Galpha Gbetagamma dissociation may be due to retraction of a buried lysine and disruption of an aromatic cluster by a GTP-sensing Arg Trp pair

Abstract

The heterotrimeric G protein alpha subunit (Galpha) functions as a molecular switch by cycling between inactive GDP-bound and active GTP-bound states. When bound to GDP, Galpha interacts with high affinity to a complex of the beta and gamma subunits (Gbetagamma), but when bound to GTP, Galpha dissociates from this complex to activate downstream signaling pathways. Galpha's state is communicated to other cellular components via conformational changes within its switch I and II regions. To identify key determinants of Galpha's function as a signaling pathway molecular switch, a Bayesian approach was used to infer the selective constraints that most distinguish Galpha and closely related Arf family GTPases from distantly related translational and metabolic GTPases. The strongest of these constraints are imposed on seven residues within or near the switch II region. Likewise, constraints imposed on Galpha but not on other, closely related molecular switches correspond to four nearby residues. These constraints are explained by a proposed mechanism for GTP-induced dissociation of Galpha from Gbetagamma where an Arg-Trp pair senses the presence of bound GTP leading to conformational retraction of a nearby lysine and to disruption of an aromatic cluster. Within a complex of Gialpha, Gibetagamma, and GDP, this lysine establishes greater surface contact with Gibeta than does any other residue in Gialpha, whereas the aromatic cluster packs against a highly conserved tryptophan in Gibeta that establishes greater surface contact with Gialpha than does any other residue in Gibeta. Other structural features associated with Galpha functional divergence further support the proposed mechanism.

Figure 1.

Schematic representation of the input and output for the Bayesian partitioning with pattern selection (BPPS) procedure (Neuwald et al. 2003) used here to characterize Gα functional divergence. The input consists of a “query alignment” of representative sequences of interest and a “main alignment” containing either all or a subset of the available sequences related to the query set. The main alignment typically contains hundreds or thousands of sequences, which need to be aligned very accurately, as previously described (Neuwald and Liu 2004). The output is defined statistically by the BPPS procedure, which optimally partitions the main alignment into a “foreground” and a “background,” such that the foreground (which includes the query sequences) exhibits a strikingly conserved pattern that is strikingly nonconserved within (and thus contrasts with) the background. For this reason the output is termed a Constrast Hierarchical (CH) alignment. In the figure, horizontal bars represent aligned sequences. Vertical colored bars represent foreground residues that strikingly diverge from the background at those positions; the colors reflect the different types of amino acids conserved. The histogram above the alignment represents the estimated strengths of the selective pressures imposed on divergent residue positions. For an overview of this approach, see Neuwald (2006). (Figure adapted from Neuwald 2006 with permission from Elsevier © 2006.)

Figure 2.

CH alignments characterizing three categories of functionally divergent constraints imposed on Gα GTPases. The proteins whose constraints are being compared are indicated above each alignment using the format “foreground vs. background.” The bars directly below each alignment correspond to the structural regions shown in Figures 3 and 4 and are color coded as follows: Walker A, purple; switch I, red; and switch II, orange. Directly below the bars, the most conserved residue patterns at each position in the foreground are shown, and directly below these the corresponding (weighted) frequencies are shown in integer tenths, where a “9,” for example, indicates that the corresponding residue occurs in 90%–100% of the sequences. Below this (in B and C) the most conserved residue patterns and their frequencies at each position in the background are shown in light gray. The relative selective pressures imposed on divergent residues are indicated by the histogram above each alignment using an approximately logarithmic scale. For the analyses in B and C, the strongest constraints in these categories all occur within the aligned region shown. (A) CH-alignment revealing the locations of key catalytic residues generally conserved within all P-loop GTPases. (B) CH-alignment showing divergent residues distinguishing the Gα and Arf families from distantly related GTPases that, like Gα and Arf, belong to the TRAFAC class (Leipe et al. 2002). The background includes the translational GTPases EF-Tu/EF-1α, IF2/eIF5B, eIF2γ/SelB, EF-G/EF-2, eRF3, LepA (Qin et al. 2006), and TypA/BipA (Owens et al. 2004) and the sulfur and iron metabolic GTPases CysN (Mougous et al. 2006) and FeoB (Cartron et al. 2006), respectively. The BPPS procedure (Neuwald et al. 2003) was used to identify and categorize these families prior to their inclusion in this analysis. (C) CH-alignment showing divergent residues distinguishing Gα from Arf family GTPases.

Figure 3.

Structural conformations of the Gα/Arl–switch II component in Arf and Arl GTPases. Color scheme for backbone traces: Walker A motif and the helix that follows it, purple; switch I region, red; switch II region and C-terminal end of the preceding β-strand, orange. Residues with magenta colored side chains distinguish all P-loop GTPases from other proteins (see Fig. 2A); residues with orange side chains distinguish the Gα and Arf families from translational and metabolic GTPases (Fig. 2B); residues with cyan side chains distinguish individual Arl subfamilies from other Arf subfamilies (corresponding CH-alignment not shown). Oxygen, nitrogen, and (predicted) hydrogen atoms that establish hydrogen bonds or ionic interactions are colored red, blue, and white, respectively. Hydrogen bonds are shown as dotted lines; ionic and aromatic–aromatic interactions are shown as dot clouds. Hydrogen atoms were added using the program REDUCE (Word et al. 1999). Figures were generated using RasMol (Sayle and Milner-White 1995). (A) Arf1 + GTP (pdb id: 1J2JA) (Shiba et al. 2003). (B) Arf1 + GDP (pdb id: 1HURA) (Amor et al. 1994). (C) Arl2 + GTP (pdb id: 1KSJA) (Hanzal-Bayer et al. 2002). (D) Arl3 + GDP (pdb id: 1FZQA) (Linari et al. 1999). (E) Arl6 + GTP (pdb id: 2H57A) (Wang et al. 2006). The canonical glycine residue of the Gα/Arl component (Gly69 in Arf1) has been replaced in Arl6 by a conserved serine (Ser71), the side chain—OH group of which appears to hydrogen bond to a buried water near the γ-phosphate of GTP. This serine thus may help position and activate this water for catalysis and presumably reflects a key (functionally divergent) feature of Arl6 GTPases. (F) Arl8A + GDP (pdb id: 2H18A) (Atanassova et al. 2006).

Figure 4.

Structural conformations of the Gα/Arl–switch II component of Gα GTPases. Residues with green side chains distinguish Gα from Arf family GTPases (see Fig. 2C). For other descriptions see the legend to Figure 3. (A) Giα + GTP analog (pdb id: 1CIPA) (Coleman and Sprang 1999). The GTP analog is designated as “GTP.” (B) Giα + GDP + PO₄ ³⁻ (pdb id: 1GITA) (Berghuis et al. 1996). (C) Gia + GDP + AlF₄, the transition state conformation (pdb id: 1GFIA) (Coleman et al. 1994). (D) Giα + GDP bound to the Gβγ complex (pdb id: 1GG2A) (Wall et al. 1995). Regions of the Gβ subunit are shown as grayish blue.