Global conformation of the Rag GTPase heterodimer governs eukaryotic amino acid sensing

TL;DR

This study reveals how the shape of a protein complex called Rag GTPase changes to sense amino acids in cells.

Contribution

The study identifies a conserved proline residue as a hinge that controls global conformational changes in the Rag GTPase heterodimer.

Findings

Single-molecule FRET reveals that nucleotides, regulators, and mutations dictate the global conformation of the Rag GTPase heterodimer.

A conserved proline residue acts as a hinge, and its mutation disrupts amino acid signaling.

Global conformation of the Rag GTPase is essential for interpreting amino acid signals in eukaryotic cells.

Abstract

The conformation of a protein—the way its amino acid chain folds into structures—determines how the protein functions and interacts with other molecules. This unique shape dictates the protein’s function, and changes in conformation may alter its biological function. This paper investigates how the Rag GTPase heterodimer, crucial for sensing amino acids, changes its overall shape (global conformation). Using single-molecule FRET, the study found that nucleotide binding, mutations, and interaction with mTORC1 alter these conformations. A conserved proline residue acts as a “hinge,” mediating these changes, and its mutation disrupts amino acid signaling. This work uncovers a checkpoint in amino acid sensing, emphasizing that the Rag GTPases’ global conformation is as vital as local nucleotide binding.

The Rag GTPase heterodimer is a central mediator of amino acid sensing in eukaryotic cells. When amino acids are abundant, it binds to the mechanistic target of rapamycin complex 1 to activate cellular programs for growth and proliferation. In its functional cycle, besides local conformational changes near the nucleotides that are commonly observed in monomeric signaling GTPases, the relative positioning of the two Rag subunits, i.e., the global conformation, is unique due to the heterodimeric architecture. Although various global conformations have been captured in static structural models, dynamic transitions between these conformations and their biological relevance remain unclear. Here, we visualize the global conformation of the Rag GTPase heterodimer using single-molecule Förster resonance energy transfer. By tracking the movement of individual protein molecules, we found that the two subunits explore a wide conformational space, which is strictly dictated by the bound nucleotides, regulators, and mutations. Additionally, we demonstrate that proper modulation of the global conformation is crucial for correctly interpreting amino acid signals. Our results defined a checkpoint of amino acid sensing in eukaryotic cells.

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