Search

Summary Protein secretion mediated by SecYEG translocon and SecA ATPase is enhanced by membrane-embedded SecDF by using proton motive force. A previous structural study of SecDF indicated that it comprises 12 transmembrane helices that can conduct protons and three periplasmic domains, which form at least two characterized transition states, termed the F and I forms. We report the structures of full-length SecDF in I form at 2.6- to 2.8-Å resolution. The structures revealed that SecDF in I form can generate a tunnel that penetrates the transmembrane region and functions as a proton pathway regulated by a conserved Asp residue of the transmembrane region. In one crystal structure, periplasmic cavity interacts with a molecule, potentially polyethylene glycol, which may mimic a substrate peptide. This study provides structural insights into the Sec protein translocation that allows future analyses to develop a more detailed working model for SecDF. [ABSTRACT FROM AUTHOR]

Summary The bacterial SecYEG translocon functions as a conserved protein-conducting channel. Conformational transitions of SecYEG allow protein translocation across the membrane without perturbation of membrane permeability. Here, we report the crystal structures of intact SecYEG at 2.7-Å resolution and of peptide-bound SecYEG at 3.6-Å resolution. The higher-resolution structure revealed that the cytoplasmic loop of SecG covers the hourglass-shaped channel, which was confirmed to also occur in the membrane by disulfide bond formation analysis and molecular dynamics simulation. The cytoplasmic loop may be involved in protein translocation. In addition, the previously unknown peptide-bound crystal structure of SecYEG implies that interactions between the cytoplasmic side of SecY and signal peptides are related to lateral gate opening at the first step of protein translocation. These SecYEG structures therefore provide a number of structural insights into the Sec machinery for further study. [ABSTRACT FROM AUTHOR]

Abstract: Although thromboxane (TX)A2 is involved in allergic rhinitis, the mechanisms inducing nasal blockage have not been elucidated. We evaluated the roles of nasal mucosal vascular changes following intranasal instillation of the TXA2 analog U-46619 or leukotriene (LT)D4 to induce nasal blockage in a guinea pig model of allergic rhinitis. Both U-46619- and LTD4-induced nasal blockages in sensitized animals were swiftly and completely suppressed by a vasoconstrictor, naphazoline. The nitric oxide synthase inhibitor N ω-nitro-l-arginine methyl ester relieved LTD4-induced nasal blockage, but not U-46619-induced nasal blockage. Although both agonists produced vasodilatation of nasal mucosa in vivo, LTD4 caused vasodilatation while U-46619 caused vasoconstriction in vitro. Both LTD4- and U-46619-induced nasal blockages in vivo should depend on vasodilatation of nasal mucosa. LTD4-induced nasal blockage is induced by direct vasodilatation via nitric oxide. In contrast, U-46619-induced nasal blockage may be associated with contraction of a certain vein that should exist at the exit of capacitance vessels, leading to congestion of the nasal mucosa. [Copyright &y& Elsevier]

With the widespread use of digital pen tablets and touch displays, sketch-based interactive computer-aided design (CAD) user interfaces have attracted considerable attention. In an existing interface called the Sketch Input Tracer (SKIT), however, the editing operation performed via an over-sketching stroke can target only a single geometric object at any given time. To overcome this limitation, in this study, we propose a new general-purpose sketch-based interface developed for use in two-dimensional CAD systems. In the proposed interface, a sketch-based editing operation can simultaneously modify the geometry and topology of multiple geometric objects via over-sketching. The interface was developed based on the fuzzy logic-based strategy of SKIT. With this interface, a user can draw in a creative manner; for example, they can begin with a rough sketch and progressively create a detailed design through repeated over-sketches. The results of experiments conducted demonstrate that experts can control the proposed interactive drawing interface and beginners can habituate to controlling it. [Display omitted] • A new general-purpose sketch-based interface for 2D CAD systems is proposed. • The interface is based on the SKIT fuzzy-logic strategy. • An editing operation via over-sketching can modify multiple geometric objects. • Users can repeatedly over-sketch on a rough drawing to obtain a detailed design. • Adequate controllability by trained users of the interface is shown quantitatively. [ABSTRACT FROM AUTHOR]

Sarco/endoplasmic reticulum (ER) Ca2+-ATPase 2b (SERCA2b) is a ubiquitously expressed membrane protein that facilitates Ca2+ uptake from the cytosol to the ER. SERCA2b includes a characteristic 11th transmembrane helix (TM11) followed by a luminal tail, but the structural basis of SERCA regulation by these C-terminal segments remains unclear. Here, we determined the crystal structures of SERCA2b and its C-terminal splicing variant SERCA2a, both in the E1-2Ca2+-adenylyl methylenediphosphonate (AMPPCP) state. Despite discrepancies with the previously reported structural model of SERCA2b, TM11 was found to be located adjacent to TM10 and to interact weakly with a part of the L8/9 loop and the N-terminal end of TM10, thereby inhibiting the SERCA2b catalytic cycle. Accordingly, mutational disruption of the interactions between TM11 and its neighboring residues caused SERCA2b to display SERCA2a-like ATPase activity. We propose that TM11 serves as a key modulator of SERCA2b activity by fine-tuning the intramolecular interactions with other transmembrane regions. • Crystal structure was solved for human SERCA2b and SERCA2a in E1-2Ca2+-AMPPCP state • The location and orientation of TM11 helix characteristic of SERCA2b were revealed • Amino acid residues involved in interaction with TM11 were identified • Molecular mechanisms of TM11-mediated SERCA2b regulation are proposed Inoue et al. present a crystal structure of human SERCA2b, a ubiquitously expressed Ca2+-ATPase that facilitates Ca2+ uptake into the endoplasmic reticulum and thereby plays central roles in intracellular calcium homeostasis. The structural and biochemical analyses clarify molecular mechanisms of SERCA2b regulation via TM helix interplay. [ABSTRACT FROM AUTHOR]

Summary The multidrug and toxic compound extrusion (MATE) family of proteins consists of transporters responsible for multidrug resistance in prokaryotes. In plants, a number of MATE proteins were identified by recent genomic and functional studies, which imply that the proteins have substrate-specific transport functions instead of multidrug extrusion. The three-dimensional structure of eukaryotic MATE proteins, including those of plants, has not been reported, preventing a better understanding of the molecular mechanism of these proteins. Here, we describe the crystal structure of a MATE protein from the plant Camelina sativa at 2.9 Å resolution. Two sets of six transmembrane α helices, assembled pseudo-symmetrically, possess a negatively charged internal pocket with an outward-facing shape. The crystal structure provides insight into the diversity of plant MATE proteins and their substrate recognition and transport through the membrane. [ABSTRACT FROM AUTHOR]

The bacterial peptidoglycan enclosing the cytoplasmic membrane is a fundamental cellular architecture. The integral membrane protein MurJ plays an essential role in flipping the cell wall building block Lipid II across the cytoplasmic membrane for peptidoglycan biosynthesis. Previously reported crystal structures of MurJ have elucidated its V-shaped inward- or outward-facing forms with an internal cavity for substrate binding. MurJ transports Lipid II using its cavity through conformational transitions between these two forms. Here, we report two crystal structures of inward-facing forms from Arsenophonus endosymbiont MurJ and an unprecedented crystal structure of Escherichia coli MurJ in a "squeezed" form, which lacks a cavity to accommodate the substrate, mainly because of the increased proximity of transmembrane helices 2 and 8. Subsequent molecular dynamics simulations supported the hypothesis that the squeezed form is an intermediate conformation. This study fills a gap in our understanding of the Lipid II flipping mechanism. [Display omitted] • Crystal structures of bacterial MurJ reveal its squeezed and inward forms • The squeezed form does not contain an internal cavity required for substrate binding • Transmembrane helices 2, 7, and 8 contribute to MurJ structural transitions • The squeezed form occurs during outward-to-inward conformational changes Kohga et al. present an unprecedented "squeezed" structure of MurJ, a Lipid II flippase that is essential for peptidoglycan biogenesis in gram-negative bacteria. The "squeezed" form is proposed to be an intermediate during outward-to-inward conformational changes after substrate release, which is further supported by molecular dynamics simulations. [ABSTRACT FROM AUTHOR]

Summary Multidrug and toxic compound extrusion (MATE) transporters efflux toxic compounds using a Na+ or H+ gradient across the membrane. Although the structures of MATE transporters have been reported, the cation-coupled substrate transport mechanism remains controversial. Here we report crystal structures of VcmN, a Vibrio cholerae MATE transporter driven by the H+ gradient. High-resolution structures in two distinct conformations associated with different pHs revealed that the rearrangement of the hydrogen-bonding network around the conserved Asp35 induces the bending of transmembrane helix 1, as in the case of the H+-coupled Pyrococcus furiosus MATE transporter. We also determined the crystal structure of the D35N mutant, which captured a unique conformation of TM1 facilitated by an altered hydrogen-bonding network. Based on the present results, we propose a common step in the transport cycle shared among prokaryotic H+-coupled MATE transporters. Graphical Abstract Highlights • Crystal structures of VcmN, an H+-coupled MATE transporter from Vibrio cholerae • Distinct conformations associated with different pHs and the D35N mutation in TM1 • Coupling of rearrangement of hydrogen bond network around Asp35 to the TM1 bending • Common step in the transport cycle shared among prokaryotic H+-driven MATE proteins Kusakizako et al. determined the crystal structures of VcmN, an H+-coupled MATE transporter from Vibrio cholerae. A structural comparison of distinct conformations associated with different pH conditions and the D35N mutation in TM1 reveals the common step in the transport cycle shared among prokaryotic H+-coupled MATE transporters. [ABSTRACT FROM AUTHOR]

Toll-like receptor (TLR) 4 mediates inflammation and is also known to trigger apoptosis in microglia. Our time-lapse observations showed that lipopolysaccharide (LPS) stimulation induced rapid death in primary cultures of rat microglia, while a portion of the microglia escaped from death and survived for much longer than 2 days, in which time, all of the control cells had died. However, it remains unclear how the LPS-stimulated microglia subpopulation could continue to survive in the absence of any supplied growth factors. In the present study, to clarify the mechanism underlying the LPS-stimulated survival, we investigated whether microglia could produce their own survival factors in response to LPS, focusing on macrophage colony-stimulating factor (M-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-34, which are mainly supplied by astrocytes or neurons. The LPS-stimulated microglia drastically induced the expression of the GM-CSF mRNA and protein, while M-CSF and IL-34 levels were unchanged. The surviving microglia also significantly upregulated the expression of GM-CSF receptor (GM-CSFR) mRNA without affecting M-CSFR. As for the GM-CSFR downstream signal, LPS resulted in the phosphorylation of STAT5 and its translocation to the nucleus in the surviving microglia. Moreover, a specific JAK2 inhibitor, NVP-BSK805, suppressed STAT5 phosphorylation and microglia survival in response to LPS, indicating a critical role of the JAK2/STAT5 pathway in this survival mechanism. Together, these results suggest that a subpopulation of TLR4-activated microglia may survive by producing GM-CSF and up-regulating GM-CSFR. This autocrine GM-CSF pathway may activate the JAK2/STAT5 signaling pathway, which controls the transcription of survival-related genes. Finally, these surviving microglia may have neuroprotective functions because the neurons remained viable in co-cultures with these microglia. [ABSTRACT FROM AUTHOR]

Summary: The FeoB family proteins are widely distributed prokaryotic membrane proteins involved in Fe2+ uptake. FeoB consists of N-terminal cytosolic and C-terminal transmembrane domains. The N-terminal region of the cytosolic domain is homologous to small GTPase (G) proteins and is considered to regulate Fe2+ uptake. The spacer region connecting the G and TM domains reportedly functions as a GDP dissociation inhibitor (GDI)–like domain that stabilizes the GDP-binding state. However, the function of the G and GDI-like domains in iron uptake remains unclear. Here, we report the structural and functional analyses of the FeoB cytosolic domain from Thermotoga maritima. The structure-based mutational analysis indicated that the interaction between the G and GDI-like domains is important for both the GDI and Fe2+ uptake activities. On the basis of these results, we propose a regulatory mechanism of Fe2+ uptake. [Copyright &y& Elsevier]

Summary Membrane proteins play important roles in various cellular functions. To analyze membrane proteins, nanodisc technology using membrane scaffold proteins allows single membrane protein units to be embedded into the lipid bilayer disc without detergents. Recent advancements in high-speed atomic force microscopy (HS-AFM) have enabled us to monitor the real-time dynamics of proteins in solution at the nanometer scale. In this study, we report HS-AFM imaging of membrane proteins reconstituted into nanodiscs using two membrane protein complexes, SecYEG complex and MgtE dimer. The observed images showed single particles of membrane protein-embedded nanodiscs in an end-up orientation whereby the membrane was fixed parallel to the supporting solid surface and in a side-on orientation whereby the membrane plane was vertically fixed to the solid surface, enabling the elucidation of domain fluctuations in membrane proteins. This technique provides a basic method for the high-resolution imaging of single membrane proteins by HS-AFM. Graphical Abstract Highlights • Membrane protein-embedded nanodiscs were used for high-speed (HS)-AFM observation • Nanodiscs in an end-up and side-on orientation were observed by HS-AFM in real time • HS-AFM combined with nanodiscs is a powerful tool for membrane protein dynamics Haruyama et al. established a procedure for the observation of membrane proteins in nanodiscs by high-speed AFM in real time. Using the two-dimensional crystal of streptavidin as well as mica as a solid support surface, dynamic structural changes of membrane proteins in the lipid environment were visualized from two distinct angles. [ABSTRACT FROM AUTHOR]

Summary The membrane protein SecDF, belonging to the RND superfamily, enhances protein translocation at the extracytoplasmic side using a proton gradient. Here, we report the crystal structure of SecDF in a form we named Super-membrane-facing ( Super F ) form, demonstrating a β-barrel architecture instead of the previously reported β-sheet structure. Through this structural insight and supporting results of an in vivo crosslinking experiment, we propose a remote coupling model in which a structural change of the transmembrane region drives a functional, extracytoplasmic conformational transition. [ABSTRACT FROM AUTHOR]