How a G Protein Binds a Membrane

Heterotrimeric G proteins interact with receptors and effectors at the membrane-cytoplasm interface. Structures of soluble forms have not revealed how they interact with membranes. We have used electron crystallography to determine the structure in ice of a helical array of the photoreceptor G protein, transducin, bound to the surface of a tubular lipid bilayer. The protein binds to the membrane with a very small area of contact, restricted to two points, between the surface of the protein and the surface of the lipids. Fitting the x-ray structure into the membrane-bound structure reveals one membrane contact near the lipidated Gγ C terminus and Gα N terminus, and another near the Gα C terminus. The narrowness of the tethers to the lipid bilayer provides flexibility for the protein to adopt multiple orientations on the membrane, and leaves most of the G protein surface area available for protein-protein interactions. Heterotrimeric G proteins interact with receptors and effectors at the membrane-cytoplasm interface. Structures of soluble forms have not revealed how they interact with membranes. We have used electron crystallography to determine the structure in ice of a helical array of the photoreceptor G protein, transducin, bound to the surface of a tubular lipid bilayer. The protein binds to the membrane with a very small area of contact, restricted to two points, between the surface of the protein and the surface of the lipids. Fitting the x-ray structure into the membrane-bound structure reveals one membrane contact near the lipidated Gγ C terminus and Gα N terminus, and another near the Gα C terminus. The narrowness of the tethers to the lipid bilayer provides flexibility for the protein to adopt multiple orientations on the membrane, and leaves most of the G protein surface area available for protein-protein interactions. G proteins mediate signal transduction pathways on the membranes of virtually all eukaryotic cells. In the inactive, GDP-bound, heterotrimeric state most G proteins are themselves tightly membrane associated. Activation takes place when GTP is exchanged for GDP in a complex with an integral membrane G protein-coupled receptor, which produces separated and active Gα-GTP and Gβγ proteins. These active subunits are then free to move along the membrane and regulate their downstream effectors that may be either peripherally membrane associated (e.g. cGMP phosphodiesterase, phospholipase C) or integral membrane proteins (e.g. adenylyl cyclase and ion channels). There have been numerous studies of G protein-membrane interactions using biochemical, functional, and mutagenic approaches. These have made it clear that membrane association and localization play a critical role in G protein function and have also revealed biochemical features of Gα and Gγ that are important for membrane binding. All Gα subunits have one or more fatty acids added at or near their N termini, and both the fatty acids and the amino acids in the N-terminal region are important for membrane binding (1Brand S.H. Holtzman E.J. Scher D.A. Ausiello D.A. Stow J.L. Am. J. Physiol. 1996; 270: C1362-C1369Crossref PubMed Google Scholar, 2Busconi L. Boutin P.M. Denker B.M. Biochem. J. 1997; 323: 239-244Crossref PubMed Scopus (10) Google Scholar, 3Chen C.A. Manning D.R. Oncogene. 2001; 20: 1643-1652Crossref PubMed Scopus (171) Google Scholar). The attachment site is either an N-terminal glycine, which is linked to fatty acid, usually myristic acid, in an amide linkage, or a cysteine at position 2, which is linked to palmitic acid in a thioester link-age. The Gγ subunits are proteolytically processed to leave a C-terminal methyl-esterified cysteine residue linked via a thioether to either a 15- or 20-carbon isoprenyl chain (4Fukada Y. 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Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1058) Google Scholar) these hydrophobic modifications and their immediately adjacent amino acids are missing; however, the resolved portions of the Gγ C terminus and the Gα N terminus are close to one another in space, suggesting a common site for membrane insertion. The only G protein structure in which a lipid modification is present and visible is the complex of Gβγt with phosducin, in which the farnesyl group is tucked into the β-propeller blades of Gβt (17Loew A. Ho Y.K. Blundell T. Bax B. Structure. 1998; 6: 1007-1019Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), and which has greatly diminished affinity for membranes. Another region thought to be near the membrane is the C terminus of the α subunit, because of its critical role in binding to receptors (18Garcia P.D. Onrust R. Bell S.M. Sakmar T.P. Bourne H.R. EMBO J. 1995; 14: 4460-4469Crossref PubMed Scopus (96) Google Scholar, 19Hamm H.E. Rarick H. Mazzoni M. Malinski J. Suh K.H. Biochem. Soc. Symp. 1990; 56: 35-44PubMed Google Scholar). A proposed membrane-binding surface for the heterotrimer was identified by adding to these constraints a criterion of electrostatic matching between positively charged or neutral regions of the protein surface and the negatively charged phospholipid (16Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1058) Google Scholar). However, this model remains in question because studies with the retinal G protein transducin have suggested that there is no electrostatic attraction to the membrane. In fact, low ionic strength is routinely used to remove this protein from membranes, suggesting that the net electrostatic forces are actually weakly repulsive in nature (20Kuhn H. Nature. 1980; 283: 587-589Crossref PubMed Scopus (326) Google Scholar, 21Stryer L. Hurley J.B. Fung B.K. 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In this approach the protein is allowed to assemble into an ordered lattice on the surface of a lipid bilayer, and electron microscopy of samples preserved in vitreous ice is used to obtain images for structural analysis. Although in most cases only low resolution structural maps are obtained from this technique, it is possible to fit high resolution structures into these maps computationally (23Jiang W. Baker M.L. Ludtke S.J. Chiu W. J. Mol. Biol. 2001; 308: 1033-1044Crossref PubMed Scopus (259) Google Scholar, 24Wu X. Milne J.L. A.V. S. J. Biol. PubMed Scopus Google Scholar, R. J. Biol. 2001; PubMed Scopus Google Scholar, P. W. J. Mol. Biol. PubMed Scopus Google Scholar), and obtain about the interactions of of the protein with the membrane. We that ordered be obtained for the photoreceptor G protein transducin either on Malinski J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (42) Google Scholar) or tubular L. J. 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Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1058) Google Scholar) using the (23Jiang W. Baker M.L. Ludtke S.J. Chiu W. J. Mol. Biol. 2001; 308: 1033-1044Crossref PubMed Scopus (259) Google the crystal structure at a resolution of is in C and D. The position of the structures is by the region of which is in the of the β-propeller of the between the two structures the in and The position of the crystal structure is in in a for the of to the for the position is and and of a of the to a a on a of the crystal structure in the The membrane surface on a the protein in and is to and the in In this position the N-terminal of and its are close to the membrane and of the regions of Gβt and visible in the crystal structure are immediately adjacent to the membrane. The visible residue on the C terminus of terminus near the in is that the the farnesyl on to the membrane is that the portions of the C terminus of and the N terminus of their most of the to the membrane at attachment site on the of The at attachment site on the of in in A and to be from the and from the C-terminal of the N-terminal of are in and The of both and the membrane surface in this that of from the membrane and of to the most from the is that the area of contact between protein and lipid is only of the protein from of the and not on the of the actually be from the by of the in the and not on the of the A model proposed (16Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. 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Cell. 1997; Full Text Full Text PDF PubMed Scopus Google both of these orientations are from the one used for membrane binding. in position of the Gα N-terminal be by electrostatic interactions between negatively charged lipid groups and the and on the near the membrane be in the fit of this of the crystal structure is not the with a position in the membrane complex to its position in or in the three-dimensional The may be for the C terminus of the position of the farnesyl group to the C-terminal cysteine of in the one structure in which it is resolved (17Loew A. Ho Y.K. Blundell T. Bax B. 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Thus is to orientations in its for which it may from a of in the of the membrane and also and from the membrane very of the surface of is by the membrane. Although we not the structure of the interface between and it that it of the surface area of the of The of lipid of the surface to that of between is whether the with heterotrimers in and the or are by There is from and studies that or Ho Y.K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. Biochem. PubMed Scopus Google Scholar, R.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: PubMed Scopus Google Scholar). force microscopy of photoreceptor membranes that of D. Y. S. D.A. A. K. Nature. PubMed Scopus Google Scholar, Y. D. S. D.A. K. A. J. Biol. Chem. 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There of in the resolution features of and the in of the x-ray However, the about which of the protein are near the membrane also for with for the fit because these have the The about a area of contact between the and protein not on the of the and at there is between the regions of the protein we with the membranes and biochemical on the role of lipid modifications and on the role in binding of the All of these that the we with to the membrane is not the one that is primarily in one that is a of the Because the structure and fit strong about the of they be by of and of electron and electron crystallography for signaling is only to be In this we have the structure of the membrane complex by the G protein heterotrimer The may a of for of structures of signaling using electron the membrane with the peripheral membrane protein cGMP and the complex of and All of these be on membranes from for structural and for of G proteins with and proteins in all eukaryotic cells. We S. W. M. and M. for and

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