this motif is not present in the S. aureus enzyme, which displays much weaker binding to PC-rich vesicles and virtually no binding to pure PC vesicles (23).…”

“…PI-PLC from B. thuringiensis binds to PC-rich vesicles; residues located on the loop connecting helices F and G (notably Trp-242), as well several tyrosines located on helix G (Tyr-246, Tyr-247, Tyr-248, Tyr-251) were implicated in binding to PC interfaces (22,28). Sequence and structural alignments of the B. thuringiensis and S. aureus PI-PLC enzymes show that whereas Tyr-246 and Tyr-248 in the Bacillus enzyme correspond to Tyr-253 and Tyr-255 in S. aureus, the other two tyrosine residues (247 and 251) in the Bacillus enzyme have been replaced with Asn-254 and His-258 in the S. aureus enzyme (Fig.…”

“…The parameters R P-e (0) and P-e along with the total spinlabeled PI-PLC concentration, [PI-PLC-SL], and phospholipid concentration, L o , are related to r P-e , the distance between the phospholipid 31 P and the nitroxide when the ligand is bound (22). To a first approximation we assume that if there is a discrete site on the protein for an individual phosphorylated molecule, it is saturated with 5 mM of the ligand.…”

“…Vesicle Binding Measured by Fluorescence Correlation Spectroscopy (FCS)-The partitioning of both wild-type recombinant S. aureus PI-PLC and the N254Y/H258Y mutant was measured by FCS as described previously (22,25). The fluorescence was monitored at 22°C with samples placed in chambered coverglass wells (Lab-Tek, Nunc), containing 10 nM labeled S. aureus PI-PLC protein and 1 mg/ml of BSA in 300 l of 50 mM MES, pH 6.5 (the same buffer as used for enzymatic assays).…”