Emerging evidence suggests that heterotrimeric G protein gamma subunits (Ggamma) are important governors of G protein signaling, a function that is mediated through GPCR- and pH-dependent combinatorial phosphorylation of their intrinsically disordered N-terminal tails (Ggamma-Nt) that controls Gbetagamma/effector interactions and signaling. Intrinsic disorder is a universally conserved structural feature of all Ggamma subunit N-termini, which prompted us to hypothesize that, beyond phosphorylation, intrinsic disorder itself is inherently important to the signal-governing roles of Ggamma subunits. To test this hypothesis we devised a strategy in which single amino acid substitutions are sequentially introduced into the Ggamma tail, producing a series of isoforms that proceed step-wise from a fully-disordered to fully-ordered (alpha-helical) Nt tail structure. As a control for the increasing mutation load, we compare these mutants to those in which the same number of amino acid substitutions are incorporated that do not alter the inherent structural disorder of the tail. These mutant isoforms were then structurally analyzed by circular dichroism (CD) in vitro, by molecular dynamics (MD) simulation in silico, and by functional analysis of Gbetagamma-dependent molecular signaling in vivo. Here, we apply this approach to the yeast Ggamma subunit, Ste18. CD and MD analyses of Ste18-Nt tail isoforms indicate that a successful transition from a fully-disordered to fully-ordered state is achievable through precise point mutation. Replacing the wild type Ggamma subunit with each of the mutant isoforms in yeast, we further show that intrinsic disorder of Ggamma-Nt controls the stability of the Ggamma subunit in a manner that is proportional to the loss of intrinsic disorder in vivo. pH-dependent phosphorylation at Ser3 in the tail is largely unaffected by these changes. However, unexpectedly, we found that the GPCR-dependent phosphorylation site, Ser7, becomes pH-sensitive in response to changes in tail structure. Ongoing experiments reveal the effects of Ste18-Nt tail structure on the interaction of yeast Gbg with its primary effector Ste5, and subsequent effects on activation of MAPKs, which have been shown to be highly sensitive to Ggamma-Nt tail phosphorylation previously. Taken together, these data provide evidence that intrinsic structural disorder plays a direct role in functionality of Ggamma subunits as governors of G proteins signaling and substantiates the rationale for exploring similar roles for these tails in mammalian Gbetagamma-dependent signaling pathways.

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Su X, Li W, Pang YT, Gumbart JC, Torres M

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