Marc Chabre

Fluoroaluminates activate transducin-GDP by mimicking the γ-phosphate of GTP in its binding site

Fluoride activation of the cGMP cascade of vision requires the presence of aluminum, and is shown to be mediated by the binding of one AlF‐4 to the GDP/GTP‐binding subunit of transducin. The presence of GDP in the site is required: AlF4 − is ineffective when the site is empty or when GDPßS is substituted for GDP. This sensitivity to the sulfur of GDPßS suggests that AlF4 − is in contact with the GDP. Striking structural similarities between AlF4 − and PO4 −1 lead us to propose that AlF4 − mimics the role of the γ‐phosphate of GTP.

Fluoride complexes of aluminium or beryllium act on G-proteins as reversibly bound analogues of the gamma phosphate of GTP.

Fluoride activation of G proteins requires the presence of aluminium or beryllium and it has been suggested that AIF4‐ acts as an analogue of the gamma‐phosphate of GTP in the nucleotide site. We have investigated the action of AIF4‐ or of BeF3‐ on transducin (T), the G protein of the retinal rods, either indirectly through the activation of cGMP phosphodiesterase, or more directly through their effects on the conformation of transducin itself. In the presence of AIF4‐ or BeF3‐, purified T alpha subunit of transducin activates purified cyclic GMP phosphodiesterase (PDE) in the absence of photoactivated rhodopsin. Activation is totally reversed by elution of fluoride or partially reversed by addition of excess T beta gamma. Activation requires that GDP or a suitable analogue be bound to T alpha: T alpha‐GDP and T alpha‐GDP alpha S are activable by fluorides, but not T alpha‐GDP beta S, nor T alpha that has released its nucleotide upon binding to photoexcited rhodopsin. Analysis of previous works on other G proteins and with other nucleotide analogues confirm that in all cases fluoride activation requires that a GDP unsubstituted at its beta phosphate be bound in T alpha. By contrast with alumino‐fluoride complexes, which can adopt various coordination geometries, all beryllium fluoride complexes are tetracoordinated, with a Be‐F bond length of 1.55 A, and strictly isomorphous to a phosphate group. Our study confirms that fluoride activation of transducin results from a reversible binding of the metal‐fluoride complex in the nucleotide site of T alpha, next to the beta phosphate of GDP, as an analogue of the gamma phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Aluminofluoride and beryllofluoride complexes: new phosphate analogs in enzymology

The action of fluoride ions on G proteins as well as on various ATPases and phosphatases, is related to their complexation with traces of aluminium or beryllium. These fluorometallic complexes act as analogs of phosphate: they bind with high affinity, but reversibly, in phosphate sites or, concomitantly with nucleoside-diphosphate, in nucleoside-triphosphate sites. The beryllofluoride complexes are strictly tetrahedral; they cannot take on the pentavalent conformation adopted by phosphate in transition states hence they interfere with phospho-transfer reaction mechanisms.

A glutamic finger in the guanine nucleotide exchange factor ARNO displaces Mg2+ and the beta-phosphate to destabilize GDP on ARF1.

The Sec7 domain of the guanine nucleotide exchange factor ARNO (ARNO‐Sec7) is responsible for the exchange activity on the small GTP‐binding protein ARF1. ARNO‐Sec7 forms a stable complex with the nucleotide‐free form of [Δ17]ARF1, a soluble truncated form of ARF1. The crystal structure of ARNO‐Sec7 has been solved recently, and a site‐directed mutagenesis approach identified a hydrophobic groove and an adjacent hydrophilic loop as the ARF1‐binding site. We show that Glu156 in the hydrophilic loop of ARNO‐Sec7 is involved in the destabilization of Mg2+ and GDP from ARF1. The conservative mutation E156D and the charge reversal mutation E156K reduce the exchange activity of ARNO‐Sec7 by several orders of magnitude. Moreover, [E156K]ARNO‐Sec7 forms a complex with the Mg2+‐free form of [Δ17]ARF1‐GDP without inducing the release of GDP. Other mutations in ARNO‐Sec7 and in [Δ17]ARF1 suggest that prominent hydrophobic residues of the switch I region of ARF1 insert into the groove of the Sec7 domain, and that Lys73 of the switch II region of ARF1 forms an ion pair with Asp183 of ARNO‐Sec7.

Role of Protein-Phospholipid Interactions in the Activation of ARF1 by the Guanine Nucleotide Exchange Factor Arno

Arno is a 47-kDa human protein recently identified as a guanine nucleotide exchange factor for ADP ribosylation factor 1 (ARF1) with a central Sec7 domain responsible for the exchange activity and a carboxyl-terminal pleckstrin homology (PH) domain (Chardin, P., Paris, S., Antonny, B., Robineau, S., Béraud-Dufour, S., Jackson, C. L., and Chabre, M. (1996)Nature 384, 481–484). Binding of the PH domain to phosphatidylinositol 4,5-bisphosphate (PIP2) greatly enhances Arno-mediated activation of myristoylated ARF1. We show here that in the absence of phospholipids, Arno promotes nucleotide exchange on [Δ17]ARF1, a soluble mutant of ARF1 lacking the first 17 amino acids. This reaction is unaffected by PIP2, which suggests that the PIP2-PH domain interaction does not directly regulate the catalytic activity of Arno but rather serves to recruit Arno to membranes. Arno catalyzes the release of GDP more efficiently than that of GTP from [Δ17]ARF1, and a stable complex between Arno Sec7 domain and nucleotide-free [Δ17]ARF1 can be isolated. In contrast to [Δ17]ARF1, full-length unmyristoylated ARF1 is not readily activated by Arno in solution. Its activation requires the presence of phospholipids and a reduction of ionic strength and Mg2+ concentration. PIP2 is strongly stimulatory, indicating that binding of Arno to phospholipids is involved, but in addition, electrostatic interactions between phospholipids and the amino-terminal portion of unmyristoylated ARF1GDP seem to be important. We conclude that efficient activation of full-length ARF1 by Arno requires a membrane surface and two distinct protein-phospholipid interactions: one between the PH domain of Arno and PIP2, and the other between amino-terminal cationic residues of ARF1 and anionic phospholipids. The latter interaction is normally induced by insertion of the amino-terminal myristate into the bilayer but can also be artificially facilitated by decreasing Mg2+ and salt concentrations.

Activation of ADP-ribosylation Factor 1 GTPase-Activating Protein by Phosphatidylcholine-derived Diacylglycerols

Disassembly of the coatomer from Golgi vesicles requires that the small GTP-binding protein ADP-ribosylation factor 1 (ARF1) hydrolyzes its bound GTP by the action of a GTPase-activating protein. In vitro, the binding of the ARF1 GTPase-activating protein to lipid vesicles and its activity on membrane-bound ARF1GTP are increased by diacylglycerols with monounsaturated acyl chains, such as those arising in vivo as secondary products from the hydrolysis of phosphatidylcholine by ARF-activated phospholipase D. Thus, the phospholipase D pathway may provide a feedback mechanism that promotes GTP hydrolysis on ARF1 and the consequent uncoating of vesicles.

Myristoylation-facilitated Binding of the G Protein ARF1 to Membrane Phospholipids Is Required for Its Activation by a Soluble Nucleotide Exchange Factor

We have investigated the role of N-myristoylation in the activation of bovine ADP-ribosylation factor 1 (ARF1). We previously showed that myristoylation allows some spontaneous GDP-to-GTP exchange to occur on ARF1 at physiological Mg levels in the presence of phospholipid vesicles (Franco, M., Chardin, P., Chabre, M., and Paris, S.(1995) J. Biol. Chem. 270, 1337-1341). Here, we report that this basal nucleotide exchange can be accelerated (by up to 5-fold) by addition of a soluble fraction obtained from bovine retinas. This acceleration is totally abolished by brefeldin A (IC = 2 μM) and by trypsin treatment of the retinal extract, as expected for an ARF-specific guanine nucleotide exchange factor. To accelerate GDP release from ARF1, this soluble exchange factor absolutely requires myristoylation of ARF1 and the presence of phospholipid vesicles. The retinal extract also stimulates guanosine 5′-3-O-(thio)triphosphate (GTPS) release from ARF1 in the presence of phospholipids, but in this case myristoylation of ARF is not required. These observations, together with our previous findings that both myristoylated and nonmyristoylated forms of ARF but only the myristoylated form of ARF bind to membrane phospholipids, suggest that (i) the retinal exchange factor acts only on membrane-bound ARF, (ii) the myristate is not involved in the protein-protein interaction between ARF1 and the exchange factor, and (iii) N-myristoylation facilitates both spontaneous and catalyzed GDP-to-GTP exchange on ARF1 simply by facilitating the binding of ARF to membrane phospholipids.

X-ray diffraction studies of retinal rods. I. Structure of the disc membrane, effect of illumination

The structure of the retinal rod disc membrane and its modifications upon bleaching have been studied by X-ray diffraction. Three types of preparations are used: functioning isolated from retina, isolated rods from frog retina, oriented by a magnetic field, and stacked discs from cattle retina. X-rays are detected by a position-sensitive linear counter. Diffraction spectra are obtained in 10-100 s. The electron density profile favors models where the rhodopsin molecule spans the whole thickness of the membrane. Upon bleaching, a small increase of electron density appears instantly at the cytoplasmic edge of the membrane. In the intact retina this structural change is accompanied by disorder and slow swelling reactions which are not observed in the isolated rod outer segment. The diffraction signal arising from the protein distribution in the plane of the membrane has been reinvestigated carefully. Patterns identical to those of Blasie (Blaise (1969) J. Mol. Biol. 39, 407 and Blaise (1972) Biophys. J. 12, 191) can be obtained but these are shown to be dominated by artefacts. The actual signal is a single broad band around (55 A)-1, upon which bleaching has a negligible effect. No measurable displacement of rhodopsin in the thickness of the membrane occurs upon bleaching. Temperature effects on the protein distribution are found to be large only for disc membranes from cattle retina. In this material from a warm-blooded animal those effects are correlated with the occurrence, upon lowering the temperature, of a partial phase transition of the paraffin chains of the lipids. The position and the slope of the transition are not sensitive to bleaching.

Neutron diffraction studies of retinal rod outer segment membranes.

Neutron diffraction measurements on isolated retinal rod outer segments show that most of the visual pigment protein, rhodopsin, is embedded in the hydrophobic core of the disk membrane. A very slight outward shift of protein at the cytoplasmic side of the membrane is associated with pigment bleaching.

The apparent cooperativity of some GPCRs does not necessarily imply dimerization

When the binding of one ligand to its receptor is influenced by a second ligand acting on a different receptor, one might assume that the receptors dimerize, enabling allosteric interactions between ligands. This reasoning is frequently used to explain the complex binding curves of ligands of class A G-protein-coupled receptors (GPCRs). Here, we argue that in classical in vitro experiments the lack of GTP makes ligand-binding properties dependent on the available pool of G protein. Under such conditions a 1:1 GPCR-G-protein complex is stabilized, in which the G protein lacks a nucleotide and ligand binding is of high affinity. In vivo, this complex, a key intermediate of G-protein activation, never accumulates because of fast and irreversible GTP binding. In vitro, this complex creates interference in ligand binding when two monomeric GPCRs compete for the same G protein. Interestingly, this competition explains some in vivo effects of orphan GPCRs.