Differences in intradomain and interdomain motion confer distinct activation properties to structurally similar Gα proteins

Abstract

Proteins with similar crystal structures can have dissimilar rates of substrate binding and catalysis. Here we used molecular dynamics simulations and biochemical analysis to determine the role of intradomain and interdomain motions in conferring distinct activation rates to two Gα proteins, Gα i1 and GPA1. Despite high structural similarity, GPA1 can activate itself without a receptor, whereas Gα i1 cannot. We found that motions in these proteins vary greatly in type and frequency. Whereas motion is greatest in the Ras domain of Gα i1 , it is greatest in helices αA and αB from the helical domain of GPA1. Using protein chimeras, we show that helix αA from GPA1 is sufficient to confer rapid activation to Gα i1 . Gα i1 has less intradomain motion than GPA1 and instead displays interdomain displacement resembling that observed in a receptor-heterotrimer crystal complex. Thus, structurally similar proteins can have distinct atomic motions that confer distinct activation mechanisms.H eterotrimeric G proteins are molecular switches that are activated in response to extracellular stimuli including hormones, light, and neurotransmitters. In animals, the G-protein heterotrimer is activated by cell-surface receptors that trigger the Gα subunit of the heterotrimer to release GDP and bind GTP. GTP binding induces conformational changes in three small switch regions that result in heterotrimer dissociation (1). The free subunits then relay signals by activating or inhibiting downstream effectors. G-protein signaling is terminated after Gα hydrolyzes GTP and the heterotrimer reassociates. Thus, Gα proteins serve as timing devices that determine the duration of signaling.Gα proteins from different organisms and subclasses share nearly identical structural features (2-4). However, G proteins exhibit a large spectrum of nucleotide exchange and hydrolysis rates. Basal nucleotide exchange in Gα q , for example, is essentially undetectable without a receptor (5), whereas the Gα protein (GPA1) from the plant Arabidopsis thaliana exchanges nucleotides at a pace of at least 4/min (6). Thus, GPA1 serves as a counterexample to slowly exchanging animal proteins and provides an opportunity to compare the molecular basis for receptor-dependent and -independent signaling.The Gα subunit is composed of two domains connected by two short linker regions: (i) a domain that resembles the monomeric G protein Ras and (ii) an all α-helical domain unique to heterotrimeric G proteins. The guanine nucleotide binds at the interface of the two domains, but nucleotide-binding residues and switch regions are contained within the Ras domain and linker regions. The recently solved cocrystal structure of a G-protein heterotrimer with an activated receptor shows a large receptorinduced displacement of the Gα helical domain relative to the Ras domain (7). It is unclear whether this interdomain rearrangement is the cause or the consequence of nucleotide release. Nonetheless this work shows that both intradomain and interdomain rearrangements are required for G-p...

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins

“…More frequent RasD-HD domain separation has previously been suggested to underlie the self-activation of the G protein GPA1 from Arabidopsis thaliana (9). GPA1 is permanently activated, has enhanced nucleotide exchange rates, and displays enhanced domain opening in simulations relative to G␣ i .…”

“…This conformational change, which effectively exposes the otherwise buried nucleotide binding site, has been linked to GPCR-mediated nucleotide exchange (4). Evidence for domain opening has also been obtained from recent electron microscopy (5), double electron-electron resonance analysis (6), hydrogen-deuterium exchange mass spectrometry (7), biochemical analysis (8), and molecular dynamics (MD) simulations (3,9,10). In addition, the structure of Rasmussen and co-workers (7) together with mass spectrometry results also confirm that both N-terminal ␤1 strand and C-terminal ␣5 helix are major interaction sites for receptors.…”

Molecular interaction of δ-conopeptide EVIA with voltage-gated Na+ channels

“…as a measure of dynamic motion, which is often linked to intrinsic domain motion. [57][58][59] As depicted in Supporting Fig. S8, the local per-residue flexibility is reduced upon δ-EVIA binding relative to the reference simulation with/without the toxin, especially in the linker region connecting the transmembrane segments of the voltage-sensor domain of Na V 1.7 and the toxin-sensitive chimera 4447.…”

Guanine Nucleotide-binding Protein (Gα) Endocytosis by a Cascade of Ubiquitin Binding Domain Proteins Is Required for Sustained Morphogenesis and Proper Mating in Yeast

“…The UD is adjacent to the A/B helix of the helical domain, a region whose dynamics were previously shown to promote the rapid, receptor-independent, nucleotide exchange activity of Arabidopsis thaliana G␣ (45). However, it is not known whether the UD regulates the enzymatic activity of S. cerevisiae Gpa1.…”

“…Functional Contributions of the Ubiquitination Domain to Gpa1 Activity-The ␣-helical domain is known to influence G protein nucleotide exchange activity (45). The UD is adjacent to the A/B helix of the helical domain, a region whose dynamics were previously shown to promote the rapid, receptor-independent, nucleotide exchange activity of Arabidopsis thaliana G␣ (45).…”