TRPML2 in distinct states reveals the activation and modulation principles of the TRPML family

Abstract

TRPML2 activity is critical for endolysosomal integrity and chemokine secretion, and can be modulated by various ligands. Interestingly, two ML-SI3 isomers regulate TRPML2 oppositely. The molecular mechanism underlying this unique isomeric preference as well as the TRPML2 agonistic mechanism remains unknown. Here, we present six cryo-EM structures of human TRPML2 in distinct states revealing that the π-bulge of the S6 undergoes a π-α transition upon agonist binding, highlighting the remarkable role of the π-bulge in ion channel regulation. Moreover, we identify that PI(3,5)P₂ allosterically affects the pose of ML2-SA1, a TRPML2 specific activator, resulting in an open channel without the π-α transition. Functional and structural studies show that mutating the S5 of TRPML1 to that of TRPML2 enables the mutated TRPML1 to be activated by (+)ML-SI3 and ML2-SA1. Thus, our work elucidates the activation mechanism of TRPML channels and paves the way for the development of selective TRPML modulators.

Fig. 1. Overview of TRPML2 structures bound to different compounds.

a–f Overall structures of human TRPML2 from the side of the membrane with respective substrates shown as sticks in darker respective colors: apo in yellow (a), (-)ML-SI3–bound in green with the molecule shown as sticks in dark green (b), (+)ML-SI3–bound in a pre-open state in light blue with molecule shown as sticks in purple (c), (+)ML-SI3–bound in an open state in blue with molecule shown as sticks in dark blue (d), ML2-SA1–bound in pink with molecule shown as sticks in dark pink (e), ML2-SA1/PI(3,5)P₂–bound in salmon with ML2-SA1 shown as sticks in ruby (f). g–l Side view of the ion pore of each structure with the pore and the α-helix or π-bulge in S6 labeled. The lower gate residues and compounds are shown as sticks and colored as in (a–f). Gray balls indicate the path of the cation pore as calculated by HOLE.

Fig. 2. Binding and interaction details of (-)ML-SI3 and (+)ML-SI3.

a Structural comparison between ML-SI3–bound TRPML1 (7MGL) in gray and (-)ML-SI3–bound TRPML2 in green with the compounds shown in their respective colors. b Zoom-in comparison of molecule orientation between the two compounds in a, with the π-bulge indicated. c Interaction details of (-)ML-SI3–bound TRPML2 with important residues shown as sticks. Residues in gray denote a neighboring subunit. d Comparison of apo (yellow), and (+)ML-SI3–bound pre-open (light blue) TRPML2 with the α-helix indicated, and the movement of the S4-S5 linker denoted by a red arrow. e Interaction details of (+)ML-SI3–bound pre-open TRPML2 with residues shown as sticks and (+)ML-SI3 shown in purple. Residues in gray denote a neighboring subunit. f Comparison of apo (yellow), and (+)ML-SI3–bound open (blue) TRPML2 with the π-bulge indicated, and the movement of the S4-S5 linker denoted by a red arrow. g Interaction details of (+)ML-SI3–bound open TRPML2 with residues shown as sticks and (+)ML-SI3 shown in dark blue. Residues in gray denote a neighboring subunit. h Comparison of the poses of ML-SI3 in three structures and Y496 labeled as sticks.

Fig. 3. Interaction details of ML2-SA1 and allosteric effects of PI(3,5)P₂.

a Structural comparison between apo TRPML2 (yellow) and ML2-SA1–bound TRPML2 (pink) with the molecule shown as sticks in dark pink, and the movement of the S4-S5 linker denoted by a red arrow. b Comparison of the same structures as shown in a rotated 90° with the movement of Y496 indicated by a red arrow. c Interaction details of ML2-SA1–bound TRPML2 with residues shown as sticks. d Pore distances through the ML2-SA1–bound TRPML2, with the different F502 orientations labeled (one in pink and one in sage). Right panel shows the same movement of F502 rotated 90°. e Comparison of ML-SA1/PI(3,5)P₂–bound TRPML1 (7SQ9, light blue) with ML2-SA1/PI(3,5)P₂–bound TRPML2 (salmon). f PI(3,5)P₂ induces the conformational changes on the S4-S5 linker to prevent the rotation of S6. The salt bridges between residues and PI(3,5)P₂ are indicated by dashed lines. g Interaction details of ML2-SA1/PI(3,5)P₂–bound TRPML2 with residues shown as sticks. Residues in gray denote a neighboring subunit.

Fig. 4. Electrophysiological characterization of TRPML2 mutants.

a Representative current density-voltage (I/C_m-V) relation of transiently expressed and localized in plasma membrane human TRPML2-eYFP variants, WT (left) or L414A mutant (right). b Representative current density-voltage (I/C_m-V) relation of transiently expressed and localized in plasma membrane human TRPML2-eYFP variants, WT (left) or F457A mutant (right). c Representative current density-voltage (I/C_m-V) relation of transiently expressed and endolysosomal localized human TRPML2-eYFP variants, WT (left) and Y496A mutant (right). For each panel (a–c), basal levels are shown in black. Channels were activated by application of (+)ML-SI3 (10 µM, blue, top) or ML2-SA1 (10 µM, pink, bottom). Statistical analysis of experiments is shown in bar graphs (far right), with each dot representing mean of biological replicate measurements withdrawn at −100 mV, with the exact n indicated above each bar, and the precise p value shown (mean ± SEM, one-way ANOVA, Tukey’s post hoc test using GraphPad Prism 10.2.3, ****p < 0.0001, **p < 0.01).

Fig. 5. Docking and electrophysiological characterization of TRPML1^VA/AG in the presence of (+)ML-SI3 and ML2-SA1.

a Docking of (+)ML-SI3 (dark blue) from the (+)ML-SI3 bound TRPML2 open conformation into ML-SI3 bound TRPML1 (PDB: 7MGL; gray). b Docking of ML2-SA1 (dark pink) from the ML2-SA1 bound TRPML2 pre-open conformation into ML-SI3 bound TRPML1 (PDB: 7MGL; gray). The TRPML1 residues of importance are labeled and represented as sticks. The red arrow indicates potential clashes between the compound and TRPML1. c Representative current density-voltage (I/C_m-V) relation of transiently expressed and endolysosomal localized human TRPML1-eYFP variants, WT (left) and V432A/A433G mutant (right). Basal levels are shown in black. Channels were activated by application of (+)ML-SI3 (10 µM, blue, top) or ML2-SA1 (10 µM, pink, bottom). Statistical analysis of experiments is shown in bar graphs (far right), with each dot representing mean of biological replicate measurements withdrawn at −100 mV, with the exact n indicated above each bar, and the precise p value shown (mean ± SEM, one-way ANOVA, Tukey’s post hoc test using GraphPad Prism 10.2.3, ****p < 0.0001).

Fig. 6. Structures of ML2-SA1 and (+)ML-SI3–bound TRPML1^VA/AG, and ML-SA5–bound TRPML1^WT.

a Side view of TRPML1^VA/AG and TRPML1^WT bound to different ligands (from left to right): ML2-SA1–bound TRPML1^VA/AG in tan with the substrate in olive; (+)ML-SI3–bound TRPML1^VA/AG in a partially open state in green; (+)ML-SI3–bound TRPML1^VA/AG in a pre-open state in teal with (+)ML-SI3 indicated in gray sticks; and ML-SA5–bound TRPML1^WT in green with ML-SA5 shown in light green sticks. b Side view of the ion pore of each structure with the pore and the α-helix or π-bulge in S6 labeled. The lower gate residues and compounds are shown in sticks and colored as in (a). Gray balls indicate the path of the cation pore as calculated by HOLE. c Interaction details of (+)ML-SI3–bound TRPML1^VA/AG pre-open state with residues shown as sticks. Residues in gray denote a neighboring subunit. d Comparison of the binding orientation of (+)ML-SI3–bound TRPML1^VA/AG (teal/gray) with (+)ML-SI3–bound TRPML2 in an open state (blue/dark blue). e Comparison of the binding orientation of ML-SA5–bound TRPML1^WT (green) with ML-SA1–bound TRPML1 (5WJ9, cyan). f Interaction details of ML-SA5–bound TRPML1^WT with residues shown as sticks.