Author Correction: Structural basis for the drug extrusion mechanism by a MATE multidrug transporter.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mammalian neurotoxins, Blarina paralytic peptides, cause hyperpolarization of human T-type Ca channel hCav3.2 activation.

Among the rare venomous mammals, the short-tailed shrew Blarina brevicauda has been suggested to produce potent neurotoxins in its saliva to effectively capture prey. Several kallikrein-like lethal proteases have been identified, but the active substances of B. brevicauda remained unclear. Here, we report Blarina paralytic peptides (BPPs) 1 and 2 isolated from its submaxillary glands. Synthetic BPP2 showed mealworm paralysis and a hyperpolarization shift (-11 mV) of a human T-type Ca2+ channel (hCav3.2) activation. The amino acid sequences of BPPs were similar to those of synenkephalins, which are precursors of brain opioid peptide hormones that are highly conserved among mammals. However, BPPs rather resembled centipede neurotoxic peptides SLPTXs in terms of disulfide bond connectivity and stereostructure. Our results suggested that the neurotoxin BPPs were the result of convergent evolution as homologs of nontoxic endogenous peptides that are widely conserved in mammals. This finding is of great interest from the viewpoint of the chemical evolution of vertebrate venoms.

The structure of MgtE in the absence of magnesium provides new insights into channel gating.

MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed-state, and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. There are no structures of the transmembrane (TM) domain for MgtE in Mg2+-free conditions, and the pore-opening mechanism has thus remained unclear. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE TM domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

Structural basis for Na(+) transport mechanism by a light-driven Na(+) pump.

Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na(+) pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na(+) transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na(+) transport. Together with the structure-based engineering of the first light-driven K(+) pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.

Id2 Represses Aldosterone-Stimulated Cardiac T-Type Calcium Channels Expression.

Aldosterone excess is a cardiovascular risk factor. Aldosterone can directly stimulate an electrical remodeling of cardiomyocytes leading to cardiac arrhythmia and hypertrophy. L-type and T-type voltage-gated calcium (Ca2+) channels expression are increased by aldosterone in cardiomyocytes. To further understand the regulation of these channels expression, we studied the role of a transcriptional repressor, the inhibitor of differentiation/DNA binding protein 2 (Id2). We found that aldosterone inhibited the expression of Id2 in neonatal rat cardiomyocytes and in the heart of adult mice. When Id2 was overexpressed in cardiomyocytes, we observed a reduction in the spontaneous action potentials rate and an arrest in aldosterone-stimulated rate increase. Accordingly, Id2 siRNA knockdown increased this rate. We also observed that CaV1.2 (L-type Ca2+ channel) or CaV3.1, and CaV3.2 (T-type Ca2+ channels) mRNA expression levels and Ca2+ currents were affected by Id2 presence. These observations were further corroborated in a heart specific Id2- transgenic mice. Taken together, our results suggest that Id2 functions as a transcriptional repressor for L- and T-type Ca2+ channels, particularly CaV3.1, in cardiomyocytes and its expression is controlled by aldosterone. We propose that Id2 might contributes to a protective mechanism in cardiomyocytes preventing the presence of channels associated with a pathological state.

Robo2 contains a cryptic binding site for neural EGFL-like (NELL) protein 1/2.

The signaling pathways that are mediated by Slit ligands and their Roundabout (Robo) family of receptors play multifunctional roles in the development of the nervous system and other organs. A recent study identified neural epidermal growth factor-like (NEL)-like 2 (NELL2) as a novel ligand for Robo3. In this study, we carried out a comprehensive analysis of the interaction between NELL1 and the Robo family of receptors and demonstrated that Robo2 contains a cryptic binding site for both NELL1 and NELL2. NELL1/2 binds to the first fibronectin type III (FNIII) domain of Robo2 but not to intact Robo2. Mutation analysis revealed that several amino acids within the first FNIII domain are critical for NELL1 binding to Robo2 but not to Robo1. The Robo2 deletion mutants without the fourth immunoglobulin domain and single amino acid substitution mutants that can influence the architecture of the ectodomain facilitated binding to NELL1/2. Acidic conditions increased the binding affinity of Robo2 for NELL1. These results suggest that Robo2 functions as a receptor for NELL1/2, particularly under circumstances where Robo2 undergoes proteolytic digestion. If this is not the case, conformational changes of the ectodomain of Robo2 may unmask the binding site for NELL1/2.

Structural basis of Sec-independent membrane protein insertion by YidC.

Newly synthesized membrane proteins must be accurately inserted into the membrane, folded and assembled for proper functioning. The protein YidC inserts its substrates into the membrane, thereby facilitating membrane protein assembly in bacteria; the homologous proteins Oxa1 and Alb3 have the same function in mitochondria and chloroplasts, respectively. In the bacterial cytoplasmic membrane, YidC functions as an independent insertase and a membrane chaperone in cooperation with the translocon SecYEG. Here we present the crystal structure of YidC from Bacillus halodurans, at 2.4 Å resolution. The structure reveals a novel fold, in which five conserved transmembrane helices form a positively charged hydrophilic groove that is open towards both the lipid bilayer and the cytoplasm but closed on the extracellular side. Structure-based in vivo analyses reveal that a conserved arginine residue in the groove is important for the insertion of membrane proteins by YidC. We propose an insertion mechanism for single-spanning membrane proteins, in which the hydrophilic environment generated by the groove recruits the extracellular regions of substrates into the low-dielectric environment of the membrane.

Structural basis for the drug extrusion mechanism by a MATE multidrug transporter.

Multidrug and toxic compound extrusion (MATE) family transporters are conserved in the three primary domains of life (Archaea, Bacteria and Eukarya), and export xenobiotics using an electrochemical gradient of H(+) or Na(+) across the membrane. MATE transporters confer multidrug resistance to bacterial pathogens and cancer cells, thus causing critical reductions in the therapeutic efficacies of antibiotics and anti-cancer drugs, respectively. Therefore, the development of MATE inhibitors has long been awaited in the field of clinical medicine. Here we present the crystal structures of the H(+)-driven MATE transporter from Pyrococcus furiosus in two distinct apo-form conformations, and in complexes with a derivative of the antibacterial drug norfloxacin and three in vitro selected thioether-macrocyclic peptides, at 2.1-3.0 Å resolutions. The structures, combined with functional analyses, show that the protonation of Asp 41 on the amino (N)-terminal lobe induces the bending of TM1, which in turn collapses the N-lobe cavity, thereby extruding the substrate drug to the extracellular space. Moreover, the macrocyclic peptides bind the central cleft in distinct manners, which correlate with their inhibitory activities. The strongest inhibitory peptide that occupies the N-lobe cavity may pave the way towards the development of efficient inhibitors against MATE transporters.

RBM20 Regulates CaV1.2 Surface Expression by Promoting Exon 9* Inclusion of CACNA1C in Neonatal Rat Cardiomyocytes.

The CACNA1C gene encodes for the CaV1.2 protein, which is the pore subunit of cardiac l-type voltage-gated calcium (Ca2+) channels (l-channels). Through alternative splicing, CACNA1C encodes for various CaV1.2 isoforms with different electrophysiological properties. Splice variants of CaV1.2 are differentially expressed during heart development or pathologies. The molecular mechanisms of CACNA1C alternative splicing still remain incompletely understood. RNA sequencing analysis has suggested that CACNA1C is a potential target of the splicing factor RNA-binding protein motif 20 (RBM20). Here, we aimed at elucidating the role of RBM20 in the regulation of CACNA1C alternative splicing. We found that in neonatal rat cardiomyocytes (NRCMs), RBM20 overexpression promoted the inclusion of CACNA1C's exon 9*, whereas the skipping of exon 9* occurred upon RBM20 siRNA knockdown. The splicing of other known alternative exons was not altered by RBM20. RNA immunoprecipitation suggested that RBM20 binds to introns flanking exon 9*. Functionally, in NRCMs, RBM20 overexpression decreased l-type Ca2+ currents, whereas RBM20 siRNA knockdown increased l-type Ca2+ currents. Finally, we found that RBM20 overexpression reduced CaV1.2 membrane surface expression in NRCMs. Taken together, our results suggest that RBM20 specifically regulates the inclusion of exon 9* in CACNA1C mRNA, resulting in reduced cell-surface membrane expression of l-channels in cardiomyocytes.

Crystal structure of the channelrhodopsin light-gated cation channel.

Channelrhodopsins (ChRs) are light-gated cation channels derived from algae that have shown experimental utility in optogenetics; for example, neurons expressing ChRs can be optically controlled with high temporal precision within systems as complex as freely moving mammals. Although ChRs have been broadly applied to neuroscience research, little is known about the molecular mechanisms by which these unusual and powerful proteins operate. Here we present the crystal structure of a ChR (a C1C2 chimaera between ChR1 and ChR2 from Chlamydomonas reinhardtii) at 2.3 Å resolution. The structure reveals the essential molecular architecture of ChRs, including the retinal-binding pocket and cation conduction pathway. This integration of structural and electrophysiological analyses provides insight into the molecular basis for the remarkable function of ChRs, and paves the way for the precise and principled design of ChR variants with novel properties.

Structure and function of a membrane component SecDF that enhances protein export.

Protein translocation across the bacterial membrane, mediated by the secretory translocon SecYEG and the SecA ATPase, is enhanced by proton motive force and membrane-integrated SecDF, which associates with SecYEG. The role of SecDF has remained unclear, although it is proposed to function in later stages of translocation as well as in membrane protein biogenesis. Here, we determined the crystal structure of Thermus thermophilus SecDF at 3.3 Å resolution, revealing a pseudo-symmetrical, 12-helix transmembrane domain belonging to the RND superfamily and two major periplasmic domains, P1 and P4. Higher-resolution analysis of the periplasmic domains suggested that P1, which binds an unfolded protein, undergoes functionally important conformational changes. In vitro analyses identified an ATP-independent step of protein translocation that requires both SecDF and proton motive force. Electrophysiological analyses revealed that SecDF conducts protons in a manner dependent on pH and the presence of an unfolded protein, with conserved Asp and Arg residues at the transmembrane interface between SecD and SecF playing essential roles in the movements of protons and preproteins. Therefore, we propose that SecDF functions as a membrane-integrated chaperone, powered by proton motive force, to achieve ATP-independent protein translocation.

Scaffold protein enigma homolog 1 overcomes the repression of myogenesis activation by inhibitor of DNA binding 2.

Enigma Homolog 1 (ENH1) is a scaffold protein for signaling proteins and transcription factors. Previously, we reported that ENH1 overexpression promotes the differentiation of C2C12 myoblasts. However, the molecular mechanism underlying the role of ENH1 in the C2C12 cells differentiation remains elusive. ENH1 was shown to inhibit the proliferation of neuroblastoma cells by sequestering Inhibitor of DNA binding protein 2 (Id2) in the cytosol. Id2 is a repressor of basic Helix-Loop-Helix transcription factors activity and prevents myogenesis. Here, we found that ENH1 overcome the Id2 repression of C2C12 cells myogenic differentiation and that ENH1 overexpression promotes mice satellite cells activation, the first step toward myogenic differentiation. In addition, we show that ENH1 interacted with Id2 in C2C12 cells and mice satellite cells. Collectively, our results suggest that ENH1 plays an important role in the activation of myogenesis through the repression of Id2 activity.

Oligomerization-induced conformational change in the C-terminal region of Nel-like molecule 1 (NELL1) protein is necessary for the efficient mediation of murine MC3T3-E1 cell adhesion and spreading.

NELL1 is a large oligomeric secretory glycoprotein that functions as an osteoinductive factor. NELL1 contains several conserved domains, has structural similarities to thrombospondin 1, and supports osteoblastic cell adhesion through integrins. To define the structural requirements for NELL1-mediated cell adhesion, we prepared a series of recombinant NELL1 proteins (intact, deleted, and cysteine-mutant) from a mammalian expression system and tested their activities. A deletion analysis demonstrated that the C-terminal cysteine-rich region of NELL1 is critical for the cell adhesion activity of NELL1. Reducing agent treatment decreased the cell adhesion activity of full-length NELL1 but not of its C-terminal fragments, suggesting that the intramolecular disulfide bonds within this region are not functionally necessary but that other disulfide linkages in the N-terminal region of NELL1 may be involved in cell adhesion activity. By replacing cysteine residues with serines around the coiled-coil domain of NELL1, which is responsible for oligomerization, we created a mutant NELL1 protein that was unable to form homo-oligomers, and this monomeric mutant showed substantially lower cell adhesion activity than intact NELL1. These results suggest that an oligomerization-induced conformational change in the C-terminal region of NELL1 is important for the efficient mediation of cell adhesion and spreading by NELL1.

Cell surface-fluorescence immunosorbent assay for real-time detection of hybridomas with efficient antibody secretion at the single-cell level.

For establishing cells that secrete antibodies most efficiently (e.g., hybridomas, CHO (Chinese hamster ovary) cells), the screening and subsequent breeding of promising cells have been performed at the single-colony level, which requires several weeks to propagate a substantial number of cells by forming colonies from single cells for evaluation by the conventional assays. However, this screening process lacks high-throughput performance in time and colony numbers. Therefore, development of novel methods is expected to identify single cells secreting higher amounts of antibodies in real-time and in a nondestructive manner without colony formation. In this study, we prepared lipid-labeled antimouse IgG Fc antibodies (capture molecules) that were uniformly displayed on the surface of candidate cells. Secreted nascent antibodies were subsequently sandwiched between capture molecules and fluorescence-labeled antimouse IgG F(ab')(2) F(ab')(2) (detection molecules). This newly developed method is hereinafter referred to as a cell surface-fluorescence immunosorbent assay (CS-FIA). The fluorescence intensity of each cell was found to correlate well with the amount of sandwiched antibodies (from 6.25 fg/cell to 6.40 pg/cell). When about 4 × 10(3) cells of mouse hybridomas were subjected to CS-FIA, we isolated 28 hybridomas showing the highest fluorescence intensity within a day. Furthermore, after propagation of single cells to about 10(5) cells (after 2 weeks), 20 hybridomas were still able to secrete higher amounts (up to 7-fold) of antibodies than parental hybridomas. Our results demonstrate that CS-FIA is a powerful method for the single-cell-based establishment of cells that secrete most efficiently not only antibodies but also various biomolecules.

Enigma homolog 1 promotes myogenic gene expression and differentiation of C2C12 cells.

The Enigma homolog (ENH) gene generates several splicing variants. The initially identified ENH1 possesses one PDZ and three LIM domains, whereas ENH2~4 lack the latter domains. The splicing switch from ENH1 to LIM-less ENHs occurs during development/maturation of skeletal and heart muscles. We examined for the roles of ENH splicing variants in muscle differentiation using C2C12 cells. Cells stably expressing ENH1 exhibited significantly higher MyoD and myogenin mRNA levels before differentiation and after 5 days in low serum-differentiating medium than mock-transfected cells. ENH1-stable transformants also retained the ability to exhibit elongated morphology with well-extended actin fibers following differentiation. In contrast, cells stably expressing ENH3 or ENH4 did not show myotube-like morphology or reorganization of actin fibers following culture in the differentiating medium. Transient overexpression of ENH1 using adenovirus supported the increased expression of muscle marker mRNAs and the formation of well-organized stress fibers, whereas ENH4 overexpression prevented these morphological changes. Furthermore, specific suppression of ENH1 expression by RNAi caused a significant reduction in MyoD mRNA level and blocked the morphological changes. These results suggest that ENH1 with multiple protein-protein interaction modules is essential for differentiation of striated muscles, whereas ectopic expression of LIM-less ENH disrupts normal muscle differentiation.

Splicing transitions of the anchoring protein ENH during striated muscle development.

The ENH (PDLIM5) protein acts as a scaffold to tether various functional proteins at subcellular sites via PDZ and three LIM domains. Splicing of the ENH primary transcript generates various products with different repertories of protein interaction modules. Three LIM-containing ENH predominates in neonatal cardiac tissue, whereas LIM-less ENHs are abundant in adult hearts, as well as skeletal muscles. Here we examine the timing of splicing transitions of ENH gene products during postnatal heart development and C2C12 myoblast differentiation. Real-time PCR analysis shows that LIM-containing ENH1 mRNA is gradually decreased during postnatal heart development, whereas transcripts with the short exon 5 appear in the late postnatal period and continues to increase until at least one month after birth. The splicing transition from LIM-containing ENH1 to LIM-less ENHs is also observed during the early period of C2C12 differentiation. This transition correlates with the emergence of ENH transcripts with the short exon 5, as well as the expression of myogenin mRNA. In contrast, the shift from the short exon 5 to the exon 7 occurs in the late differentiation period. The timing of this late event corresponds to the appearance of mRNA for the skeletal myosin heavy chain MYH4. Thus, coordinated and stepwise splicing transitions result in the production of specific ENH transcripts in mature striated muscles.

Conformational Change of the Hairpin-like-structured Robo2 Ectodomain Allows NELL1/2 Binding.

Since neural epidermal growth factor-like-like (NELL) 2 was identified as a novel ligand for the roundabout (Robo) 3 receptor, research on NELL-Robo signaling has become increasingly important. We have previously reported that Robo2 can bind to NELL1/2 in acidic conditions but not at neutral pH. The NELL1/2-binding site that is occluded in neutral conditions is thought to be exposed by a conformational change of the Robo2 ectodomain upon exposure to acidic pH; however, the underlying structural mechanisms are not well understood. Here, we investigated the interaction between the immunoglobulin-like domains and fibronectin type III domains that form hairpin-like structure of the Robo2 ectodomain, and demonstrated that acidic pH attenuates the interaction between them. Alternative splicing isoforms of Robo2, which affect the conformation of the hairpin-like structure, were found to have distinct NELL1/2-binding affinities. We developed Förster resonance energy transfer-based indicators for monitoring conformational change of the Robo2 ectodomain by individually inserting donor and acceptor fluorescent proteins at its ends. These experiments revealed that the ends of the Robo2 ectodomain are close to each other in acidic conditions. By combining these findings with the results of size exclusion chromatography analysis, we suggest that, in acidic conditions, the Robo2 ectodomain has a compact conformation with a loose hairpin-like structure. These results may help elucidate the signaling mechanisms resulting from the interaction between Robo2 and NELL1/2 in acidic conditions.

Cryo-EM structures of thylakoid-located voltage-dependent chloride channel VCCN1.

In the light reaction of plant photosynthesis, modulation of electron transport chain reactions is important to maintain the efficiency of photosynthesis under a broad range of light intensities. VCCN1 was recently identified as a voltage-gated chloride channel residing in the thylakoid membrane, where it plays a key role in photoreaction tuning to avoid the generation of reactive oxygen species (ROS). Here, we present the cryo-EM structures of Malus domestica VCCN1 (MdVCCN1) in nanodiscs and detergent at 2.7 Å and 3.0 Å resolutions, respectively, and the structure-based electrophysiological analyses. VCCN1 structurally resembles its animal homolog, bestrophin, a Ca2+-gated anion channel. However, unlike bestrophin channels, VCCN1 lacks the Ca2+-binding motif but instead contains an N-terminal charged helix that is anchored to the lipid membrane through an additional amphipathic helix. Electrophysiological experiments demonstrate that these structural elements are essential for the channel activity, thus revealing the distinct activation mechanism of VCCN1.

Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin.

Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore.

Functions of fasciculation and elongation protein zeta-1 (FEZ1) in the brain.

Fasciculation and elongation protein zeta-1 (FEZ1) is a mammalian ortholog of the Caenorhabditis elegans UNC-76 protein that possesses four coiled-coil domains and a nuclear localization signal. It is mainly expressed in the brain. Suppression of FEZ1 expression in cultured embryonic neurons causes deficiency of neuronal differentiation. Recently, proteomic techniques revealed that FEZ1 interacts with various intracellular partners, such as signaling, motor, and structural proteins. FEZ1 was shown to act as an antiviral factor. The findings reported so far indicate that FEZ1 is associated with neuronal development, neuropathologies, and viral infection. Based on these accumulating evidences, we herein review the biological functions of FEZ1.