Structural basis of respiratory complexes adaptation to cold temperatures.

In response to cold, mammals activate brown fat for respiratory-dependent thermogenesis reliant on the electron transport chain (1, 2). Yet, the structural basis of respiratory complex adaptation to cold remains elusive. Herein we combined thermoregulatory physiology and cryo-EM to study endogenous respiratory supercomplexes exposed to different temperatures. A cold-induced conformation of CI:III 2 (termed type 2) was identified with a ∼25° rotation of CIII 2 around its inter-dimer axis, shortening inter-complex Q exchange space, and exhibiting different catalytic states which favor electron transfer. Large-scale supercomplex simulations in lipid membrane reveal how unique lipid-protein arrangements stabilize type 2 complexes to enhance catalytic activity. Together, our cryo-EM studies, multiscale simulations and biochemical analyses unveil the mechanisms and dynamics of respiratory adaptation at the structural and energetic level.

Auto-inhibition and activation of a short Argonaute-associated TIR-APAZ defense system.

Short prokaryotic Ago accounts for most prokaryotic Argonaute proteins (pAgos) and is involved in defending bacteria against invading nucleic acids. Short pAgo associated with TIR-APAZ (SPARTA) has been shown to oligomerize and deplete NAD+ upon guide-mediated target DNA recognition. However, the molecular basis of SPARTA inhibition and activation remains unknown. In this study, we determined the cryogenic electron microscopy structures of Crenotalea thermophila SPARTA in its inhibited, transient and activated states. The SPARTA monomer is auto-inhibited by its acidic tail, which occupies the guide-target binding channel. Guide-mediated target binding expels this acidic tail and triggers substantial conformational changes to expose the Ago-Ago dimerization interface. As a result, SPARTA assembles into an active tetramer, where the four TIR domains are rearranged and packed to form NADase active sites. Together with biochemical evidence, our results provide a panoramic vision explaining SPARTA auto-inhibition and activation and expand understanding of pAgo-mediated bacterial defense systems.

Analysing an allelic series of rare missense variants of CACNA1I in a Swedish schizophrenia cohort.

CACNA1I is implicated in the susceptibility to schizophrenia by large-scale genetic association studies of single nucleotide polymorphisms. However, the channelopathy of CACNA1I in schizophrenia is unknown. CACNA1I encodes CaV3.3, a neuronal voltage-gated calcium channel that underlies a subtype of T-type current that is important for neuronal excitability in the thalamic reticular nucleus and other regions of the brain. Here, we present an extensive functional characterization of 57 naturally occurring rare and common missense variants of CACNA1I derived from a Swedish schizophrenia cohort of more than 10 000 individuals. Our analysis of this allelic series of coding CACNA1I variants revealed that reduced CaV3.3 channel current density was the dominant phenotype associated with rare CACNA1I coding alleles derived from control subjects, whereas rare CACNA1I alleles from schizophrenia patients encoded CaV3.3 channels with altered responses to voltages. CACNA1I variants associated with altered current density primarily impact the ionic channel pore and those associated with altered responses to voltage impact the voltage-sensing domain. CaV3.3 variants associated with altered voltage dependence of the CaV3.3 channel and those associated with peak current density deficits were significantly segregated across affected and unaffected groups (Fisher's exact test, P = 0.034). Our results, together with recent data from the SCHEMA (Schizophrenia Exome Sequencing Meta-Analysis) cohort, suggest that reduced CaV3.3 function may protect against schizophrenia risk in rare cases. We subsequently modelled the effect of the biophysical properties of CaV3.3 channel variants on thalamic reticular nucleus excitability and found that compared with common variants, ultrarare CaV3.3-coding variants derived from control subjects significantly decreased thalamic reticular nucleus excitability (P = 0.011). When all rare variants were analysed, there was a non-significant trend between variants that reduced thalamic reticular nucleus excitability and variants that either had no effect or increased thalamic reticular nucleus excitability across disease status. Taken together, the results of our functional analysis of an allelic series of >50 CACNA1I variants in a schizophrenia cohort reveal that loss of function of CaV3.3 is a molecular phenotype associated with reduced disease risk burden, and our approach may serve as a template strategy for channelopathies in polygenic disorders.

TRP Channels as Emerging Therapeutic Targets for Neurodegenerative Diseases.

The development of treatment for neurodegenerative diseases (NDs) such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis is facing medical challenges due to the increasingly aging population. However, some pharmaceutical companies have ceased the development of therapeutics for NDs, and no new treatments for NDs have been established during the last decade. The relationship between ND pathogenesis and risk factors has not been completely elucidated. Herein, we review the potential involvement of transient receptor potential (TRP) channels in NDs, where oxidative stress and disrupted Ca2+ homeostasis consequently lead to neuronal apoptosis. Reactive oxygen species (ROS) -sensitive TRP channels can be key risk factors as polymodal sensors, since progressive late onset with secondary pathological damage after initial toxic insult is one of the typical characteristics of NDs. Recent evidence indicates that the dysregulation of TRP channels is a missing link between disruption of Ca2+ homeostasis and neuronal loss in NDs. In this review, we discuss the latest findings regarding TRP channels to provide insights into the research and quests for alternative therapeutic candidates for NDs. As the structures of TRP channels have recently been revealed by cryo-electron microscopy, it is necessary to develop new TRP channel antagonists and reevaluate existing drugs.

Analysis of interaction between intracellular spermine and transient receptor potential canonical 4 channel: multiple candidate sites of negatively charged amino acids for the inward rectification of transient receptor potential canonical 4.

Transient receptor potential canonical 4 (TRPC4) channel is a nonselective calcium-permeable cation channels. In intestinal smooth muscle cells, TRPC4 currents contribute more than 80% to muscarinic cationic current (mIcat). With its inward-rectifying current-voltage relationship and high calcium permeability, TRPC4 channels permit calcium influx once the channel is opened by muscarinic receptor stimulation. Polyamines are known to inhibit nonselective cation channels that mediate the generation of mIcat. Moreover, it is reported that TRPC4 channels are blocked by the intracellular spermine through electrostatic interaction with glutamate residues (E728, E729). Here, we investigated the correlation between the magnitude of channel inactivation by spermine and the magnitude of channel conductance. We also found additional spermine binding sites in TRPC4. We evaluated channel activity with electrophysiological recordings and revalidated structural significance based on Cryo-EM structure, which was resolved recently. We found that there is no correlation between magnitude of inhibitory action of spermine and magnitude of maximum current of the channel. In intracellular region, TRPC4 attracts spermine at channel periphery by reducing access resistance, and acidic residues contribute to blocking action of intracellular spermine; channel periphery, E649; cytosolic space, D629, D649, and E687.

Differential PI(4,5)P2 sensitivities of TRPC4, C5 homomeric and TRPC1/4, C1/5 heteromeric channels.

Transient receptor potential canonical (TRPC) 4 and TRPC5 channels are modulated by the Gαq-PLC pathway. Since phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) maintains TRPC4 and TRPC5 channel function, the Gαq-PLC pathway inhibits channel activity by depleting PI(4,5)P2. Here we investigated the difference in PI(4,5)P2 sensitivity between homomeric and heteromeric TRPC channels. First, by using a Danio rerio voltage-sensing phosphatase (DrVSP), we show that PI(4,5)P2 dephosphorylation robustly inhibits TRPC4α, TRPC4ß, and TRPC5 homotetramer currents and also TRPC1/4α, TRPC1/4ß, and TRPC1/5 heterotetramer currents. Secondly, sensitivity of channels to PI(4,5)P2 dephosphorylation was suggested through the usage of FRET in combination with patch clamping. The sensitivity increased in the sequence TRPC4ß < TRPC4α < TRPC5 in homotetramers, whereas when forming heterotetramers with TRPC1, the sensitivity was approximately equal between the channels. Thirdly, we determined putative PI(4,5)P2 binding sites based on a TRPC4 prediction model. By neutralization of basic residues, we identified putative PI(4,5)P2 binding sites because the mutations reduced FRET to a PI(4,5)P2 sensor and reduced the current amplitude. Therefore, one functional TRPC4 has 8 pockets with the two main binding regions; K419, K664/R511, K518, H630. We conclude that TRPC1 channel function as a regulator in setting PI(4,5)P2 affinity for TRPC4 and TRPC5 that changes PI(4,5)P2 sensitivity.

Structural dynamics of the transaminase active site revealed by the crystal structure of a co-factor free omega-transaminase from Vibrio fluvialis JS17.

Omega (ω)-transaminase catalyzes the transfer of an amino group from a non-α position amino acid, or an amine compound with no carboxylic group, to an amino acceptor, and has been studied intensively because of its high potential utility in industry and pharmatheutics. The ω-transaminase from Vibrio fluvialis JS17 (Vfat) is an amine:pyruvate transaminase capable of the stereo-selective transamination of arylic chiral amines. This enzyme exhibits extraordinary enantio-selectivity, and has a rapid reaction rate for chiral amine substrates. In this study, we report the crystal structure of the apo form of Vfat. The overall structure of Vfat was typical of other class III aminotransferase exhibiting an N-terminal helical domain, a small domain, and a large domain. Interestingly, the two subunits of apo Vfat in the asymmetric unit had different structures. A comparison of the overall structure to other transaminases, revealed that the structures of the N-terminal helical domain and the large domain can be affected by cofactor occupancy, but the structural rearrangement in these regions can occur independently.

Molecular mechanism of constitutively active Rab11A was revealed by crystal structure of Rab11A S20V.

Rab11A is a small GTP-binding protein involved in the regulation of vesicle trafficking during recycling of endosomes. Substitution of S20 to V (S20V) at Rab11A inhibits the GTP hydrolysis activity of Rab11A. This mutation is known to be constitutively in an active form. Here, we report the crystal structure of the human Rab11A S20V mutant form complexed with GTP at a resolution of 2.4 Å. Without adding any substrate, Rab11A contained non-hydrolyzed natural substrate GTP in the nucleotide binding pocket with Mg(2+). In our observations, substituted V20 of Rab11A was found to interfere with proper localization of the water molecule, which mediated GTP hydrolysis, resulting in GTP being locked in an active form of Rab11A S20V.

Occupation of nucleotide in the binding pocket is critical to the stability of Rab11A.

The Ras superfamily of small G proteins is a family of guanosine triphosphatases (GTPases) and each GTPase has conserved amino acid sequences in the enzymatic active site that are responsible for specific interactions with GDP and GTP molecules. Rab GTPases, which belong to the Ras superfamily, are key regulators of intracellular vesicle trafficking via the recruitment of effector molecules. Here, we purified wild type, active mutant and inactive mutant of Rab11A. In this process, we found that the inactive mutant (Rab11A S25N) had low stability compared with wild type and other mutants. Further analysis revealed that the stability of Rab11A S25N is dependent on the occupation of GDP in the nucleotide binding pocket of the protein. We found that the stability of Rab11A S25N is affected by the presence of GDP, not other nucleotides, and is independent of pH or salt in FPLC buffer. Our results provide a better understanding of how GTPase can be stable under in vitro conditions without effector proteins and how proper substrate/cofactor coordination is crucial to the stability of Rab11A. Successful purification and proposed purification methods will provide a valuable guide for investigation of other small GTPase proteins.

Intracellular spermine blocks TRPC4 channel via electrostatic interaction with C-terminal negative amino acids.

Transient receptor potential canonical (TRPC) 4 channels are calcium-permeable, nonselective cation channels and are widely expressed in mammalian tissue, especially in the GI tract and brain. TRPC4 channels are known to be involved in neurogenic contraction of ileal smooth muscle cells via generating cationic current after muscarinic stimulation (muscarinic cationic current (mIcat)). Polyamines exist in numerous tissues and are believed to be involved in cell proliferation, differentiation, scar formation, wound healing, and carcinogenesis. Besides, physiological polyamines are essential to maintain inward rectification of cardiac potassium channels (Kir2.1). At membrane potentials more positive than equilibrium potential, intracellular polyamines plug the cytosolic surface of the Kir2.1 so that potassium ions cannot pass through the pore. Recently, it was reported that polyamines inhibit not only cardiac potassium channels but also nonselective cation channels that mediate the generation of mIcat. Here, we report that TRPC4, a definite mIcat mediator, is inhibited by intracellular spermine with great extent. The inhibition was specific to TRPC4 and TRPC5 channels but was not effective to TRPC1/4, TRPC1/5, and TRPC3 channels. For this inhibition to occur, we found that glutamates at 728th and 729th position of TRPC4 channels are essential whereby we conclude that spermine blocks the TRPC4 channel with electrostatic interaction between negative amino acids at the C-terminus of the channel.

A network perspective on unraveling the role of TRP channels in biology and disease.

Transient receptor potential (TRP) channels are a large family of non-selective cation channels that mediate numerous physiological and pathophysiological processes; however, still largely unknown are the underlying molecular mechanisms. With data generated on an unprecedented scale, network-based approaches have been revolutionizing the way in which we understand biology and disease, discover disease genes, and develop therapeutic strategies. These circumstances have created opportunities to encounter TRP channel research to data-intensive science. In this review, we provide an introduction of network-based approaches in biomedical science, describe the current state of TRP channel network biology, and discuss the future direction of TRP channel research. Network perspective will facilitate the discovery of latent roles and underlying mechanisms of TRP channels in biology and disease.

Crystallization and preliminary X-ray crystallographic studies of the coiled-coil domain of PIST.

PIST [PDZ (PSD-95, Discs-large and ZO-1) protein interacting specifically with TC10] functions as a regulator of membrane trafficking with Rab6A. Recently, the involvement of the fusion of PIST with ROS1 in cancer development has been identified. In this study, the coiled-coil domain of PIST, which is the domain responsible for interaction with Rab6A and fusion with ROS1, corresponding to amino acids 29-133, was overexpressed in Escherichia coli using engineered C-terminal His tags. The coiled-coil domain of PIST was then purified to homogeneity and crystallized at 293 K. Finally, X-ray diffraction data were collected to a resolution of 4.0 Å from a crystal belonging to the hexagonal space group P6(2)22 or P6(4)22, with unit-cell parameters a = b = 85.19, c = 240.09 Å, γ = 120.00°.

TRIP database 2.0: a manually curated information hub for accessing TRP channel interaction network.

Transient receptor potential (TRP) channels are a family of Ca(2+)-permeable cation channels that play a crucial role in biological and disease processes. To advance TRP channel research, we previously created the TRIP (TRansient receptor potential channel-Interacting Protein) Database, a manually curated database that compiles scattered information on TRP channel protein-protein interactions (PPIs). However, the database needs to be improved for information accessibility and data utilization. Here, we present the TRIP Database 2.0 (http://www.trpchannel.org) in which many helpful, user-friendly web interfaces have been developed to facilitate knowledge acquisition and inspire new approaches to studying TRP channel functions: 1) the PPI information found in the supplementary data of referred articles was curated; 2) the PPI summary matrix enables users to intuitively grasp overall PPI information; 3) the search capability has been expanded to retrieve information from 'PubMed' and 'PIE the search' (a specialized search engine for PPI-related articles); and 4) the PPI data are available as sif files for network visualization and analysis using 'Cytoscape'. Therefore, our TRIP Database 2.0 is an information hub that works toward advancing data-driven TRP channel research.

Crystallization and preliminary X-ray crystallographic studies of Rab6A'(Q72L): a GTP-locked form.

Rab6A, a member of the Ras superfamily of small G proteins, is involved in the regulation of vesicle trafficking, which is critical for endocytosis, cell differentiation and cell growth. Rab6A can exist in two isoforms termed Rab6A and Rab6A'. The substitution of Gln72 by Leu (Q72L) in the Rab6A family blocks GTP-hydrolysis activity, and this mutation usually causes the Rab6A protein to be in a constitutively active form. In this study, in order to understand the functional uniqueness of Rab6A' and the molecular mechanism of the control of activity by GTP and GDP from the crystal structure, a Rab6A'(Q72L) mutant form was overexpressed in Escherichia coli with an engineered N-terminal His tag. Rab6A'(Q72L) was then purified to homogeneity and crystallized at 293 K. X-ray diffraction data were collected to a resolution of 1.9 Å from a crystal belonging to space group P22(1)2(1) with unit-cell parameters a = 36.84, b = 96.78, c = 109.99 Å. The asymmetric unit was estimated to contain two molecules.

Crystal structure of Rab6A'(Q72L) mutant reveals unexpected GDP/Mg²âº binding with opened GTP-binding domain.

The Ras small G protein-superfamily is a family of GTP hydrolases whose activity is regulated by GTP/GDP binding states. Rab6A, a member of the Ras superfamily, is involved in the regulation of vesicle trafficking, which is critical for endocytosis, biosynthesis, secretion, cell differentiation and cell growth. Rab6A exists in two isoforms, termed RabA and Rab6A'. Substitution of Gln72 to Leu72 (Q72L) at Rab6 family blocks GTP hydrolysis activity and this mutation usually causes the Rab6 protein to be constitutively in an active form. Here, we report the crystal structure of the human Rab6A'(Q72L) mutant form at 1.9Å resolution. Unexpectedly, we found that Rab6A'(Q72L) possesses GDP/Mg(2+) in the GTP binding pockets, which is formed by a flexible switch I and switch II. Large conformational changes were also detected in the switch I and switch II regions. Our structure revealed that the non-hydrolysable, constitutively active form of Rab6A' can accommodate GDP/Mg(2+) in the open conformation.

TRIP Database: a manually curated database of protein-protein interactions for mammalian TRP channels.

Transient receptor potential (TRP) channels are a superfamily of Ca(2+)-permeable cation channels that translate cellular stimuli into electrochemical signals. Aberrant activity of TRP channels has been implicated in a variety of human diseases, such as neurological disorders, cardiovascular disease and cancer. To facilitate the understanding of the molecular network by which TRP channels are associated with biological and disease processes, we have developed the TRIP (TRansient receptor potential channel-Interacting Protein) Database (http://www.trpchannel.org), a manually curated database that aims to offer comprehensive information on protein-protein interactions (PPIs) of mammalian TRP channels. The TRIP Database was created by systematically curating 277 peer-reviewed literature; the current version documents 490 PPI pairs, 28 TRP channels and 297 cellular proteins. The TRIP Database provides a detailed summary of PPI data that fit into four categories: screening, validation, characterization and functional consequence. Users can find in-depth information specified in the literature on relevant analytical methods and experimental resources, such as gene constructs and cell/tissue types. The TRIP Database has user-friendly web interfaces with helpful features, including a search engine, an interaction map and a function for cross-referencing useful external databases. Our TRIP Database will provide a valuable tool to assist in understanding the molecular regulatory network of TRP channels.