Skip to Content
Merck

Cryo-EM Structure of the Human FLCN-FNIP2-Rag-Ragulator Complex.

Cell (2019-11-11)
Kuang Shen, Kacper B Rogala, Hui-Ting Chou, Rick K Huang, Zhiheng Yu, David M Sabatini
ABSTRACT

mTORC1 controls anabolic and catabolic processes in response to nutrients through the Rag GTPase heterodimer, which is regulated by multiple upstream protein complexes. One such regulator, FLCN-FNIP2, is a GTPase activating protein (GAP) for RagC/D, but despite its important role, how it activates the Rag GTPase heterodimer remains unknown. We used cryo-EM to determine the structure of FLCN-FNIP2 in a complex with the Rag GTPases and Ragulator. FLCN-FNIP2 adopts an extended conformation with two pairs of heterodimerized domains. The Longin domains heterodimerize and contact both nucleotide binding domains of the Rag heterodimer, while the DENN domains interact at the distal end of the structure. Biochemical analyses reveal a conserved arginine on FLCN as the catalytic arginine finger and lead us to interpret our structure as an on-pathway intermediate. These data reveal features of a GAP-GTPase interaction and the structure of a critical component of the nutrient-sensing mTORC1 pathway.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Imidazole, for molecular biology, ≥99% (titration), free-flowing, Redi-Dri
Sigma-Aldrich
Guanosine 5′-triphosphate sodium salt hydrate, ≥95% (HPLC), powder
Sigma-Aldrich
Tris(2-carboxyethyl)phosphine hydrochloride, powder
Sigma-Aldrich
DL-Dithiothreitol, BioXtra, ≥99.0% (titration)
Sigma-Aldrich
HEPES, ≥99.5% (titration)
Sigma-Aldrich
Imidazole, for molecular biology, ≥99% (titration)
Sigma-Aldrich
CHAPS hydrate, BioXtra, ≥98% (TLC)
Sigma-Aldrich
Guanosine 5′-diphosphate sodium salt, Type I, ≥96% (HPLC)
Sigma-Aldrich
Potassium acetate, for molecular biology, ≥99.0%
Sigma-Aldrich
Magnesium chloride, anhydrous, ≥98%
Sigma-Aldrich
Triton X-100, BioXtra