Structure and inhibition of the human lysosomal transporter Sialin.

Sialin, a member of the solute carrier 17 (SLC17) transporter family, is unique in its ability to transport not only sialic acid using a pH-driven mechanism, but also transport mono and diacidic neurotransmitters, such as glutamate and N-acetylaspartylglutamate (NAAG), into synaptic vesicles via a membrane potential-driven mechanism. While most transporters utilize one of these mechanisms, the structural basis of how Sialin transports substrates using both remains unclear. Here, we present the cryogenic electron-microscopy structures of human Sialin: apo cytosol-open, apo lumen-open, NAAG-bound, and inhibitor-bound. Our structures show that a positively charged cytosol-open vestibule accommodates either NAAG or the Sialin inhibitor Fmoc-Leu-OH, while its luminal cavity potentially binds sialic acid. Moreover, functional analyses along with molecular dynamics simulations identify key residues in binding sialic acid and NAAG. Thus, our findings uncover the essential conformational states in NAAG and sialic acid transport, demonstrating a working model of SLC17 transporters.

Structure and mechanism of human cystine exporter cystinosin.

Lysosomal amino acid efflux by proton-driven transporters is essential for lysosomal homeostasis, amino acid recycling, mTOR signaling, and maintaining lysosomal pH. To unravel the mechanisms of these transporters, we focus on cystinosin, a prototypical lysosomal amino acid transporter that exports cystine to the cytosol, where its reduction to cysteine supplies this limiting amino acid for diverse fundamental processes and controlling nutrient adaptation. Cystinosin mutations cause cystinosis, a devastating lysosomal storage disease. Here, we present structures of human cystinosin in lumen-open, cytosol-open, and cystine-bound states, which uncover the cystine recognition mechanism and capture the key conformational states of the transport cycle. Our structures, along with functional studies and double electron-electron resonance spectroscopic investigations, reveal the molecular basis for the transporter's conformational transitions and protonation switch, show conformation-dependent Ragulator-Rag complex engagement, and demonstrate an unexpected activation mechanism. These findings provide molecular insights into lysosomal amino acid efflux and a potential therapeutic strategy.

Expression, Purification, and Structure Determination of Human PTCH1-HH-N Complexes.

Patched-1 (PTCH1), a tumor suppressor, serves as the receptor of Hedgehog (HH) ligand and negatively regulates the HH signaling pathway. Mutations of PTCH1 are implicated in many human cancers. Structural investigation revealed the mechanism of PTCH1-mediated HH signal regulation, further facilitating the therapeutic development of cancers. Here, we describe the expression and purification of a nearly full-length functional PTCH1 variant, PTCH1*. With purified PTCH1* protein, two forms of PTCH1*-Sonic Hedgehog (SHH) complexes were assembled, and their structures subsequently determined by cryo-electron microscope (cryo-EM).

Atomic insights into ML-SI3 mediated human TRPML1 inhibition.

Transient receptor potential mucolipin 1 (TRPML1) regulates lysosomal calcium signaling, lipid trafficking, and autophagy-related processes. This channel is regulated by phosphoinositides and the low pH environment of the lysosome, maintaining calcium levels essential for proper lysosomal function. Recently, several small molecules specifically targeting the TRPML family have been demonstrated to modulate channel activity. One of these, a synthetic antagonist ML-SI3, can prevent lysosomal calcium efflux and has been reported to block downstream TRPML1-mediated induction of autophagy. Here, we report a cryo-electron microscopy structure of human TRPML1 with ML-SI3 at 2.9-Å resolution. ML-SI3 binds to the hydrophobic cavity created by S5, S6, and PH1, the same cavity where the synthetic agonist ML-SA1 binds. Electrophysiological characterizations show that ML-SI3 can compete with ML-SA1, blocking channel activation yet does not inhibit PI(3,5)P2-dependent activation of the channel. Consequently, this work provides molecular insight into how ML-SI3 and native lipids regulate TRPML1 activity.

Marked structural rearrangement of mannose 6-phosphate/IGF2 receptor at different pH environments.

Many cell surface receptors internalize their ligands and deliver them to endosomes, where the acidic pH causes the ligand to dissociate. The liberated receptor returns to the cell surface in a process called receptor cycling. The structural basis for pH-dependent ligand dissociation is not well understood. In some receptors, the ligand binding domain is composed of multiple repeated sequences. The insulin-like growth factor 2 receptor (IGF2R) contains 15 ß strand-rich repeat domains. The overall structure and the mechanism by which IGF2R binds IGF2 and releases it are unknown. We used cryo-EM to determine the structures of the IGF2R at pH 7.4 with IGF2 bound and at pH 4.5 in the ligand-dissociated state. The results reveal different arrangements of the receptor in different pH environments mediated by changes in the interactions between the repeated sequences. These results have implications for our understanding of ligand release from receptors in endocytic compartments.

Structural basis for PtdInsP2-mediated human TRPML1 regulation.

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.

Two Patched molecules engage distinct sites on Hedgehog yielding a signaling-competent complex.

Aberrant Hedgehog (HH) signaling leads to various types of cancer and birth defects. N-terminally palmitoylated HH initiates signaling by binding its receptor Patched-1 (PTCH1). A recent 1:1 PTCH1-HH complex structure visualized a palmitate-mediated binding site on HH, which was inconsistent with previous studies that implied a distinct, calcium-mediated binding site for PTCH1 and HH co-receptors. Our 3.5-angstrom resolution cryo-electron microscopy structure of native Sonic Hedgehog (SHH-N) in complex with PTCH1 at a physiological calcium concentration reconciles these disparate findings and demonstrates that one SHH-N molecule engages both epitopes to bind two PTCH1 receptors in an asymmetric manner. Functional assays using PTCH1 or SHH-N mutants that disrupt the individual interfaces illustrate that simultaneous engagement of both interfaces is required for efficient signaling in cells.

Structures of human Patched and its complex with native palmitoylated sonic hedgehog.

Hedgehog (HH) signalling governs embryogenesis and adult tissue homeostasis in mammals and other multicellular organisms1-3. Whereas deficient HH signalling leads to birth defects, unrestrained HH signalling is implicated in human cancers2,4-6. N-terminally palmitoylated HH releases the repression of Patched to the oncoprotein smoothened (SMO); however, the mechanism by which HH recognizes Patched is unclear. Here we report cryo-electron microscopy structures of human patched 1 (PTCH1) alone and in complex with the N-terminal domain of 'native' sonic hedgehog (native SHH-N has both a C-terminal cholesterol and an N-terminal fatty-acid modification), at resolutions of 3.5 Å and 3.8 Å, respectively. The structure of PTCH1 has internal two-fold pseudosymmetry in the transmembrane core, which features a sterol-sensing domain and two homologous extracellular domains, resembling the architecture of Niemann-Pick C1 (NPC1) protein7. The palmitoylated N terminus of SHH-N inserts into a cavity between the extracellular domains of PTCH1 and dominates the PTCH1-SHH-N interface, which is distinct from that reported for SHH-N co-receptors8. Our biochemical assays show that SHH-N may use another interface, one that is required for its co-receptor binding, to recruit PTCH1 in the absence of a covalently attached palmitate. Our work provides atomic insights into the recognition of the N-terminal domain of HH (HH-N) by PTCH1, offers a structural basis for cooperative binding of HH-N to various receptors and serves as a molecular framework for HH signalling and its malfunction in disease.

The regulatory mechanism of mammalian TRPMLs revealed by cryo-EM.

Transient receptor potential mucolipin (TRPML) channels are the most recently identified subfamily of TRP channels and have seen a surge of new reports revealing both structural and functional insight. In 2017, several groups published multiple conformations of TRPML channels using cryo-EM. Similar to other TRP channels, the ML subfamily consists of six transmembrane helices (S1-S6), and a pore region including S5, S6, and two pore helices (PH1 and PH2). However, these reports also reveal distinct structural characteristics of the ML subfamily. Asp residues within the luminal pore may function to control calcium/pH regulation. A synthetic agonist, ML-SA1, can bind to the pore region of TRPMLs to force a direct dilation of the lower gate. Finally, biophysical and electrophysiological characterizations reveal another natural agonist binding site in the unique domain of TRPMLs, presumably regulating the conformation of the S4-S5 linker to open the channel. This work elucidates the molecular architecture and provides insights into how multiple ligands regulate TRPMLs.

Human TRPML1 channel structures in open and closed conformations.

Transient receptor potential mucolipin 1 (TRPML1) is a Ca2+-releasing cation channel that mediates the calcium signalling and homeostasis of lysosomes. Mutations in TRPML1 lead to mucolipidosis type IV, a severe lysosomal storage disorder. Here we report two electron cryo-microscopy structures of full-length human TRPML1: a 3.72-Å apo structure at pH 7.0 in the closed state, and a 3.49-Å agonist-bound structure at pH 6.0 in an open state. Several aromatic and hydrophobic residues in pore helix 1, helices S5 and S6, and helix S6 of a neighbouring subunit, form a hydrophobic cavity to house the agonist, suggesting a distinct agonist-binding site from that found in TRPV1, a TRP channel from a different subfamily. The opening of TRPML1 is associated with distinct dilations of its lower gate together with a slight structural movement of pore helix 1. Our work reveals the regulatory mechanism of TRPML channels, facilitates better understanding of TRP channel activation, and provides insights into the molecular basis of mucolipidosis type IV pathogenesis.

3.3 Å structure of Niemann-Pick C1 protein reveals insights into the function of the C-terminal luminal domain in cholesterol transport.

Niemann-Pick C1 (NPC1) and NPC2 proteins are indispensable for the export of LDL-derived cholesterol from late endosomes. Mutations in these proteins result in Niemann-Pick type C disease, a lysosomal storage disease. Despite recent reports of the NPC1 structure depicting its overall architecture, the function of its C-terminal luminal domain (CTD) remains poorly understood even though 45% of NPC disease-causing mutations are in this domain. Here, we report a crystal structure at 3.3 Å resolution of NPC1* (residues 314-1,278), which-in contrast to previous lower resolution structures-features the entire CTD well resolved. Notably, all eight cysteines of the CTD form four disulfide bonds, one of which (C909-C914) enforces a specific loop that in turn mediates an interaction with a loop of the N-terminal domain (NTD). Importantly, this loop and its interaction with the NTD were not observed in any previous structures due to the lower resolution. Our mutagenesis experiments highlight the physiological relevance of the CTD-NTD interaction, which might function to keep the NTD in the proper orientation for receiving cholesterol from NPC2. Additionally, this structure allows us to more precisely map all of the disease-causing mutations, allowing future molecular insights into the pathogenesis of NPC disease.

Structural and biochemical analyses of the DEAD-box ATPase Sub2 in association with THO or Yra1.

mRNA is cotranscrptionally processed and packaged into messenger ribonucleoprotein particles (mRNPs) in the nucleus. Prior to export through the nuclear pore, mRNPs undergo several obligatory remodeling reactions. In yeast, one of these reactions involves loading of the mRNA-binding protein Yra1 by the DEAD-box ATPase Sub2 as assisted by the hetero-pentameric THO complex. To obtain molecular insights into reaction mechanisms, we determined crystal structures of two relevant complexes: a THO hetero-pentamer bound to Sub2 at 6.0 Å resolution; and Sub2 associated with an ATP analogue, RNA, and a C-terminal fragment of Yra1 (Yra1-C) at 2.6 Å resolution. We found that the 25 nm long THO clamps Sub2 in a half-open configuration; in contrast, when bound to the ATP analogue, RNA and Yra1-C, Sub2 assumes a closed conformation. Both THO and Yra1-C stimulated Sub2's intrinsic ATPase activity. We propose that THO surveys common landmarks in each nuclear mRNP to localize Sub2 for targeted loading of Yra1.