Mechanisms of neurotransmitter transport and drug inhibition in human VMAT2.

Monoamine neurotransmitters such as dopamine and serotonin control important brain pathways, including movement, sleep, reward and mood1. Dysfunction of monoaminergic circuits has been implicated in various neurodegenerative and neuropsychiatric disorders2. Vesicular monoamine transporters (VMATs) pack monoamines into vesicles for synaptic release and are essential to neurotransmission3-5. VMATs are also therapeutic drug targets for a number of different conditions6-9. Despite the importance of these transporters, the mechanisms of substrate transport and drug inhibition of VMATs have remained elusive. Here we report cryo-electron microscopy structures of the human vesicular monoamine transporter VMAT2 in complex with the antichorea drug tetrabenazine, the antihypertensive drug reserpine or the substrate serotonin. Remarkably, the two drugs use completely distinct inhibition mechanisms. Tetrabenazine binds VMAT2 in a lumen-facing conformation, locking the luminal gating lid in an occluded state to arrest the transport cycle. By contrast, reserpine binds in a cytoplasm-facing conformation, expanding the vestibule and blocking substrate access. Structural analyses of VMAT2 also reveal the conformational changes following transporter isomerization that drive substrate transport into the vesicle. These findings provide a structural framework for understanding the physiology and pharmacology of neurotransmitter packaging by synaptic vesicular transporters.

Structural and functional insights into Spns2-mediated transport of sphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) is an important signaling sphingolipid that regulates the immune system, angiogenesis, auditory function, and epithelial and endothelial barrier integrity. Spinster homolog 2 (Spns2) is an S1P transporter that exports S1P to initiate lipid signaling cascades. Modulating Spns2 activity can be beneficial in treatments of cancer, inflammation, and immune diseases. However, the transport mechanism of Spns2 and its inhibition remain unclear. Here, we present six cryo-EM structures of human Spns2 in lipid nanodiscs, including two functionally relevant intermediate conformations that link the inward- and outward-facing states, to reveal the structural basis of the S1P transport cycle. Functional analyses suggest that Spns2 exports S1P via facilitated diffusion, a mechanism distinct from other MFS lipid transporters. Finally, we show that the Spns2 inhibitor 16d attenuates the transport activity by locking Spns2 in the inward-facing state. Our work sheds light on Spns2-mediated S1P transport and aids the development of advanced Spns2 inhibitors.

SPTSSA variants alter sphingolipid synthesis and cause a complex hereditary spastic paraplegia.

Sphingolipids are a diverse family of lipids with critical structural and signalling functions in the mammalian nervous system, where they are abundant in myelin membranes. Serine palmitoyltransferase, the enzyme that catalyses the rate-limiting reaction of sphingolipid synthesis, is composed of multiple subunits including an activating subunit, SPTSSA. Sphingolipids are both essential and cytotoxic and their synthesis must therefore be tightly regulated. Key to the homeostatic regulation are the ORMDL proteins that are bound to serine palmitoyltransferase and mediate feedback inhibition of enzymatic activity when sphingolipid levels become excessive. Exome sequencing identified potential disease-causing variants in SPTSSA in three children presenting with a complex form of hereditary spastic paraplegia. The effect of these variants on the catalytic activity and homeostatic regulation of serine palmitoyltransferase was investigated in human embryonic kidney cells, patient fibroblasts and Drosophila. Our results showed that two different pathogenic variants in SPTSSA caused a hereditary spastic paraplegia resulting in progressive motor disturbance with variable sensorineural hearing loss and language/cognitive dysfunction in three individuals. The variants in SPTSSA impaired the negative regulation of serine palmitoyltransferase by ORMDLs leading to excessive sphingolipid synthesis based on biochemical studies and in vivo studies in Drosophila. These findings support the pathogenicity of the SPTSSA variants and point to excessive sphingolipid synthesis due to impaired homeostatic regulation of serine palmitoyltransferase as responsible for defects in early brain development and function.

Molecular mechanism of substrate recognition by folate transporter SLC19A1.

Folate (vitamin B9) is the coenzyme involved in one-carbon transfer biochemical reactions essential for cell survival and proliferation, with its inadequacy causing developmental defects or severe diseases. Notably, mammalian cells lack the ability to de novo synthesize folate but instead rely on its intake from extracellular sources via specific transporters or receptors, among which SLC19A1 is the ubiquitously expressed one in tissues. However, the mechanism of substrate recognition by SLC19A1 remains unclear. Here we report the cryo-EM structures of human SLC19A1 and its complex with 5-methyltetrahydrofolate at 3.5-3.6 Å resolution and elucidate the critical residues for substrate recognition. In particular, we reveal that two variant residues among SLC19 subfamily members designate the specificity for folate. Moreover, we identify intracellular thiamine pyrophosphate as the favorite coupled substrate for folate transport by SLC19A1. Together, this work establishes the molecular basis of substrate recognition by this central folate transporter.

Specificity of TGF-ß1 signal designated by LRRC33 and integrin αVß8.

Myeloid lineage cells present the latent form of transforming growth factor-ß1 (L-TGF-ß1) to the membrane using an anchor protein LRRC33. Integrin αVß8 activates extracellular L-TGF-ß1 to trigger the downstream signaling functions. However, the mechanism designating the specificity of TGF-ß1 presentation and activation remains incompletely understood. Here, we report cryo-EM structures of human L-TGF-ß1/LRRC33 and integrin αVß8/L-TGF-ß1 complexes. Combined with biochemical and cell-based analyses, we demonstrate that LRRC33 only presents L-TGF-ß1 but not the -ß2 or -ß3 isoforms due to difference of key residues on the growth factor domains. Moreover, we reveal a 2:2 binding mode of integrin αVß8 and L-TGF-ß1, which shows higher avidity and more efficient L-TGF-ß1 activation than previously reported 1:2 binding mode. We also uncover that the disulfide-linked loop of the integrin subunit ß8 determines its exquisite affinity to L-TGF-ß1. Together, our findings provide important insights into the specificity of TGF-ß1 signaling achieved by LRRC33 and integrin αVß8.

Activation and closed-state inactivation mechanisms of the human voltage-gated KV4 channel complexes.

The voltage-gated ion channel activity depends on both activation (transition from the resting state to the open state) and inactivation. Inactivation is a self-restraint mechanism to limit ion conduction and is as crucial to membrane excitability as activation. Inactivation can occur when the channel is open or closed. Although open-state inactivation is well understood, the molecular basis of closed-state inactivation has remained elusive. We report cryo-EM structures of human KV4.2 channel complexes in inactivated, open, and closed states. Closed-state inactivation of KV4 involves an unprecedented symmetry breakdown for pore closure by only two of the four S4-S5 linkers, distinct from known mechanisms of open-state inactivation. We further capture KV4 in a putative resting state, revealing how voltage sensor movements control the pore. Moreover, our structures provide insights regarding channel modulation by KChIP2 and DPP6 auxiliary subunits. Our findings elucidate mechanisms of closed-state inactivation and voltage-dependent activation of the KV4 channel.

Childhood amyotrophic lateral sclerosis caused by excess sphingolipid synthesis.

Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disease of the lower and upper motor neurons with sporadic or hereditary occurrence. Age of onset, pattern of motor neuron degeneration and disease progression vary widely among individuals with ALS. Various cellular processes may drive ALS pathomechanisms, but a monogenic direct metabolic disturbance has not been causally linked to ALS. Here we show SPTLC1 variants that result in unrestrained sphingoid base synthesis cause a monogenic form of ALS. We identified four specific, dominantly acting SPTLC1 variants in seven families manifesting as childhood-onset ALS. These variants disrupt the normal homeostatic regulation of serine palmitoyltransferase (SPT) by ORMDL proteins, resulting in unregulated SPT activity and elevated levels of canonical SPT products. Notably, this is in contrast with SPTLC1 variants that shift SPT amino acid usage from serine to alanine, result in elevated levels of deoxysphingolipids and manifest with the alternate phenotype of hereditary sensory and autonomic neuropathy. We custom designed small interfering RNAs that selectively target the SPTLC1 ALS allele for degradation, leave the normal allele intact and normalize sphingolipid levels in vitro. The role of primary metabolic disturbances in ALS has been elusive; this study defines excess sphingolipid biosynthesis as a fundamental metabolic mechanism for motor neuron disease.

Molecular basis of V-ATPase inhibition by bafilomycin A1.

Pharmacological inhibition of vacuolar-type H+-ATPase (V-ATPase) by its specific inhibitor can abrogate tumor metastasis, prevent autophagy, and reduce cellular signaling responses. Bafilomycin A1, a member of macrolide antibiotics and an autophagy inhibitor, serves as a specific and potent V-ATPases inhibitor. Although there are many V-ATPase structures reported, the molecular basis of specific inhibitors on V-ATPase remains unknown. Here, we report the cryo-EM structure of bafilomycin A1 bound intact bovine V-ATPase at an overall resolution of 3.6-Å. The structure reveals six bafilomycin A1 molecules bound to the c-ring. One bafilomycin A1 molecule engages with two c subunits and disrupts the interactions between the c-ring and subunit a, thereby preventing proton translocation. Structural and sequence analyses demonstrate that the bafilomycin A1-binding residues are conserved in yeast and mammalian species and the 7'-hydroxyl group of bafilomycin A1 acts as a unique feature recognized by subunit c.

Structural insights into the regulation of human serine palmitoyltransferase complexes.

Sphingolipids are essential lipids in eukaryotic membranes. In humans, the first and rate-limiting step of sphingolipid synthesis is catalyzed by the serine palmitoyltransferase holocomplex, which consists of catalytic components (SPTLC1 and SPTLC2) and regulatory components (ssSPTa and ORMDL3). However, the assembly, substrate processing and regulation of the complex are unclear. Here, we present 8 cryo-electron microscopy structures of the human serine palmitoyltransferase holocomplex in various functional states at resolutions of 2.6-3.4 Å. The structures reveal not only how catalytic components recognize the substrate, but also how regulatory components modulate the substrate-binding tunnel to control enzyme activity: ssSPTa engages SPTLC2 and shapes the tunnel to determine substrate specificity. ORMDL3 blocks the tunnel and competes with substrate binding through its amino terminus. These findings provide mechanistic insights into sphingolipid biogenesis governed by the serine palmitoyltransferase complex.

The NAD+-mediated self-inhibition mechanism of pro-neurodegenerative SARM1.

Pathological degeneration of axons disrupts neural circuits and represents one of the hallmarks of neurodegeneration1-4. Sterile alpha and Toll/interleukin-1 receptor motif-containing protein 1 (SARM1) is a central regulator of this neurodegenerative process5-8, and its Toll/interleukin-1 receptor (TIR) domain exerts its pro-neurodegenerative action through NADase activity9,10. However, the mechanisms by which the activation of SARM1 is stringently controlled are unclear. Here we report the cryo-electron microscopy structures of full-length SARM1 proteins. We show that NAD+ is an unexpected ligand of the armadillo/heat repeat motifs (ARM) domain of SARM1. This binding of NAD+ to the ARM domain facilitated the inhibition of the TIR-domain NADase through the domain interface. Disruption of the NAD+-binding site or the ARM-TIR interaction caused constitutive activation of SARM1 and thereby led to axonal degeneration. These findings suggest that NAD+ mediates self-inhibition of this central pro-neurodegenerative protein.

Voltage Sensor Movements during Hyperpolarization in the HCN Channel.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channel is a voltage-gated cation channel that mediates neuronal and cardiac pacemaker activity. The HCN channel exhibits reversed voltage dependence, meaning it closes with depolarization and opens with hyperpolarization. Different from Na+, Ca2+, and Kv1-Kv7 channels, the HCN channel does not have domain-swapped voltage sensors. We introduced a reversible, metal-mediated cross bridge into the voltage sensors to create the chemical equivalent of a hyperpolarized conformation and determined the structure using cryoelectron microscopy (cryo-EM). Unlike the depolarized HCN channel, the S4 helix is displaced toward the cytoplasm by two helical turns. Near the cytoplasm, the S4 helix breaks into two helices, one running parallel to the membrane surface, analogous to the S4-S5 linker of domain-swapped voltage-gated channels. These findings suggest a basis for allosteric communication between voltage sensors and the gate in this kind of channel. They also imply that voltage sensor movements are not the same in all voltage-gated channels.

Activation mechanism of a human SK-calmodulin channel complex elucidated by cryo-EM structures.

Small-conductance Ca2+-activated K+ (SK) channels mediate neuron excitability and are associated with synaptic transmission and plasticity. They also regulate immune responses and the size of blood cells. Activation of SK channels requires calmodulin (CaM), but how CaM binds and opens SK channels has been unclear. Here we report cryo-electron microscopy (cryo-EM) structures of a human SK4-CaM channel complex in closed and activated states at 3.4- and 3.5-angstrom resolution, respectively. Four CaM molecules bind to one channel tetramer. Each lobe of CaM serves a distinct function: The C-lobe binds to the channel constitutively, whereas the N-lobe interacts with the S4-S5 linker in a Ca2+-dependent manner. The S4-S5 linker, which contains two distinct helices, undergoes conformational changes upon CaM binding to open the channel pore. These structures reveal the gating mechanism of SK channels and provide a basis for understanding SK channel pharmacology.

Mechanism of NMDA receptor channel block by MK-801 and memantine.

The NMDA (N-methyl-D-aspartate) receptor transduces the binding of glutamate and glycine, coupling it to the opening of a calcium-permeable ion channel 1 . Owing to the lack of high-resolution structural studies of the NMDA receptor, the mechanism by which ion-channel blockers occlude ion permeation is not well understood. Here we show that removal of the amino-terminal domains from the GluN1-GluN2B NMDA receptor yields a functional receptor and crystals with good diffraction properties, allowing us to map the binding site of the NMDA receptor blocker, MK-801. This crystal structure, together with long-timescale molecular dynamics simulations, shows how MK-801 and memantine (a drug approved for the treatment of Alzheimer's disease) bind within the vestibule of the ion channel, promote closure of the ion channel gate and lodge between the M3-helix-bundle crossing and the M2-pore loops, physically blocking ion permeation.

Structures of the Human HCN1 Hyperpolarization-Activated Channel.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels underlie the control of rhythmic activity in cardiac and neuronal pacemaker cells. In HCN, the polarity of voltage dependence is uniquely reversed. Intracellular cyclic adenosine monophosphate (cAMP) levels tune the voltage response, enabling sympathetic nerve stimulation to increase the heart rate. We present cryo-electron microscopy structures of the human HCN channel in the absence and presence of cAMP at 3.5 Å resolution. HCN channels contain a K+ channel selectivity filter-forming sequence from which the amino acids create a unique structure that explains Na+ and K+ permeability. The voltage sensor adopts a depolarized conformation, and the pore is closed. An S4 helix of unprecedented length extends into the cytoplasm, contacts the C-linker, and twists the inner helical gate shut. cAMP binding rotates cytoplasmic domains to favor opening of the inner helical gate. These structures advance understanding of ion selectivity, reversed polarity gating, and cAMP regulation in HCN channels.

Mechanism of NMDA Receptor Inhibition and Activation.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated, calcium-permeable ion channels that mediate synaptic transmission and underpin learning and memory. NMDAR dysfunction is directly implicated in diseases ranging from seizure to ischemia. Despite its fundamental importance, little is known about how the NMDAR transitions between inactive and active states and how small molecules inhibit or activate ion channel gating. Here, we report electron cryo-microscopy structures of the GluN1-GluN2B NMDA receptor in an ensemble of competitive antagonist-bound states, an agonist-bound form, and a state bound with agonists and the allosteric inhibitor Ro25-6981. Together with double electron-electron resonance experiments, we show how competitive antagonists rupture the ligand binding domain (LBD) gating "ring," how agonists retain the ring in a dimer-of-dimers configuration, and how allosteric inhibitors, acting within the amino terminal domain, further stabilize the LBD layer. These studies illuminate how the LBD gating ring is fundamental to signal transduction and gating in NMDARs.

Screening and large-scale expression of membrane proteins in mammalian cells for structural studies.

Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His8-tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI(-) (N-acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4-6 weeks.

NMDA receptor structures reveal subunit arrangement and pore architecture.

N-methyl-d-aspartate (NMDA) receptors are Hebbian-like coincidence detectors, requiring binding of glycine and glutamate in combination with the relief of voltage-dependent magnesium block to open an ion conductive pore across the membrane bilayer. Despite the importance of the NMDA receptor in the development and function of the brain, a molecular structure of an intact receptor has remained elusive. Here we present X-ray crystal structures of the Xenopus laevis GluN1-GluN2B NMDA receptor with the allosteric inhibitor, Ro25-6981, partial agonists and the ion channel blocker, MK-801. Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino-terminal and ligand-binding domains. The transmembrane domains harbour a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ∼twofold symmetric arrangement of ion channel pore loops. These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors.

Coactivation of multiple tightly coupled calcium channels triggers spontaneous release of GABA.

Voltage-activated Ca(2+) channels (VACCs) mediate Ca(2+) influx to trigger action potential-evoked neurotransmitter release, but the mechanism by which Ca(2+) regulates spontaneous transmission is unclear. We found that VACCs are the major physiological triggers for spontaneous release at mouse neocortical inhibitory synapses. Moreover, despite the absence of a synchronizing action potential, we found that spontaneous fusion of a GABA-containing vesicle required the activation of multiple tightly coupled VACCs of variable type.