Structural basis for PtdInsP2-mediated human TRPML1 regulation

Abstract

Transient receptor potential mucolipin 1 (TRPML1), a lysosomal channel, maintains the low pH and calcium levels for lysosomal function. Several small molecules modulate TRPML1 activity. ML-SA1, a synthetic agonist, binds to the pore region and phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P₂), a natural lipid, stimulates channel activity to a lesser extent than ML-SA1; moreover, PtdIns(4,5)P₂, another natural lipid, prevents TRPML1-mediated calcium release. Notably, PtdIns(3,5)P₂ and ML-SA1 cooperate further increasing calcium efflux. Here we report the structures of human TRPML1 at pH 5.0 with PtdIns(3,5)P₂, PtdIns(4,5)P₂, or ML-SA1 and PtdIns(3,5)P₂, revealing a unique lipid-binding site. PtdIns(3,5)P₂ and PtdIns(4,5)P₂ bind to the extended helices of S1, S2, and S3. The phosphate group of PtdIns(3,5)P₂ induces Y355 to form a π-cation interaction with R403, moving the S4-S5 linker, thus allosterically activating the channel. Our structures and electrophysiological characterizations reveal an allosteric site and provide molecular insight into how lipids regulate TRP channels.

Fig. 1

The overall structures of hTRPML1 with different ligands. a The secondary structure of hTRPML1. The major structural elements are labeled and the key residues for the PtdInsP₂ activation or in the pore regions are indicated. b The structure of hTRPML1 with PtdIns(3,5)P₂ (yellow sticks). c The structure of hTRPML1 with PtdIns(4,5)P₂ (green sticks). d The structure of hTRPML1 with PtdIns(3,5)P₂ (yellow sticks) and ML-SA1 (blue sticks)

Fig. 2

The molecular details of hTRPML1 bound to PtdInsP₂. a The sequence alignment of the key residues for PtdInsP₂ recognition among multiple TRPMLs. b The interaction details of hTRPML1 bound to PtdIns(3,5)P₂. c The interaction details of hTRPML1 bound to PtdIns(4,5)P₂. d The structural comparison of hTRPML1 with PtdIns(3,5)P₂ or PtdIns(4,5)P₂. e Interaction details of R403 and Y355 (pink) in mouse TRPML1 closed conformation in nanodiscs (PDB: 5WPV)

Fig. 3

TRPML1 channel activation for WT-L/A and Inositide binding mutants. a Current densities at −100 mV for various TRPML1 constructs with or without cytoplasmic PtdIns(3,5)P₂ reveal significant stimulation for WT but not the inositide pore binding mutants Y355A and R403A when 50 µM PtdIns(3,5)P₂ is present in the pipette solution. (*p = 0.038; **p < 0.005); (n = 8, 7, 5, 5, 4, 4, resp). b Stimulation with the synthetic agonist ML-SA1 (10 µM); (n = 11, 6, 5 resp). c Structural view of the R403 and Y355 residues (blue) and the R403K mutant (magenta). d Current densities at −100 mV for various WT-L/A and R403K TRPML1 mutant with or without 50 µM PtdIns(3,5)P₂ or 10 µM ML-SA1 at pH 4.6 and 7.4. The R403K mutation leads to a significant increase in current densities when ML-SA1 is applied regardless of the presence of inositide yet does not significantly increase channel current densities when ML-SA1 is not present. (*p = 0.023; #p = 0.001; **p < 0.001; ##p = 0.008); (n = 11, 8, 8, 6, 9, 6, 11, 11, resp). Values are mean ± s.e.m

Fig. 4

The putative mechanism of PtdIns(3,5)P₂ and ML-SA1 cooperation. a PtdIns(3,5)P₂ induces the π-cation interaction of Y355 and R403 in PtdIns(3,5)P₂/ML-SA1 bound structure. b The molecular detail of Y355 and R403 in the ML-SA1 bound structure (PDB: 5WJ9). c Structural comparison of both agonists bound (cyan) and the ML-SA1 bound (gray) hTRMPL1 structures. d The comparison of the pore region of both agonists bound and the ML-SA1 bound (gray) hTRMPL1 structures