scholarly journals Structural basis of pH-dependent client binding by ERp44, a key regulator of protein secretion at the ER–Golgi interface

2017 ◽  
Vol 114 (16) ◽  
pp. E3224-E3232 ◽  
Author(s):  
Satoshi Watanabe ◽  
Manami Harayama ◽  
Shingo Kanemura ◽  
Roberto Sitia ◽  
Kenji Inaba
Keyword(s):  
Conformational Changes ◽  
Low Ph ◽  
Structural Basis ◽  
Secretory Proteins ◽  
Hydrogen Bonding Pattern ◽  
Mixed Disulfides ◽  
Loop Regions ◽  
Ph Dependent ◽  
Α Helix ◽  
Dynamics Simulations

ERp44 retrieves some endoplasmic reticulum (ER)-resident enzymes and immature oligomers of secretory proteins from the Golgi. Association of ERp44 with its clients is regulated by pH-dependent mechanisms, but the molecular details are not fully understood. Here we report high-resolution crystal structures of human ERp44 at neutral and weakly acidic pH. These structures reveal key regions in the C-terminal tail (C tail) missing in the original crystal structure, including a regulatory histidine-rich region and a subsequent extended loop. The former region forms a short α-helix (α16), generating a histidine-clustered site (His cluster). At low pH, the three Trx-like domains of ERp44 (“a,” “b,” and “b′”) undergo significant rearrangements, likely induced by protonation of His157 located at the interface between the a and b domains. The α16-helix is partially unwound and the extended loop is disordered in weakly acidic conditions, probably due to electrostatic repulsion between the protonated histidines in the His cluster. Molecular dynamics simulations indicated that helix unwinding enhances the flexibility of the C tail, disrupting its normal hydrogen-bonding pattern. The observed pH-dependent conformational changes significantly enlarge the positively charged regions around the client-binding site of ERp44 at low pH. Mutational analyses showed that ERp44 forms mixed disulfides with specific cysteines residing on negatively charged loop regions of Ero1α. We propose that the protonation states of the essential histidines regulate the ERp44–client interaction by altering the C-tail dynamics and surface electrostatic potential of ERp44.

scholarly journals Structural Basis for pH-Gating of the K+ Channel TWIK1 at the Selectivity Filter

2021 ◽  
Author(s):  
Toby S Turney ◽  
Vivian Li ◽  
Stephen G Brohawn
Keyword(s):  
Conformational Changes ◽  
Pancreatic Beta Cells ◽  
Low Ph ◽  
Selectivity Filter ◽  
Structural Basis ◽  
K Channel ◽  
Open Conformation ◽  
Gating Mechanism ◽  
Lipid Nanodiscs ◽  
Pore Domain

TWIK1 is a widely expressed pH-gated two-pore domain K+ channel (K2P) that contributes to cardiac rhythm generation and insulin release from pancreatic beta cells. TWIK1 displays unique properties among K2Ps including low basal activity and inhibition by extracellular protons through incompletely understood mechanisms. Here, we present cryo-EM structures of TWIK1 in lipid nanodiscs at high and low pH that reveal a novel gating mechanism at the K+ selectivity filter. At high pH, TWIK1 adopts an open conformation. At low pH, protonation of an extracellular histidine results in a cascade of conformational changes that close the channel by sealing the top of the selectivity filter, displacing the helical cap to block extracellular ion access pathways, and opening gaps for lipid block of the intracellular cavity. These data provide a mechanistic understanding for extracellular pH-gating of TWIK1 and show how diverse mechanisms have evolved to gate the selectivity filter of K+ channels.

scholarly journals Proteomic analysis of monolayer-integrated proteins on lipid droplets identifies amphipathic interfacial α-helical membrane anchors

2018 ◽  
Vol 115 (35) ◽  
pp. E8172-E8180 ◽  
Author(s):  
Camille I. Pataki ◽  
João Rodrigues ◽  
Lichao Zhang ◽  
Junyang Qian ◽  
Bradley Efron ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Coarse Grained ◽  
Structural Basis ◽  
Endoplasmic Reticulum Membrane ◽  
Membrane Surfaces ◽  
Common Motif ◽  
Cysteine Scanning Mutagenesis ◽  
Α Helix ◽  
Dynamics Simulations ◽  
Integral Proteins

Despite not spanning phospholipid bilayers, monotopic integral proteins (MIPs) play critical roles in organizing biochemical reactions on membrane surfaces. Defining the structural basis by which these proteins are anchored to membranes has been hampered by the paucity of unambiguously identified MIPs and a lack of computational tools that accurately distinguish monolayer-integrating motifs from bilayer-spanning transmembrane domains (TMDs). We used quantitative proteomics and statistical modeling to identify 87 high-confidence candidate MIPs in lipid droplets, including 21 proteins with predicted TMDs that cannot be accommodated in these monolayer-enveloped organelles. Systematic cysteine-scanning mutagenesis showed the predicted TMD of one candidate MIP, DHRS3, to be a partially buried amphipathic α-helix in both lipid droplet monolayers and the cytoplasmic leaflet of endoplasmic reticulum membrane bilayers. Coarse-grained molecular dynamics simulations support these observations, suggesting that this helix is most stable at the solvent–membrane interface. The simulations also predicted similar interfacial amphipathic helices when applied to seven additional MIPs from our dataset. Our findings suggest that interfacial helices may be a common motif by which MIPs are integrated into membranes, and provide high-throughput methods to identify and study MIPs.

Structure of a double-domain phosphagen kinase reveals an asymmetric arrangement of the tandem domains

2015 ◽  
Vol 71 (4) ◽  
pp. 779-789 ◽  
Author(s):  
Zhiming Wang ◽  
Zhu Qiao ◽  
Sheng Ye ◽  
Rongguang Zhang
Keyword(s):  
Bound States ◽  
Hydrophobic Interactions ◽  
Crystal Packing ◽  
Protein Structures ◽  
Structural Basis ◽  
Phosphagen Kinase ◽  
Domain Arrangement ◽  
Α Helix ◽  
Dynamics Simulations ◽  
Shaped Domain

Tandem duplications and fusions of single genes have led to magnificent expansions in the divergence of protein structures and functions over evolutionary timescales. One of the possible results is polydomain enzymes with interdomain cooperativities, few examples of which have been structurally characterized at the full-length level to explore their innate synergistic mechanisms. This work reports the crystal structures of a double-domain phosphagen kinase in both apo and ligand-bound states, revealing a novel asymmetric L-shaped arrangement of the two domains. Unexpectedly, the interdomain connections are not based on a flexible hinge linker but on a rigid secondary-structure element: a long α-helix that tethers the tandem domains in relatively fixed positions. Besides the connective helix, the two domains also contact each other directly and form an interdomain interface in which hydrogen bonds and hydrophobic interactions further stabilize the L-shaped domain arrangement. Molecular-dynamics simulations show that the interface is generally stable, suggesting that the asymmetric domain arrangement crystallographically observed in the present study is not a conformational state simply restrained by crystal-packing forces. It is possible that the asymmetrically arranged tandem domains could provide a structural basis for further studies of the interdomain synergy.

scholarly journals The Avian Coronavirus Infectious Bronchitis Virus Undergoes Direct Low-pH-Dependent Fusion Activation during Entry into Host Cells

2006 ◽  
Vol 80 (7) ◽  
pp. 3180-3188 ◽  
Author(s):  
Victor C. Chu ◽  
Lisa J. McElroy ◽  
Vicky Chu ◽  
Beverley E. Bauman ◽  
Gary R. Whittaker
Keyword(s):  
Cell Fusion ◽  
Conformational Changes ◽  
Infectious Bronchitis Virus ◽  
Fusion Reaction ◽  
Low Ph ◽  
Surface Proteins ◽  
Host Cells ◽  
Dependent Manner ◽  
Infectious Bronchitis ◽  
Ph Dependent

ABSTRACT Coronaviruses are the causative agents of respiratory disease in humans and animals, including severe acute respiratory syndrome. Fusion of coronaviruses is generally thought to occur at neutral pH, although there is also evidence for a role of acidic endosomes during entry of a variety of coronaviruses. Therefore, the molecular basis of coronavirus fusion during entry into host cells remains incompletely defined. Here, we examined coronavirus-cell fusion and entry employing the avian coronavirus infectious bronchitis virus (IBV). Virus entry into cells was inhibited by acidotropic bases and by other inhibitors of pH-dependent endocytosis. We carried out fluorescence-dequenching fusion assays of R18-labeled virions and show that for IBV, coronavirus-cell fusion occurs in a low-pH-dependent manner, with a half-maximal rate of fusion occurring at pH 5.5. Fusion was reduced, but still occurred, at lower temperatures (20°C). We observed no effect of inhibitors of endosomal proteases on the fusion event. These data are the first direct measure of virus-cell fusion for any coronavirus and demonstrate that the coronavirus IBV employs a direct, low-pH-dependent virus-cell fusion activation reaction. We further show that IBV was not inactivated, and fusion was unaffected, by prior exposure to pH 5.0 buffer. Virions also showed evidence of reversible conformational changes in their surface proteins, indicating that aspects of the fusion reaction may be reversible in nature.

scholarly journals Mechanisms of activation and desensitization of full-length glycine receptor in membranes

2019 ◽  
Author(s):  
Arvind Kumar ◽  
Sandip Basak ◽  
Shanlin Rao ◽  
Yvonne Gicheru ◽  
Megan L. Mayer ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Single Channel ◽  
Glycine Receptor ◽  
Full Length ◽  
Structural Basis ◽  
Molecular Architecture ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
The Central Nervous System ◽  
Dynamics Simulations

AbstractGlycinergic synapses play a central role in motor control and pain processing in the central nervous system. Glycine receptors (GlyR) are key players in mediating fast inhibitory neurotransmission at these synapses. While previous high-resolution structural studies have provided insights into the molecular architecture of GlyR, several mechanistic questions pertaining to channel function are still unknown. Here, we present Cryo-EM structures of the full-length GlyR protein reconstituted into lipid nanodiscs that are captured in the unliganded (closed) and glycine-bound (open and desensitized) conformations. A comparison of the three states reveals global conformational changes underlying GlyR channel gating. The functional state assignments were validated by molecular dynamics simulations of the structures incorporated in a lipid bilayer. Observed permeation events are in agreement with the anion selectivity of the channel and the reported single-channel conductance of GlyR. These studies establish the structural basis for gating, selectivity, and single-channel conductance of GlyR in a physiological environment.

scholarly journals Coupling of HSP72 α-Helix Subdomains by the Unexpected Irreversible Targeting of Lysine-56 over Cysteine-17; Coevolution of Covalent Bonding

Molecules ◽  
2020 ◽  
Vol 25 (18) ◽  
pp. 4239
Author(s):  
Aimen Aljoundi ◽  
Ahmed El Rashedy ◽  
Patrick Appiah-Kubi ◽  
Mahmoud E. S. Soliman
Keyword(s):  
Conformational Changes ◽  
Conformational Dynamics ◽  
Salt Bridge ◽  
Structural Basis ◽  
Irreversible Inhibitor ◽  
Closed Conformation ◽  
Covalent Inhibition ◽  
Covalent Inhibitors ◽  
Biological Target ◽  
Α Helix

Covalent inhibition has recently gained a resurgence of interest in several drug discovery areas. The expansion of this approach is based on evidence elucidating the selectivity and potency of covalent inhibitors when bound to particular amino acids of a biological target. The unexpected covalent inhibition of heat shock protein 72 (HSP72) by covalently targeting Lys-56 instead of Cys-17 was an interesting observation. However, the structural basis and conformational changes associated with this preferential coupling to Lys-56 over Cys-17 remain unclear. To resolve this mystery, we employed structural and dynamic analyses to investigate the structural basis and conformational dynamics associated with the unexpected covalent inhibition. Our analyses reveal that the coupling of the irreversible inhibitor to Lys-56 is intrinsically less dynamic than Cys-17. Conformational dynamics analyses further reveal that the coupling of the inhibitor to Lys-56 induced a closed conformation of the nucleotide-binding subdomain (NBD) α-helices, in contrast, an open conformation was observed in the case of Cys-17. The closed conformation maintained the crucial salt-bridge between Glu-268 and Lys-56 residues, which strengthens the interaction affinity of the inhibitor nearly identical to adenosine triphosphate (ADP/Pi) bound to the HSP72-NBD. The outcome of this report provides a substantial shift in the conventional direction for the design of more potent covalent inhibitors.

Structural basis for pH-dependent retrieval of ER proteins from the Golgi by the KDEL receptor

Science ◽  
2019 ◽  
Vol 363 (6431) ◽  
pp. 1103-1107 ◽  
Author(s):  
Philipp Bräuer ◽  
Joanne L. Parker ◽  
Andreas Gerondopoulos ◽  
Iwan Zimmermann ◽  
Markus A. Seeger ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Secretory Pathway ◽  
Cell Function ◽  
Interaction Network ◽  
Eukaryotic Cell ◽  
Structural Basis ◽  
Ph Dependent ◽  
Kdel Receptor

Selective export and retrieval of proteins between the endoplasmic reticulum (ER) and Golgi apparatus is indispensable for eukaryotic cell function. An essential step in the retrieval of ER luminal proteins from the Golgi is the pH-dependent recognition of a carboxyl-terminal Lys-Asp-Glu-Leu (KDEL) signal by the KDEL receptor. Here, we present crystal structures of the chicken KDEL receptor in the apo ER state, KDEL-bound Golgi state, and in complex with an antagonistic synthetic nanobody (sybody). These structures show a transporter-like architecture that undergoes conformational changes upon KDEL binding and reveal a pH-dependent interaction network crucial for recognition of the carboxyl terminus of the KDEL signal. Complementary in vitro binding and in vivo cell localization data explain how these features create a pH-dependent retrieval system in the secretory pathway.

scholarly journals Structural basis for the geometry-driven localization of a small protein

2015 ◽  
Vol 112 (15) ◽  
pp. E1908-E1915 ◽  
Author(s):  
Richard L. Gill ◽  
Jean-Philippe Castaing ◽  
Jen Hsin ◽  
Irene S. Tan ◽  
Xingsheng Wang ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Structural Basis ◽  
Supported Lipid Bilayers ◽  
Curvature Sensing ◽  
Preferential Localization ◽  
Acyl Chains ◽  
Shape Sensing ◽  
Α Helix ◽  
Dynamics Simulations ◽  
Curved Membranes

In bacteria, certain shape-sensing proteins localize to differently curved membranes. During sporulation in Bacillus subtilis, the only convex (positively curved) surface in the cell is the forespore, an approximately spherical internal organelle. Previously, we demonstrated that SpoVM localizes to the forespore by preferentially adsorbing onto slightly convex membranes. Here, we used NMR and molecular dynamics simulations of SpoVM and a localization mutant (SpoVMP9A) to reveal that SpoVM’s atypical amphipathic α-helix inserts deeply into the membrane and interacts extensively with acyl chains to sense packing differences in differently curved membranes. Based on binding to spherical supported lipid bilayers and Monte Carlo simulations, we hypothesize that SpoVM’s membrane insertion, along with potential cooperative interactions with other SpoVM molecules in the lipid bilayer, drives its preferential localization onto slightly convex membranes. Such a mechanism, which is distinct from that used by high curvature-sensing proteins, may be widely conserved for the localization of proteins onto the surface of cellular organelles.

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