Designed mini-Ga proteins capable of molecular switch functionalities

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Designed mini-Ga proteins capable of molecular switch functionalities

ABSTRACT (UP TO one hundred fifty words)

Heterotrimeric (aßy) G proteins regulate intracellular signaling as molecular switches governed by bound guanine nucleotides. This functionally requires enzymatic GTPase activity and interactions with multiple regulator or downstream "effectors". Truncated "mini-Ga" proteins have been previously engineered and used to bind G protein-coupled receptors, mostly in structural studies. Here, we design novel mini-Ga proteins with molecular switch functionalities in terms of regulation and partner interactions. These constructs are able to bind and hydrolyze guanine nucleotides in vitro, and, notably, be inactivated by RGS proteins with high efficiency. In cells, these mini-Ga proteins can also bind GBy and interact with downstream effectors. Such functional mini-G's can be used to investigate G protein function and interactions and can be utilized in synthetic biology efforts.

INTRODUCTION

Heterotrimeric G proteins (aßy) are molecular switches that regulate numerous intracellular signaling cascades in response to G protein-coupled receptors, interacting with downstream effectors that regulate diverse physiological systems. Within the heterotrimer, the Ga subunit is the actual molecular switch whose functionality depends on binding and hydrolysis of guanine nucleotides. In the inactive conformation, the Ga subunit is bound to GDP and associates with the GBy dimer, which prevents Ga interactions with downstream effectors. The Ga switch is activated by the exchange of the bound GDP with GTP, leading to dissociation of the GTP-bound Ga from GBy, with either moiety activating downstream partners. Ga subunits are "turned off" by GTP hydrolysis (GTPase) catalyzed by the Ga subunit itself, yielding the inactive form of Ga-GDP. The intrinsically slow Ga GTPase activity can be accelerated by Regulator of G protein Signaling proteins that determine the actual duration of G protein mediated signaling. RGS proteins bind activated Ga-GTP, accelerating GTP hydrolysis allosterically, thereby acting as GTPase activating proteins.

Engineered "mini-Ga" proteins have been developed, originally for structural studies, by truncating more than forty percent of the entire Ga subunit and mutating several amino acids to increase stability. These mini-Ga proteins contain only the central Ga "GTPase domain", following deletions of the Ga N-terminus, the entire Ga "helical domain", and additional segments within the GTPase domain. Additionally, six to eight point mutations were introduced into the GTPase domain to stabilize flexible regions, improve thermostability, and increase affinity to G protein-coupled receptors. These mini-Ga proteins were optimized for G protein-coupled receptor-binding, so we term them here "G protein-coupled receptor-binding mini-Ga" (GB-mini-Ga) proteins. Over the last decade GB-mini-Ga proteins have been used to solve experimentally dozens of G protein-coupled receptor complexes with GB-mini-Ga proteins, mostly using GB-mini-Gas and GB-mini-Goo. Notably, most of the GB-mini-Ga-G protein-coupled receptor complexes that have been solved include By subunits, and are nucleotide-free. GB-mini-Ga proteins have also been used as biosensors that selectively bind active G protein-coupled receptors in living cells. Previous studies have shown that fusion of these proteins to fluorescent or luminescent tags, combined with bioluminescence resonance energy transfer assays or NanoLuc Binary Technology, enables measurement of receptor activation and monitoring of subcellularly localized signaling events. All of these previous studies focus on the ability of mini Ga proteins to strongly bind activated G protein-coupled receptors, however, they do not demonstrate functional downstream interactions. Moreover, previous studies suggested that the GB-mini-Ga proteins are unable to facilitate nucleotide exchange. To our knowledge, GB-mini-Ga proteins have not been tested for their ability to bind and hydrolyze nucleotides or their ability to be regulated by RGS proteins.

Previous structural studies reveal that the functionality of GTP binding and hydrolysis by Ga subunits is mostly encoded within the GTPase domain of Ga subunits, which is common across all members of the G protein superfamily. On the other hand, the helical domain plays a role in enabling nucleotide exchange and contributes only to interactions with a limited number of effectors and RGS proteins. The Ga GTPase domain includes three flexible regions designated switch one, two, and three, which "switch" conformation depending on whether GTP or GDP is bound in the active site. Nucleotide binding was shown to be mediated by conserved motifs in the Ga GTPase domain that include the phosphate-binding loop, switch one and two, the five-aG region, and the a5-06 loop. Nucleotide hydrolysis requires two conserved Ga catalytic residues, an arginine in switch one and a glutamine in switch two, that are crucial for stabilizing the transition state of GTP hydrolysis. Note that the Ga switch one connects the Ga helical domain to the GTPase domain, therefore reasonably to assume that the positioning of the catalytic arginine requires both domains. The Ga active site also contains a magnesium ion that is needed for nucleotide binding and hydrolysis; this magnesium ion is coordinated by residues from the Ga P-loop, switch one and two. An additional region that plays a role in GTP binding and hydrolysis is switch three, which functions indirectly via interactions with switch two to position the catalytic glutamine for GTP hydrolysis. Importantly, the GTPase domain in general, and the three switch regions in particular, are central to interactions with RGS proteins. However, The Ga helical domain was shown to determine specific interactions with RGS proteins, which have been suggested to play a role in conferring RGS specificity. The GTPase domain also provides all of the Ga binding surface for GBy subunits and is critical in interactions with effectors. In particular, Ga interactions with effectors, are mediated not only by the Ga switch regions, but also by an "effector-binding region" that includes the C-terminal half of switch two, the a3 helix, and the loop that connects the latter to the beta five strand. Taken together, it remains unclear which structural motifs in the Ga GTPase domain are sufficient to enable nucleotide binding and hydrolysis, as well as interaction with RGS proteins and downstream effectors, and whether GB-mini-Ga proteins contain sufficient functional regions to enable some or all of these functions.

Here, we designed novel mini-Gao proteins that can act as molecular switches that can be regulated by RGS proteins and interact with downstream effectors. We used modeling to select mini-Gao proteins predicted to be compatible with nucleotide binding and hydrolysis. We tested in-vitro which mini-Goo constructs can bind and hydrolyze nucleotides, and can be inactivated by RGS proteins. We then used BRET-based assays in cells to assess the ability of mini-Gao proteins to bind GBy and downstream effectors, and extended this approach to mini-Gail. In this study, we therefore developed, for the first time, mini-Ga proteins that function as molecular switches in terms of nucleotide binding and hydrolysis, regulation by RGS proteins, and interaction with downstream effectors. These new proteins can be used to better understand the mechanisms of G protein activation and inactivation and serve as synthetic tools to study cellular communication.

RESULTS

Designing functional mini-Gao proteins that can bind and hydrolyze nucleotides

Identification of designed mini-Gao constructs with molecular switch functionality

Mini-Gao proteins can be efficiently inactivated by RGS sixteen

Mini-Gao can bind GBy subunits and interact with the Rap one Gap effector

DISCUSSION

AUTHOR CONTRIBUTIONS

MATERIALS AND METHODS

Structure-based definition of the consensus Ga nucleotide binding site

Protein expression, purification, and mutagenesis

Protein thermostability analysis using Differential Scanning Fluorimetry

Guanine nucleotide binding measurements

Single-turnover GTPase assays and RGS GAP activity measurements

Overview

The study develops and characterizes new mini-Ga proteins that act as molecular switches, showing their capacity for nucleotide binding and hydrolysis, and regulation by RGS proteins. These findings provide insights into G protein functionality and applications in synthetic biology.

Key Points

1Novel mini-Ga proteins were engineered to bind and hydrolyze guanine nucleotides
2The proteins can be regulated by RGS proteins and interact with downstream effectors
3The research establishes a method for designing functional molecular switches
4Mini-Ga proteins may be utilized in synthetic biology for cellular communication studies
5The findings offer new approaches to investigate G protein signaling mechanisms

Details

Authors: Sabreen Higazy-Mreih, Rojeh Eid, Lyudmila Getmanenko, Irina Nekrasova, Meirav Avital- Shacham, Mickey Kosloff
Category: Biology and Natural Sciences

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