G protein ß subunits regulate Cav3.3 T-type channel activity and current kinetics via interaction with the Cav3.3 C-terminus.

Ca2+ influx through Cav3.3 T-type channel plays crucial roles in neuronal excitability and is subject to regulation by various signaling molecules. However, our understanding of the partners of Cav3.3 and the related regulatory pathways remains largely limited. To address this quest, we employed the rat Cav3.3 C-terminus as bait in yeast-two-hybrid screenings of a cDNA library, identifying rat Gß2 as an interaction partner. Subsequent assays revealed that the interaction of Gß2 subunit was specific to the Cav3.3 C-terminus. Through systematic dissection of the C-terminus, we pinpointed a 22 amino acid sequence (amino acids 1789-1810) as the Gß2 interaction site. Coexpression studies of rat Cav3.3 with various Gßγ compositions were conducted in HEK-293 cells. Patch clamp recordings revealed that coexpression of Gß2γ2 reduced Cav3.3 current density and accelerated inactivation kinetics. Interestingly, the effects were not unique to Gß2γ2, but were mimicked by Gß2 alone as well as other Gßγ dimers, with similar potencies. Deletion of the Gß2 interaction site abolished the effects of Gß2γ2. Importantly, these Gß2 effects were reproduced in human Cav3.3. Overall, our findings provide evidence that Gß(γ) complexes inhibit Cav3.3 channel activity and accelerate the inactivation kinetics through the Gß interaction with the Cav3.3 C-terminus.

A unifying mechanism for seipin-mediated lipid droplet formation.

Lipid droplets (LDs) are dynamic organelles essential for cellular lipid homeostasis. Assembly of LDs occurs in the endoplasmic reticulum (ER), and the conserved ER membrane protein seipin emerged as a key player in this process. Here, we review recent advances provided by structural, biochemical, and in silico analysis that revealed mechanistic insights into the molecular role of the seipin complexes and led to an updated model for LD biogenesis. We further discuss how other ER components cooperate with seipin during LD biogenesis. Understanding the molecular mechanisms underlying seipin-mediated LD assembly is important to uncover the fundamental aspects of lipid homeostasis and organelle biogenesis and to provide hints on the pathogenesis of lipid storage disorders.

Molecular dissection of PI3Kß synergistic activation by receptor tyrosine kinases, GßGγ, and Rho-family GTPases.

Phosphoinositide 3-kinase (PI3K) beta (PI3Kß) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kß prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kß localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kß when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kß membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kß prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GßGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kß to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GßGγ or pY/Rac1(GTP), PI3Kß activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kß is synergistically activated by pY/GßGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.

Molecular basis for Gßγ-mediated activation of phosphoinositide 3-kinase γ.

The conversion of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-triphosphate by phosphoinositide 3-kinase γ (PI3Kγ) is critical for neutrophil chemotaxis and cancer metastasis. PI3Kγ is activated by Gßγ heterodimers released from G protein-coupled receptors responding to extracellular signals. Here we determined cryo-electron microscopy structures of Sus scrofa PI3Kγ-human Gßγ complexes in the presence of substrates/analogs, revealing two Gßγ binding sites: one on the p110γ helical domain and another on the p101 C-terminal domain. Comparison with PI3Kγ alone reveals conformational changes in the kinase domain upon Gßγ binding that are similar to Ras·GTP-induced changes. Assays of variants perturbing the Gßγ binding sites and interdomain contacts altered by Gßγ binding suggest that Gßγ recruits the enzyme to membranes and allosterically regulates activity via both sites. Studies of zebrafish neutrophil migration align with these findings, paving the way for in-depth investigation of Gßγ-mediated activation mechanisms in this enzyme family and drug development for PI3Kγ.

Structure of adenylyl cyclase 5 in complex with Gßγ offers insights into ADCY5-related dyskinesia.

The nine different membrane-anchored adenylyl cyclase isoforms (AC1-9) in mammals are stimulated by the heterotrimeric G protein, Gαs, but their response to Gßγ regulation is isoform specific. In the present study, we report cryo-electron microscope structures of ligand-free AC5 in complex with Gßγ and a dimeric form of AC5 that could be involved in its regulation. Gßγ binds to a coiled-coil domain that links the AC transmembrane region to its catalytic core as well as to a region (C1b) that is known to be a hub for isoform-specific regulation. We confirmed the Gßγ interaction with both purified proteins and cell-based assays. Gain-of-function mutations in AC5 associated with human familial dyskinesia are located at the interface of AC5 with Gßγ and show reduced conditional activation by Gßγ, emphasizing the importance of the observed interaction for motor function in humans. We propose a molecular mechanism wherein Gßγ either prevents dimerization of AC5 or allosterically modulates the coiled-coil domain, and hence the catalytic core. As our mechanistic understanding of how individual AC isoforms are uniquely regulated is limited, studies such as this may provide new avenues for isoform-specific drug development.

Seipin forms a flexible cage at lipid droplet formation sites.

Lipid droplets (LDs) form in the endoplasmic reticulum by phase separation of neutral lipids. This process is facilitated by the seipin protein complex, which consists of a ring of seipin monomers, with a yet unclear function. Here, we report a structure of S. cerevisiae seipin based on cryogenic-electron microscopy and structural modeling data. Seipin forms a decameric, cage-like structure with the lumenal domains forming a stable ring at the cage floor and transmembrane segments forming the cage sides and top. The transmembrane segments interact with adjacent monomers in two distinct, alternating conformations. These conformations result from changes in switch regions, located between the lumenal domains and the transmembrane segments, that are required for seipin function. Our data indicate a model for LD formation in which a closed seipin cage enables triacylglycerol phase separation and subsequently switches to an open conformation to allow LD growth and budding.

Mechanism of lipid droplet formation by the yeast Sei1/Ldb16 Seipin complex.

Lipid droplets (LDs) are universal lipid storage organelles with a core of neutral lipids, such as triacylglycerols, surrounded by a phospholipid monolayer. This unique architecture is generated during LD biogenesis at endoplasmic reticulum (ER) sites marked by Seipin, a conserved membrane protein mutated in lipodystrophy. Here structural, biochemical and molecular dynamics simulation approaches reveal the mechanism of LD formation by the yeast Seipin Sei1 and its membrane partner Ldb16. We show that Sei1 luminal domain assembles a homooligomeric ring, which, in contrast to other Seipins, is unable to concentrate triacylglycerol. Instead, Sei1 positions Ldb16, which concentrates triacylglycerol within the Sei1 ring through critical hydroxyl residues. Triacylglycerol recruitment to the complex is further promoted by Sei1 transmembrane segments, which also control Ldb16 stability. Thus, we propose that LD assembly by the Sei1/Ldb16 complex, and likely other Seipins, requires sequential triacylglycerol-concentrating steps via distinct elements in the ER membrane and lumen.

A double bilayer to study the nonequilibrium environmental response of GIRK2 in complex states.

G protein-gated inwardly rectifying potassium (GIRK) channels play essential roles in electrical signaling in neurons and muscle cells. Nonequilibrium environments provide crucial driving forces behind many cellular events. Here, we apply the antiparallel alignment double bilayer model to study GIRK2 in response to the time-dependent membrane potential. Using molecular dynamics and umbrella sampling, we examined the time-dependent environmental impact on the ion conduction, energy basis, and primary motions of GIRK2 in different complex states with phosphatidylinositol-4,5-bisphosphate (PIP2) and G-protein ßγ subunits (Gßγ). The antiparallel alignment double bilayer model enables us to study the transport performance in inward and outward K+ and mixed K+ and Na+. We obtained the recoverable discharge process of GIRK2 complexed with both PIP2 and Gßγ, compared with occasional conduction under PIP2-only regulation. Calculations of potential of mean force suggest different regulation by the helix bundle crossing (HBC) gate and G-loop gate regarding different complex states and under a membrane potential. In a nonequilibrium environment, distinct functional rocking motions of GIRK2 were identified under strengthened correlations between the transmembrane helices and downstream cytoplasmic domains with binding of PIP2, cations, and Gßγ. The findings suggest the potential domain motions and dynamics associated with a nonequilibrium environment and highlight the application of the antiparallel alignment double bilayer model to investigate factors in an asymmetric environment.

Seipin accumulates and traps diacylglycerols and triglycerides in its ring-like structure.

Lipid droplets (LDs) are intracellular organelles responsible for lipid storage, and they emerge from the endoplasmic reticulum (ER) upon the accumulation of neutral lipids, mostly triglycerides (TG), between the two leaflets of the ER membrane. LD biogenesis takes place at ER sites that are marked by the protein seipin, which subsequently recruits additional proteins to catalyze LD formation. Deletion of seipin, however, does not abolish LD biogenesis, and its precise role in controlling LD assembly remains unclear. Here, we use molecular dynamics simulations to investigate the molecular mechanism through which seipin promotes LD formation. We find that seipin clusters TG, as well as its precursor diacylglycerol, inside its unconventional ring-like oligomeric structure and that both its luminal and transmembrane regions contribute to this process. This mechanism is abolished upon mutations of polar residues involved in protein-TG interactions into hydrophobic residues. Our results suggest that seipin remodels the membrane of specific ER sites to prime them for LD biogenesis.

Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs.

G-protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR-G protein complexes, few are in a lipid membrane environment. Here, we report cryo-EM structures of complexes of neurotensin, neurotensin receptor 1 and Gαi1ß1γ1 in two conformational states, resolved to resolutions of 4.1 and 4.2 Å. The structures, determined in a lipid bilayer without any stabilizing antibodies or nanobodies, reveal an extended network of protein-protein interactions at the GPCR-G protein interface as compared to structures obtained in detergent micelles. The findings show that the lipid membrane modulates the structure and dynamics of complex formation and provide a molecular explanation for the stronger interaction between GPCRs and G proteins in lipid bilayers. We propose an allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling.

Seipin traps triacylglycerols to facilitate their nanoscale clustering in the endoplasmic reticulum membrane.

Seipin is a disk-like oligomeric endoplasmic reticulum (ER) protein important for lipid droplet (LD) biogenesis and triacylglycerol (TAG) delivery to growing LDs. Here we show through biomolecular simulations bridged to experiments that seipin can trap TAGs in the ER bilayer via the luminal hydrophobic helices of the protomers delineating the inner opening of the seipin disk. This promotes the nanoscale sequestration of TAGs at a concentration that by itself is insufficient to induce TAG clustering in a lipid membrane. We identify Ser166 in the α3 helix as a favored TAG occupancy site and show that mutating it compromises the ability of seipin complexes to sequester TAG in silico and to promote TAG transfer to LDs in cells. While the S166D-seipin mutant colocalizes poorly with promethin, the association of nascent wild-type seipin complexes with promethin is promoted by TAGs. Together, these results suggest that seipin traps TAGs via its luminal hydrophobic helices, serving as a catalyst for seeding the TAG cluster from dissolved monomers inside the seipin ring, thereby generating a favorable promethin binding interface.

Structure of the class D GPCR Ste2 dimer coupled to two G proteins.

G-protein-coupled receptors (GPCRs) are divided phylogenetically into six classes1,2, denoted A to F. More than 370 structures of vertebrate GPCRs (belonging to classes A, B, C and F) have been determined, leading to a substantial understanding of their function3. By contrast, there are no structures of class D GPCRs, which are found exclusively in fungi where they regulate survival and reproduction. Here we determine the structure of a class D GPCR, the Saccharomyces cerevisiae pheromone receptor Ste2, in an active state coupled to the heterotrimeric G protein Gpa1-Ste4-Ste18. Ste2 was purified as a homodimer coupled to two G proteins. The dimer interface of Ste2 is formed by the N terminus, the transmembrane helices H1, H2 and H7, and the first extracellular loop ECL1. We establish a class D1 generic residue numbering system (CD1) to enable comparisons with orthologues and with other GPCR classes. The structure of Ste2 bears similarities in overall topology to class A GPCRs, but the transmembrane helix H4 is shifted by more than 20 Å and the G-protein-binding site is a shallow groove rather than a cleft. The structure provides a template for the design of novel drugs to target fungal GPCRs, which could be used to treat numerous intractable fungal diseases4.

The structural basis for an on-off switch controlling Gßγ-mediated inhibition of TRPM3 channels.

TRPM3 channels play important roles in the detection of noxious heat and in inflammatory thermal hyperalgesia. The activity of these ion channels in somatosensory neurons is tightly regulated by µ-opioid receptors through the signaling of Gßγ proteins, thereby reducing TRPM3-mediated pain. We show here that Gßγ directly binds to a domain of 10 amino acids in TRPM3 and solve a cocrystal structure of this domain together with Gßγ. Using these data and mutational analysis of full-length proteins, we pinpoint three amino acids in TRPM3 and their interacting partners in Gß1 that are individually necessary for TRPM3 inhibition by Gßγ. The 10-amino-acid Gßγ-interacting domain in TRPM3 is subject to alternative splicing. Its inclusion in or exclusion from TRPM3 channel proteins therefore provides a mechanism for switching on or off the inhibitory action that Gßγ proteins exert on TRPM3 channels.

Sterols in an intramolecular channel of Smoothened mediate Hedgehog signaling.

Smoothened (SMO), a class Frizzled G protein-coupled receptor (class F GPCR), transduces the Hedgehog signal across the cell membrane. Sterols can bind to its extracellular cysteine-rich domain (CRD) and to several sites in the seven transmembrane helices (7-TMs) of SMO. However, the mechanism by which sterols regulate SMO via multiple sites is unknown. Here we determined the structures of SMO-Gi complexes bound to the synthetic SMO agonist (SAG) and to 24(S),25-epoxycholesterol (24(S),25-EC). A novel sterol-binding site in the extracellular extension of TM6 was revealed to connect other sites in 7-TMs and CRD, forming an intramolecular sterol channel from the middle side of 7-TMs to CRD. Additional structures of two gain-of-function variants, SMOD384R and SMOG111C/I496C, showed that blocking the channel at its midpoints allows sterols to occupy the binding sites in 7-TMs, thereby activating SMO. These data indicate that sterol transport through the core of SMO is a major regulator of SMO-mediated signaling.

Oligomerization of A. thaliana Heterotrimeric G Protein Subunits AGB1 and AGG2 In Vitro.

Plant heterotrimeric G proteins are a major group of signaling molecules involved in regulation of critical processes including stress adaptation, seed size, grain quality and immune responses. Despite an abundance of in situ functional studies; purification of the individual subunits of the plant heterotrimer for biophysical and structural characterization and for studies on their interactions are lacking. In this study cloning of the genes encoding the ß subunit AGB1 of A. thaliana and its γ-subunits AGG1 and AGG2 using different E. coli expression vectors and screening of expression in several strains are reported. AGB1 could be expressed albeit at very low levels and in all cases it was accompanied by overexpression of E. coli chaperone proteins. AGG1 could only be detected in inclusion body fractions, whereas AGG2 was obtained in soluble fractions and was purified. Purified AGB1 and AGG2 subunits were shown to dimerize in vitro. Further characterization of AGG2 by small angle X-ray scattering measurements and by dynamic light scattering revealed that AGG2 formed homodimers with extended shape in solution. These results are also consistent with those from circular dichroism spectroscopy which yielded 39.4% helical and 50% random coil content for AGG2. This is the first study showing heterologous expression of a plant heterotrimeric G protein ß subunit individually and presenting its interaction with a plant γ-subunit in vitro. Results also show that the AGG2 subunit has a disordered structure, which would account for its role in diverse interactions for establishing selectivity in signal propagation.

Activation of Phospholipase C ß by Gßγ and Gαq Involves C-Terminal Rearrangement to Release Autoinhibition.

Phospholipase C (PLC) enzymes hydrolyze phosphoinositide lipids to inositol phosphates and diacylglycerol. Direct activation of PLCß by Gαq and/or Gßγ subunits mediates signaling by Gq and some Gi coupled G-protein-coupled receptors (GPCRs), respectively. PLCß isoforms contain a unique C-terminal extension, consisting of proximal and distal C-terminal domains (CTDs) separated by a flexible linker. The structure of PLCß3 bound to Gαq is known, however, for both Gαq and Gßγ; the mechanism for PLCß activation on membranes is unknown. We examined PLCß2 dynamics on membranes using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Gßγ caused a robust increase in dynamics of the distal C-terminal domain (CTD). Gαq showed decreased deuterium incorporation at the Gαq binding site on PLCß. In vitro Gßγ-dependent activation of PLC is inhibited by the distal CTD. The results suggest that disruption of autoinhibitory interactions with the CTD leads to increased PLCß hydrolase activity.

Come a little bit closer! Lipid droplet-ER contact sites are getting crowded.

Not so long ago, contact sites between the endoplasmic reticulum (ER) and lipid droplets (LDs) were largely unexplored on a molecular level. In recent years however, numerous proteins have been identified that are enriched or exclusively located at the interfaces between LDs and the ER. These comprise members of protein classes typically found in diverse types of contacts, such as organelle tethers and lipid transfer proteins, but also proteins that have no similarities to known contact site machineries. This structurally heterogeneous group of contact site residents might be required to fulfill unique aspects of LD-ER contact biology, such as de novo LD biogenesis, and maintenance of lipidic connections between LDs and ER. Here, we summarize the current knowledge on the molecular components of this special organelle contact site, and discuss their features and functions.

Systematic analysis of F-box proteins reveals a new branch of the yeast mating pathway.

The mating pathway in yeast Saccharomyces cerevisiae has long been used to reveal new mechanisms of signal transduction. The pathway comprises a pheromone receptor, a heterotrimeric G protein, and intracellular effectors of morphogenesis and transcription. Polarized cell growth, in the direction of a potential mating partner, is accomplished by the G-protein ßγ subunits and the small G-protein Cdc42. Transcription induction, needed for cell-cell fusion, is mediated by Gßγ and the mitogen-activated protein kinase (MAPK) scaffold protein Ste5. A potential third pathway is initiated by the G-protein α subunit Gpa1. Gpa1 signaling was shown previously to involve the F-box adaptor protein Dia2 and an endosomal effector protein, the phosphatidylinositol 3-kinase Vps34. Vps34 is also required for proper vacuolar sorting and autophagy. Here, using a panel of reporter assays, we demonstrate that mating pheromone stimulates vacuolar targeting of a cytoplasmic reporter protein and that this process depends on Vps34. Through a systematic analysis of F-box deletion mutants, we show that Dia2 is required to sustain pheromone-induced vacuolar targeting. We also found that other F-box proteins selectively regulate morphogenesis (Ydr306, renamed Pfu1) and transcription (Ucc1). These findings point to the existence of a new and distinct branch of the pheromone-signaling pathway, one that likely leads to vacuolar engulfment of cytoplasmic proteins and recycling of cellular contents in preparation for mating.

Structural basis for KCTD-mediated rapid desensitization of GABAB signalling.

The GABAB (γ-aminobutyric acid type B) receptor is one of the principal inhibitory neurotransmitter receptors in the brain, and it signals through heterotrimeric G proteins to activate a variety of effectors, including G-protein-coupled inwardly rectifying potassium channels (GIRKs)1,2. GABAB-receptor signalling is tightly regulated by auxiliary subunits called KCTDs, which control the kinetics of GIRK activation and desensitization3-5. However, the mechanistic basis for KCTD modulation of GABAB signalling remains incompletely understood. Here, using a combination of X-ray crystallography, electron microscopy, and functional and biochemical experiments, we reveal the molecular details of KCTD binding to both GABAB receptors and G-protein ßγ subunits. KCTDs associate with the receptor by forming an asymmetric pentameric ring around a region of the receptor carboxy-terminal tail, while a second KCTD domain, H1, engages in a symmetric interaction with five copies of Gßγ in which the G-protein subunits also interact directly with one another. We further show that KCTD binding to Gßγ is highly cooperative, defining a model in which KCTD proteins cooperatively strip G proteins from GIRK channels to induce rapid desensitization following receptor activation. These results provide a framework for understanding the molecular basis for the precise temporal control of GABAB signalling by KCTD proteins.

Identification of Heterotrimeric G Protein γ3 Subunit in Rice Plasma Membrane.

Heterotrimeric G proteins are important molecules for regulating plant architecture and transmitting external signals to intracellular target proteins in higher plants and mammals. The rice genome contains one canonical α subunit gene (RGA1), four extra-large GTP-binding protein genes (XLGs), one canonical ß subunit gene (RGB1), and five γ subunit genes (tentatively named RGG1, RGG2, RGG3/GS3/Mi/OsGGC1, RGG4/DEP1/DN1/OsGGC3, and RGG5/OsGGC2). RGG1 encodes the canonical γ subunit; RGG2 encodes the plant-specific type of γ subunit with additional amino acid residues at the N-terminus; and the remaining three γ subunit genes encode the atypical γ subunits with cysteine abundance at the C-terminus. We aimed to identify the RGG3/GS3/Mi/OsGGC1 gene product, Gγ3, in rice tissues using the anti-Gγ3 domain antibody. We also analyzed the truncated protein, Gγ3∆Cys, in the RGG3/GS3/Mi/OsGGC1 mutant, Mi, using the anti-Gγ3 domain antibody. Based on nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, the immunoprecipitated Gγ3 candidates were confirmed to be Gγ3. Similar to α (Gα) and ß subunits (Gß), Gγ3 was enriched in the plasma membrane fraction, and accumulated in the flower tissues. As RGG3/GS3/Mi/OsGGC1 mutants show the characteristic phenotype in flowers and consequently in seeds, the tissues that accumulated Gγ3 corresponded to the abnormal tissues observed in RGG3/GS3/Mi/OsGGC1 mutants.