Cell signalling: Binding the receptor at both ends

G-protein-coupled receptors initiate a wide range of signalling pathways in cells. It seems that both a G protein and an agonist molecule must bind to the receptors to persistently activate them

How Ras works: structure of a Rap-Raf complex

The three-dimensional structure of the complex between Rap and the \u27Ras-binding domain\u27 of Raf could be the prototype for a G protein-effector interaction

Activation of G proteins by GTP and the mechanism of Gα-catalyzed GTP hydrolysis

This review addresses the regulatory consequences of the binding of GTP to the alpha subunits (Gα) of heterotrimeric G proteins, the reaction mechanism of GTP hydrolysis catalyzed by Gα and the means by which GTPase activating proteins (GAPs) stimulate the GTPase activity of Gα. The high energy of GTP binding is used to restrain and stabilize the conformation of the Gα switch segments, particularly switch II, to afford stable complementary to the surfaces of Gα effectors, while excluding interaction with Gβγ, the regulatory binding partner of GDP-bound Gα. Upon GTP hydrolysis, the energy of these conformational restraints is dissipated and the two switch segments, particularly switch II, become flexible and are able to adopt a conformation suitable for tight binding to Gβγ. Catalytic site pre-organization presents a significant activation energy barrier to Gα GTPase activity. The glutamine residue near the N-terminus of switch II (Glncat ) must adopt a conformation in which it orients and stabilizes the γ phosphate and the water nucleophile for an in-line attack. The transition state is probably loose with dissociative character; phosphoryl transfer may be concerted. The catalytic arginine in switch I (Argcat ), together with amide hydrogen bonds from the phosphate binding loop, stabilize charge at the β-γ bridge oxygen of the leaving group. GAPs that harbor regulator of protein signaling (RGS) domains, or structurally unrelated domains within G protein effectors that function as GAPs, accelerate catalysis by stabilizing the pre-transition state for Gα-catalyzed GTP hydrolysis, primarily by restraining Argcat and Glncat to their catalytic conformations. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 449-462, 2016

An acid test for g proteins

Heterotrimeric G proteins are cytoplasmic transducers of signals generated by ligandactivated 7-transmembrane receptors (G protein-coupled receptors, or GPCRs) embedded in the plasma membranes of cells. GPCRs activate G proteins by catalyzing exchange of GDP for GTP at the alpha subunit (Gα) with the concomitant release of the Gβγ heterodimer. Separately or together Gα•GTP and Gβγ stimulate enzymes that produce second messengers (e.g. cyclic AMP), control ion channels, Ras-family G proteins and regulate transcription (Cabrera-Vera et al., 2003). In yeast, GPCRs intercept mating pheromones and monitor extracellular glucose concentration, and so generate intracellular signals either to arrest growth or to proliferate. In this issue, Isom et al. show that Gpa1, the Gα subunit involved in the mating pheromone response, is also regulated by intracellular pH, reaffirming a growing recognition that protons, like other mono- and divalent cations, have roles as cellular second messengers (Isom et al., 2013). The path to this discovery began with the structural problem, how proteins can accommodate buried ionizable amino acid side chains in their interiors, and now arrives at a better understanding of the mechanisms by which regulatory proteins evolve properties as transducers of proton-mediated signaling

Thermodynamic characterization of the binding of activator of G protein signaling 3 (AGS3) and peptides derived from AGS3 with G alpha i1

Activator of G protein signaling 3 (AGS3) is a guanine nucleotide dissociation inhibitor (GDI) that contains four G protein regulatory (GPR) or GoLoco motifs in its C-terminal domain. The entire C-terminal domain (AGS3-C) as well as certain peptides corresponding to individual GPR motifs of AGS3 bound to G alpha i1 and inhibited the binding of GTP by stabilizing the GDP-bound conformation of G alpha i1. The stoichiometry, free energy, enthalpy, and dissociation constant for binding of AGS3-C to G alpha i1 were determined using isothermal titration calorimetry. AGS3-C possesses two apparent high affinity (Kd approximately 20 nm) and two apparent low affinity (Kd approximately 300 nm) binding sites for G alpha i1. Upon deletion of the C-terminal GPR motif from AGS3-C, the remaining sites were approximately equivalent with respect to their affinity (Kd approximately 400 nm) for G alpha i1. Peptides corresponding to each of the four GPR motifs of AGS3 (referred to as GPR1, GPR2, GPR3, and GPR4, respectively, going from N to C terminus) bound to G alpha i1 with Kd values in the range of 1-8 microm. Although GPR1, GPR2, and GPR4 inhibited the binding of the fluorescent GTP analog BODIPY-FL-guanosine 5\u27-3-O-(thio)triphosphate to G alpha i1, GPR3 did not. However, addition of N- and C-terminal flanking residues to the GPR3 GoLoco core increased its affinity for G alpha i1 and conferred GDI activity similar to that of AGS3-C itself. Similar increases were observed for extended GPR2 and extended GPR1 peptides. Thus, while the tertiary structure of AGS3 may affect the affinity and activity of the GPR motifs contained within its sequence, residues outside of the GPR motifs strongly potentiate their binding and GDI activity toward G alpha i1 even though the amino acid sequences of these residues are not conserved among the GPR repeats

Transition state structures and the roles of catalytic residues in GAP-facilitated GTPase of Ras as elucidated by (18)O kinetic isotope effects

Ras-catalyzed guanosine 5\u27 triphosphate (GTP) hydrolysis proceeds through a loose transition state as suggested in our previous study of (18)O kinetic isotope effects (KIE) [ Du , X. et al. ( 2004 ) Proc. Natl. Acad. Sci. U.S.A. 101 , 8858 - 8863 ]. To probe the mechanisms of GTPase activation protein (GAP)-facilitated GTP hydrolysis reactions, we measured the (18)O KIEs in GTP hydrolysis catalyzed by Ras in the presence of GAP(334) or NF1(333), the catalytic fragment of p120GAP or NF1. The KIEs in the leaving group oxygens (the beta nonbridge and the beta-gamma bridge oxygens) reveal that chemistry is rate-limiting in GAP(334)-facilitated GTP hydrolysis but only partially rate-limiting in the NF1(333)-facilitated GTP hydrolysis reaction. The KIEs in the gamma nonbridge oxygens and the leaving group oxygens reveal that the GAP(334) or NF1(333)-facilitated GTP hydrolysis reaction proceeds through a loose transition state that is similar in nature to the transition state of the GTP hydrolysis catalyzed by Ras alone. However, the KIEs in the pro-S beta, pro-R beta, and beta-gamma oxygens suggest that charge increase on the beta-gamma bridge oxygen is more prominent in the transition states of GAP(334)- and NF1(333)-facilitated reactions than that catalyzed by the intrinsic GTPase activity of Ras. The charge distribution on the two beta nonbridge oxygens is also very asymmetric. The catalytic roles of active site residues were inferred from the effect of mutations on the reaction rate and KIEs. Our results suggest that the arginine finger of GAP and amide protons in the P-loop of Ras stabilize the negative charge on the beta-gamma bridge oxygen and the pro-S beta nonbridge oxygen of a loose transition state, whereas Lys-16 of Ras and Mg(2+) are only involved in substrate binding

Structure of the protein kinase Cβ phospholipid-binding C2 domain complexed with Ca\u3csup\u3e2+\u3c/sup\u3e

Background: Conventional isoforms (α, β and γ) of protein kinase C (PKC) are synergistically activated by phosphatidylserine and Ca2+; both bind to C2 domains located within the PKC amino-terminal regulatory regions. C2 domains contain a bipartite or tripartite Ca2+-binding site formed by opposing loops at one end of the protein. Neither the structural basis for cooperativity between phosphatidylserine and Ca2+, nor the binding site for phosphatidylserine are known. Results: The structure of the C2 domain from PKCβ complexed with Ca2+ and o-phospho-L-serine has been determined to 2.7 Å resolution using X-ray crystallography. The eight-stranded, Greek key β-sandwich fold of PKCβ-C2 is similar to that of the synaptotagmin I type I C2 domain. Three Ca2+ ions, one at a novel site, were located, each sharing common aspartate ligands. One of these ligands is donated by a dyad-related C2 molecule. A phosphoserine molecule binds to a lysine-rich cluster in C2. Conclusions: Shared ligation among the three Ca2+ ions suggests that they bind cooperatively to PKCβ-C2. Cooperativity may be compromised by the accumulation of positive charge in the binding site as successive ions are bound. Model building shows that the C1 domain could provide carboxylate and carbonyl ligands for two of the three Ca2+ sites. Ca2+-mediated interactions between the two domains could contribute to enzyme activation as well as to the creation of a positively charged phosphatidylserine-binding site