The Role of Electrostatic Interactions in the Regulation of the Membrane Association of G Protein βγ Heterodimers

Abstract

In this paper we report calculations of electrostatic interactions between the transducin (G t ) ␤␥ heterodimer (G t ␤␥) and phospholipid membranes. Although membrane association of G t ␤␥ is due primarily to the hydrophobic penetration into the membrane interior of a farnesyl chain attached to the ␥ subunit, structural studies have revealed that there is a prominent patch of basic residues on the surface of the ␤ subunit surrounding the site of farnesylation that is exposed upon dissociation from the G t ␣ subunit. Moreover, phosducin, which produces dissociation of G t ␤␥ from membranes, interacts directly with G t ␤␥ and introduces a cluster of acidic residues into this region. The calculations, which are based on the finite difference Poisson-Boltzmann method, account for a number of experimental observations and suggest that charged residues play a role in mediating protein-membrane interactions. Specifically, the calculations predict the following. 1) Favorable electrostatic interactions enhance the membrane partitioning due to the farnesyl group by an order of magnitude although G t ␤␥ has a large net negative charge (؊12). 2) This electrostatic attraction positions G t ␤␥ so that residues implicated in mediating the interaction of G t ␤␥ with its membrane-bound effectors are close to the membrane surface.3) The binding of phosducin to G t ␤␥ diminishes the membrane partitioning of G t ␤␥ by an order of magnitude. 4) Lowering the ionic strength of the solution converts the electrostatic attraction into a repulsion. Sequence analysis and homology model building suggest that our conclusions may be generalized to other G␤␥ and phosducin isoforms as well.The interaction of proteins with cellular membranes is a central feature of signal transduction. Many proteins involved in interfacial signaling contain a lipophilic modification (e.g. myristate and farnesyl) that contributes to membrane association by partitioning hydrophobically into the membrane interior (1, 2). Examples of such proteins include heterotrimeric G proteins, small GTPases, and nonreceptor tyrosine kinases. A lipid modification alone usually cannot provide enough energy to keep a protein anchored to a cellular membrane (3) and often occurs in conjunction with additional membrane binding motifs. For example, most Src family kinases (1), H-and N-Ras (4), and many G␣ subunits (5) have dual lipophilic modifications, while other proteins, e.g. K-Ras4B (6, 7) and Src itself (8, 9), have both a lipophilic attachment and an adjacent cluster of basic residues that are attracted electrostatically to the charged surface of lipid bilayers. Heterotrimeric G proteins are acylated on the ␣ subunit and prenylated on the ␥ subunit, and the membrane association of the inactive heterotrimer is due primarily to the penetration of both of these lipophilic modifications into the hydrocarbon region of the membrane (10). The exchange of GTP for GDP on the ␣ subunit, catalyzed by an activated G protein-coupled receptor, results in the dissociation of G␣ from the G␤␥ h...

Plant G protein heterotrimers require dual lipidation motifs of Gα and Gγ and do not dissociate upon activation

“…Since monomeric AGG1 does not localize to the plasma membrane, we believe that an, as yet, unidentified region in AG␤1 must have membrane affinity. Interestingly, in Journal of Cell Science 119 (24) mammalian G␤ t an electrostatic region close to the farnesyl group of G␥ in crystal structures (Sondek et al, 1996) was implicated to increase membrane partitioning of the heterodimer (Murray et al, 2001). Possibly, a similar region in AG␤1 may serve as a membrane-targeting signal that synergistically interacts with the putative lipid group(s) attached to the AGG1 subunit.…”

Both Acidic and Basic Amino Acids in an Amphitropic Enzyme, CTP:Phosphocholine Cytidylyltransferase, Dictate Its Selectivity for Anionic Membranes

“…100 mM). This is a feature of the binding of some other membrane surface interacting peptides containing mixed charges (11,44). If peptide folding into an ␣-helix precedes surface attraction and insertion, this would place strips of basic charge flanking the hydrophobic wedge, and would position the negatively charged polar face away from the membrane surface (Fig.…”

Prediction of protein orientation upon immobilization on biological and nonbiological surfaces

“…Finally, the protein͞membrane model is then mapped onto a 3D lattice of points. We assume that the distance between the adsorbed protein and the membrane is 3 Å, because the interaction is most favorable at this distance (6,45). At shorter distances, both the protein and membrane are increasingly desolvated, which is energetically unfavorable because of Born repulsion (5,46,47).…”