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

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.

Figure 1.. Structural determination of the FLCN-FNIP2-Rag-Ragulator nonamer

A. Gel filtration profiles for the assembled FLCN-FNIP2-Rag-Ragulator supercomplex on a Hiload 16/60 Superdex 200 column. Cyan, FLCN-FNIP2 heterodimer only. Orange, FLCN-FNIP2 in complex with the Rag GTPases. Red, FLCN-FNIP2 in complex with the Rag GTPases and Ragulator. B. Coomassie blue stained gel analyses to identify the protein subunits within the peaks of the gel filtration profiles in A. Lanes are labeled based on the coloring pattern in A. C & D. Cryo-EM density map (C) and segmented map (D) for the FLCN-FNIP2-Rag-Ragulator nonamer. Subunits within the FLCN-FNIP2-Rag-Ragulator nonamer are differentiated by color as indicated: FLCN, purple; FNIP2, orange; RagA, pink; RagC, cyan; Ragulator, light brown.

Figure 2.. General architecture of the FLCN-FNIP2-Rag-Ragulator supercomplex

A. Atomic model, cartoon model, and domain assignment for the FLCN-FNIP2-Rag-Ragulator nonamer. Subunits of the FLCN-FNIP2-Rag-Ragulator complex are colored as following: FLCN, purple; FNIP2, orange; RagA, pink; RagC, cyan; Ragulator, brown. B. Domain arrangement for the FLCN-FNIP2-Rag-Ragulator supercomplex. Inter- and intra-subunit interactions are shown by gray bars between domains. The DENN domain within FNIP2 is split in two, denoted as DENNn (DENN domain N-terminal fragment) and DENNc (DENN domain C-terminal fragment). C. Structural model for the Longin domain heterodimer within the FLCN-FNIP2 complex. D. Structural model for the DENN domain heterodimer within the FLCN-FNIP2 complex.

Figure 3.. Structure of the Rag GTPase heterodimer within the FLCN-FNIP2-Rag-Ragulator supercomplex

A. FLCN-FNIP2 contacts the Rag GTPases through its Longin domains. A Longin domain heterodimer inserts inbetween the NBDs of RagA and RagC like a wedge. B. The aL1 helices of the FLCN- and FNIP2-Longin domain mediate interactions with the NBDs of RagA and RagC. C. Residues on the αL1 helix of the FLCN-Longin domain contact the NBD of RagA near the guanine base of the bound GDP. D. Residues on the αL1 helix of the FNIP2-Longin domain contact the NBD of RagC near the guanine base of the bound GppNHp. E. Cartoon model for the two parameters used to characterize the conformation of the Rag GTPase heterodimer. Distance d measures the distance between the N-terminal tips of the αG5 helices on Rag subunits. Angle θ measures the angel formed between the orientation of the aG5 helix of RagA with the N-terminal tip of the αG5 helix of RagC. F. Conformations of the Rag GTPase heterodimer and its yeast homolog, Gtr1p-Gtr2p. The available structures are aligned based on their CRDs but only the NBDs are shown. A variety of conformations can be observed when the subunits bind different nucleotides, or carry different mutations.

Figure 4.. Arg164 of FLCN is necessary for the GAP activity

A. Cryo-EM density map (colored surface) and atomic model (colored ribbon) around the nucleotide binding pocket of RagC. Clear boundaries between RagC and FLCN-FNIP2 are observed with no EM density extending into the nucleotide binding pocket of RagC. B. Sequence conservation of Arg164 of FLCN. Arg164 is conserved to yeast (Lst7) and localizes between the two β-strands of the Longin domain of FLCN. C. Structural comparison between Arg164 of FLCN and Arg78 of Nprl2. Arg78 of Nprl2 is the catalytic residue for GATOR1’s GAP function on RagA. These two arginines localize at similar positions on FLCN and Nprl2, respectively. D. Co-immunoprecipitation experiment to probe the interaction between FLCN-FNIP2 and the Rag GTPases. FLCN-FNIP2 carrying the R164A mutation binds to the Rag GTPases to a similar extent as wild-type FLCN-FNIP2. This experiment was repeated twice, and a representative data set is shown here. E. Coomassie blue stained gel of wild-type FLCN-FNIP2 and the mutant carrying the FLCN(R164A) mutation. F. Single turnover GTP hydrolysis assay to determine the effect of FLCN-FNIP2. The Rag GTPase heterodimer was first loaded with radiolabeled GTP. FLCN-FNIP2 was then added to stimulate the hydrolysis. Time points were taken to track the reaction process and were fitted to extract the observed reaction constants. G & H. Time courses of GTP hydrolysis by the Rag GTPases under single turnover conditions, stimulated by wild-type FLCN-FNIP2 (G) or the mutant carrying the FLCN(R164A) mutation (H). I. Concentration dependence of stimulated GTP hydrolysis by FLCN-FNIP2 under single turnover conditions. A 100-fold decrease was observed with the FLCN(R164A)-FNIP2 mutant under single turnover conditions. Gray numbers in parenthesis denote the SDs of the reported values calculated from at least three independent experiments. J. Multiple turnover GTP hydrolysis assay to determine the effect of FLCN-FNIP2. The Rag GTPase heterodimer was doubly-loaded with GTP. FLCN-FNIP2 was then added to stimulate hydrolysis. Time points were taken to track the reaction process and were fitted to extract the observed reaction constants. K & L. Time courses of GTP hydrolysis by the Rag GTPases under multiple turnover conditions, stimulated by wild-type FLCN-FNIP2 (K) or the mutant carrying the FLCN(R164A) mutation (L). M. Concentration dependence of stimulated GTP hydrolysis by FLCN-FNIP2 under multiple turnover conditions. A 7-fold decrease was observed with the FLCN(R164A)-FNIP2 mutant under these conditions. Gray numbers in parenthesis denote the SDs of the reported values calculated from at least three independent experiments.

Figure 5.. The resolved FLCN-FNIP2-Rag-Ragulator complex represents an on-pathway intermediate during GTP hydrolysis.

A. Relative positioning of the catalytic arginine and the nucleotide binding pocket of RagC. The distance between Arg164 and the phosphate of the GppNHp molecule bound to RagC is 14.7 Å. Nucleotide binding pocket of RagC is mis-oriented to allow for insertion of Arg164. B. Model for stimulated GTP hydrolysis of the Rag GTPases by FLCN-FNIP2. We interpret our structure as an on-pathway intermediate. Subsequent local conformational changes are required to insert the catalytic residue into RagC-NBD. C. Model for stimulated GTP hydrolysis of the Rag GTPases by GATOR1. We interpret our previous structure as an off-pathway intermediate during GTP hydrolysis. Global conformational change is required to access the catalytic arginine on Nprl2.