Overlapping interaction sites on the surface of the Gα subunits of G-proteins Introduction: G-protein coupled receptors (GPCRs) are one of the largest and most diverse families of membrane receptors in eukaryotes. They play a pivotal role in numerous cell responses to extracellular signals, and have been implicated in a large number of diseases. Heterotrimeric G-proteins, composed of Gα, Gβ and Gγ subunits, act as "molecular switches" for most GPCR functions. Mammalian Gα subunits are grouped into four families depending on sequence homology (Gα s , Gα i/o , Gα q/11 and Gα 12/13 ). Activation of GPCRs after the binding of a suitable ligand leads to G-protein heterotrimer activation and dissociation into Gα and Gβγ. The subunits then regulate the function of various effectors, leading to many kinds of cellular and physiological responses ( Figure 1) until the inactivation of Gα through GTP hydrolysis. RGS (regulators of G-protein signaling) proteins often regulate Gα signalling via stimulation of its GTPase activity (Sprang et al, 2007; Oldham et al, 2008). Effectors form a diverse group of proteins that, through their interactions with Gα and Gβγ, either act as second messengers or lead to direct physiological responses. Many proteins can act as effectors including enzymes, adhesion proteins, and cytoskeletal components, and various studies over the years have identified a large number of novel G-protein effectors (Kristiansen, 2004; Woehler et al, 2009). Each Gα family and Gβγ dimer bind to a number of different effectors, and many effectors can be regulated by more than one G- proteins. Motivation: •Crystallographic data, biochemical and in silico studies have provided useful information regarding interactions between G-proteins, RGS domains and effectors (Sprang et al, 2007), however until recently little was known concerning the GPCR - Gα interaction complex. Information is still scarce concerning the interactions of Gα subunits with most GPCRs, as well as various novel effectors. •Our goal was to study the interactions between GPCRs, Gα subunits and their effectors, and arrive at general structural implications for these interactions. Fotis A. Baltoumas 1 , Margarita C. Theodoropoulou 1 and Stavros J. Hamodrakas 1 * 1 Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Athens 157 01, Greece ∗ Correspondence to: shamodr@biol.uoa.gr Discussion :  We have identified a number of parts of the Gα surface that may participate in interactions both with receptors and with effectors. These parts include residues in the N- and C-termini, the α4-β6 loop and, possibly, the α3-β5 loop.  These sites differentiate between inactive (GDP-bound), empty state and active (GTP- bound) subunits. Their behavior during Gα activation may account for their role in various G-protein interactions, while the diversity of their sequence and structural properties can be a factor in the Gα subunit’s selectivity towards its binding partners.  Gα subunits display significant diversity of electrostatic properties among the four families, as well as subfamilies. This diversity suggests that electrostatic complementarity might be an important factor for Gα interactions.  Information provided by this study may be applicable to more detailed studies of the structural basis of G-protein interactions with GPCRs and novel effectors. References : • Baker, N.A., et al. (2001) Electrostatics of nanosystems: application to microtubules and the ribosome, Proc Natl Acad Sci U S A, 98, 10037-10041. • Berman, H.M., et al. (2000) The Protein Data Bank, Nucleic Acids Res, 28, 235-242. • DeLano, W.L. (2002) The PyMOL Molecular Graphics System. • Dolinsky, T.J., et al. 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Methods: •Initially, we performed an extensive literature search on Gα interactions, gathering information from crystal structures, mutagenesis and computational studies. • We then retrieved all relevant structures from PDB (Berman et al, 2000) and identified interacting sites and residues. Sequences of Gα subunits with solved structures were retrieved from UniProt (UniProt_Consortium, 2012). •Gα subunits were compared through multiple sequence alignment, structural alignments, and calculation of the electrostatic potential ( Graph 1). Figure 1. A general overview of signaling pathways mediated by GPCRs and G- proteins (Woehler et al, 2009). Figure 3. A. Structural elements of Gα subunits. B. Effector (green) and GPCR (blue) interacting sites on the surface of Gα, according to crystal structures, mutagenesis and computational studies. The structure of Gα is Gα i1 (1GP2). Results (Part 1): Overlapping interaction sites on the surface of Gα subunits. Figure 2. Sequence alignment of Gα subunits with solved structures. Secondary structure is represented by red cylinders for helices and green arrows for strands. The Switch regions are identified with cyan boxes. Gα residues participating in interactions, as are identified in the crystal structures of complexes are orange for RGS proteins, green for effectors, purple for both and blue for GPCRs. Additional interactions suggested by complexes of receptors or G- proteins with peptides, mutagenesis and computational studies are colored grey for GPCRs and black for effectors. •Early studies had identified several elements of the Gα GTPase domain as possible interaction sites in Gα – GPCR interactions. •The recently solved β 2 receptor – Gα s -Gβγ complex (Rasmussen et al, 2011) shows interactions between the cytoplasmic segment of the receptor and the C-terminus, the β2-β3 and α4-β6 loops, and residues in the N-terminus and α-helical domain of Gα (Table 1, Figure 2). Several mutagenesis studies suggest that the α3-β5 loop may also play a part in the formation of the complex, although there are no structural data supporting this interface yet. Table 1. Gα – GPCR and – effector interacting sites, as identified by crystal structures. Thus, we see that certain surfaces of Gα can often participate in binding both effectors and GPCRs ( Figure 3). These sites differentiate significantly in terms of sequence between the four Gα families, while other areas remain conserved. It should be noted that certain residues in the C-terminus and α4-β6, and perhaps the N-terminus and α3-β5 sites, have been implicated in forming contacts both with effectors and with GPCRs. Results (Part 2): Structural and electrostatic diversity among Gα subunits. Figure 4. Comparison of active and inactive Gα i1 , Gα t , Gα q and Gα 13 through structural alignment. Active subunits are green and inactive subunits are red. Important structural elements are indicated in Gα i1 ; all other subunits are shown in the same orientation. Distances (in Å) were measured between CA atoms of residues interacting with effectors, RGS proteins or GPCRs. Inactive Gα 13 ’s Switch II is disordered, therefore no measurement was taken. Structures used are those listed in Table 2. Table 2. RMSD values of structural alignments between active and inactive subunits. *: alignment between active and empty state Gα s . Figure 5. Comparison of GPCR & effector contact sites of Gα i1 , Gα s , Gα q and Gα 12 in cartoon (A) and ribbon (B) representation through structural alignment. Switch II, α3 and α3-β5, which form the common effector site, as well as the α4-β6 loop, are compared. Gα i1 (1GIA) is green, Gα s (1AZT) is blue, Gα q (3AH8) is purple and Gα 12 (1ZCA) is red. Figure 6. Electrostatic molecular surfaces of Gα subunits. Important structural elements are indicated in Gα i1 ; all other subunits are shown in the same orientation, the same way as in Figure 5. Subunit surfaces are contoured from -5 (red) to +5 (blue) kT/e - based on the potential of the solvent accessible surface. Crystal structures are Gα i1 (1GIA), Gα i3 (2V4Z), Gα t (1TND), Gα o (3C7K), Gα s (1AZT), Gα q (3AH8), Gα 12 (1ZCA) and Gα 13 (3CX8). •The formation of the GPCR – G-proteins complex results to the opening of the nucleotide cleft for GDP to GTP exchange, by movement of the α-helical domain. Gα activation also causes rearrangements in distinct sites of the GTPase domain. •Comparison of inactive and active Gα subunits through structural alignment (Figure 4, Table 2) displays extensive conformational shifts of the Switch regions. On the other hand, the α3 helix and the α3-β5 loop show little to no movement. The α4-β6 loop and C-terminus also differentiate between active and inactive subunits, although the extent of their shifting varies between different families and subfamilies. •Alignments of different Gα subunits show that the GTPase domain structure is conserved among the four Gα families; still, a few deviations are observed. The α3-β5 and especially the α4-β6 loops differentiate not only in sequence but also in structural conformation (Figure 5). On the other hand, members of the same Gα family show little difference in most of their sequence and structure features. •Many binding partners, including various effectors and RGS proteins, display specificity in their interactions with Gα subunits, even on the level of subfamily. •Calculation and comparison of electrostatic properties ( Figure 6) show that the potential of Gα surfaces is significantly diverse among different families, and in some cases among members of the same family. •Structural studies show that RGS proteins interact with residues in the three Switch regions, the N-terminus and the α-helical region of Gα to stimulate its GTPase activity (Figure 2). • Several crystal structures (Table 1) as well as mutagenesis and computational studies, provide information regarding the the structural basis of Gα – effector interactions. Effectors interact with a conserved, common interaction surface on all Gα subunits, formed by Switch II, α3 and the α3- β5 loop (Sprang et al, 2007). Several effectors also interact with other sites, including the N- and C-termini, the α4-β6 loop, Switch I and III regions, as well as the α-helical region (Figure 2). Graph 1. Our methodology.