scholarly journals The endoplasmic reticulum P5A-ATPase is a transmembrane helix dislocase

Science ◽  
2020 ◽  
Vol 369 (6511) ◽  
pp. eabc5809 ◽  
Author(s):  
Michael J. McKenna ◽  
Sue Im Sim ◽  
Alban Ordureau ◽  
Lianjie Wei ◽  
J. Wade Harper ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Targeting ◽  
Transmembrane Helix ◽  
Protein Composition ◽  
Binding Pocket ◽  
Transmembrane Segment ◽  
Substrate Binding Pocket ◽  
Cryo Electron Microscopy ◽  
Endogenous Substrate ◽  
P Type

Organelle identity depends on protein composition. How mistargeted proteins are selectively recognized and removed from organelles is incompletely understood. Here, we found that the orphan P5A–adenosine triphosphatase (ATPase) transporter ATP13A1 (Spf1 in yeast) directly interacted with the transmembrane segment (TM) of mitochondrial tail–anchored proteins. P5A-ATPase activity mediated the extraction of mistargeted proteins from the endoplasmic reticulum (ER). Cryo–electron microscopy structures of Saccharomyces cerevisiae Spf1 revealed a large, membrane-accessible substrate-binding pocket that alternately faced the ER lumen and cytosol and an endogenous substrate resembling an α-helical TM. Our results indicate that the P5A-ATPase could dislocate misinserted hydrophobic helices flanked by short basic segments from the ER. TM dislocation by the P5A-ATPase establishes an additional class of P-type ATPase substrates and may correct mistakes in protein targeting or topogenesis.

scholarly journals Systematic Comparison of Molecular Conformations of H+,K+-ATPase Reveals an Important Contribution of the A-M2 Linker for the Luminal Gating

2014 ◽  
Vol 289 (44) ◽  
pp. 30590-30601 ◽  
Author(s):  
Kazuhiro Abe ◽  
Kazutoshi Tani ◽  
Yoshinori Fujiyoshi
Keyword(s):  
Molecular Conformation ◽  
Transmembrane Helix ◽  
Transmembrane Helices ◽  
Competitive Antagonist ◽  
Molecular Conformations ◽  
Systematic Comparison ◽  
Azimuthal Position ◽  
Cryo Electron Microscopy ◽  
Open Conformation ◽  
P Type

Gastric H+,K+-ATPase, an ATP-driven proton pump responsible for gastric acidification, is a molecular target for anti-ulcer drugs. Here we show its cryo-electron microscopy (EM) structure in an E2P analog state, bound to magnesium fluoride (MgF), and its K+-competitive antagonist SCH28080, determined at 7 Å resolution by electron crystallography of two-dimensional crystals. Systematic comparison with other E2P-related cryo-EM structures revealed that the molecular conformation in the (SCH)E2·MgF state is remarkably distinguishable. Although the azimuthal position of the A domain of the (SCH)E2·MgF state is similar to that in the E2·AlF (aluminum fluoride) state, in which the transmembrane luminal gate is closed, the arrangement of transmembrane helices in the (SCH)E2·MgF state shows a luminal-open conformation imposed on by bound SCH28080 at its luminal cavity, based on observations of the structure in the SCH28080-bound E2·BeF (beryllium fluoride) state. The molecular conformation of the (SCH)E2·MgF state thus represents a mixed overall structure in which its cytoplasmic and luminal half appear to be independently modulated by a phosphate analog and an antagonist bound to the respective parts of the enzyme. Comparison of the molecular conformations revealed that the linker region connecting the A domain and the transmembrane helix 2 (A-M2 linker) mediates the regulation of luminal gating. The mechanistic rationale underlying luminal gating observed in H+,K+-ATPase is consistent with that observed in sarcoplasmic reticulum Ca2+-ATPase and other P-type ATPases and is most likely conserved for the P-type ATPase family in general.

scholarly journals Transmembrane Helix 12 of the Staphylococcus aureus Multidrug Transporter QacA Lines the Bivalent Cationic Drug Binding Pocket

2007 ◽  
Vol 189 (24) ◽  
pp. 9131-9134 ◽  
Author(s):  
Karl A. Hassan ◽  
Ronald A. Skurray ◽  
Melissa H. Brown
Keyword(s):  
Staphylococcus Aureus ◽  
Transmembrane Helix ◽  
Binding Pocket ◽  
Drug Binding ◽  
Cation Binding ◽  
Multidrug Transporter ◽  
Transmembrane Segment ◽  
Binding Region ◽  
Bivalent Cation ◽  
Helix 12

ABSTRACT An acidic residue in transmembrane segment (TMS) 10 is important for recognition of bivalent cationic substrates by the QacA multidrug transporter. Remarkably, an acidic residue in TMS 12 compensated for the absence of such a residue in TMS 10, suggesting that TMS 12 is a component of the bivalent cation-binding region.

scholarly journals Alternative redox forms of ASNA-1 separate insulin signaling from tail-anchored protein targeting and cisplatin resistance in C. elegans

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Dorota Raj ◽  
Ola Billing ◽  
Agnieszka Podraza-Farhanieh ◽  
Bashar Kraish ◽  
Oskar Hemmingsson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Insulin Secretion ◽  
Protein Targeting ◽  
Cisplatin Resistance ◽  
Acquired Resistance ◽  
Endoplasmic Reticulum Membrane ◽  
C Elegans ◽  
Cisplatin Sensitivity ◽  
Tumor Environment ◽  
The One

AbstractCisplatin is a frontline cancer therapeutic, but intrinsic or acquired resistance is common. We previously showed that cisplatin sensitivity can be achieved by inactivation of ASNA-1/TRC40 in mammalian cancer cells and in Caenorhabditis elegans. ASNA-1 has two more conserved functions: in promoting tail-anchored protein (TAP) targeting to the endoplasmic reticulum membrane and in promoting insulin secretion. However, the relation between its different functions has remained unknown. Here, we show that ASNA-1 exists in two redox states that promote TAP-targeting and insulin secretion separately. The reduced state is the one required for cisplatin resistance: an ASNA-1 point mutant, in which the protein preferentially was found in the oxidized state, was sensitive to cisplatin and defective for TAP targeting but had no insulin secretion defect. The same was true for mutants in wrb-1, which we identify as the C. elegans homolog of WRB, the ASNA1/TRC40 receptor. Finally, we uncover a previously unknown action of cisplatin induced reactive oxygen species: cisplatin induced ROS drives ASNA-1 into the oxidized form, and selectively prevents an ASNA-1-dependent TAP substrate from reaching the endoplasmic reticulum. Our work suggests that ASNA-1 acts as a redox-sensitive target for cisplatin cytotoxicity and that cisplatin resistance is likely mediated by ASNA-1-dependent TAP substrates. Treatments that promote an oxidizing tumor environment should be explored as possible means to combat cisplatin resistance.

scholarly journals Crystal structure of steroid reductase SRD5A reveals conserved steroid reduction mechanism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yufei Han ◽  
Qian Zhuang ◽  
Bo Sun ◽  
Wenping Lv ◽  
Sheng Wang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Biochemical Characterization ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Transmembrane Segments ◽  
Therapeutic Molecules ◽  
Binding Cavity ◽  
Immune System Regulation ◽  
Mediated Reduction

AbstractSteroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ4 structure, such as testosterone or progesterone, are catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. SRD5A2 is already a target of clinically relevant drugs. However, the detailed mechanism of SRD5A-mediated reduction remains elusive. Here we report the crystal structure of PbSRD5A from Proteobacteria bacterium, a homolog of both SRD5A1 and SRD5A2, in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic substrate binding cavity, whereas TM5-7 coordinate cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with biochemical characterization, define the substrate binding pocket of SRD5As, explain the properties of disease-related mutants and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible.

scholarly journals Sigma 1 Receptor, Cholesterol and Endoplasmic Reticulum Contact Sites

Contact ◽  
2021 ◽  
Vol 4 ◽  
pp. 251525642110265
Author(s):  
Vladimir Zhemkov ◽  
Jen Liou ◽  
Ilya Bezprozvanny
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Composition ◽  
Sigma 1 Receptor ◽  
Functional Roles ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Sigma 1 ◽  
Membrane Contact ◽  
Organelle Communication ◽  
Cellular Organelles

Recent studies indicated potential importance of membrane contact sites (MCS) between the endoplasmic reticulum (ER) and other cellular organelles. These MCS have unique protein and lipid composition and serve as hubs for inter-organelle communication and signaling. Despite extensive investigation of MCS protein composition and functional roles, little is known about the process of MCS formation. In this perspective, we propose a hypothesis that MCS are formed not as a result of random interactions between membranes of ER and other organelles but on the basis of pre-existing cholesterol-enriched ER microdomains.

scholarly journals Cryo-EM structure of the human histamine H1 receptor/Gq complex

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ruixue Xia ◽  
Na Wang ◽  
Zhenmei Xu ◽  
Yang Lu ◽  
Jing Song ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Binding Pocket ◽  
Receptor Activation ◽  
Intracellular Loop ◽  
Cryo Electron Microscopy ◽  
Allergy Treatment ◽  
Domain 3 ◽  
Extracellular Side ◽  
Key Residues ◽  
Loop 2

AbstractHistamine receptors play important roles in various pathophysiological conditions and are effective targets for anti-allergy treatment, however the mechanism of receptor activation remain elusive. Here, we present the cryo-electron microscopy (cryo-EM) structure of the human H1R in complex with a Gq protein in an active conformation via a NanoBiT tethering strategy. The structure reveals that histamine activates receptor via interacting with the key residues of both transmembrane domain 3 (TM3) and TM6 to squash the binding pocket on the extracellular side and to open the cavity on the intracellular side for Gq engagement in a model of “squash to activate and expand to deactivate”. The structure also reveals features for Gq coupling, including the interaction between intracellular loop 2 (ICL2) and the αN-β junction of Gq/11 protein. The detailed analysis of our structure will provide a framework for understanding G-protein coupling selectivity and clues for designing novel antihistamines.

scholarly journals Structure of the Lectin Mannose 6-Phosphate Receptor Homology (MRH) Domain of Glucosidase II, an Enzyme That Regulates Glycoprotein Folding Quality Control in the Endoplasmic Reticulum

2013 ◽  
Vol 288 (23) ◽  
pp. 16460-16475 ◽  
Author(s):  
Linda J. Olson ◽  
Ramiro Orsi ◽  
Solana G. Alculumbre ◽  
Francis C. Peterson ◽  
Ivan D. Stigliano ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Dimensional Structure ◽  
Mannose Residue ◽  
Glucosidase Ii ◽  
Mannose 6 Phosphate ◽  
First Time ◽  
Conserved Residue

Here we report for the first time the three-dimensional structure of a mannose 6-phosphate receptor homology (MRH) domain present in a protein with enzymatic activity, glucosidase II (GII). GII is involved in glycoprotein folding in the endoplasmic reticulum. GII removes the two innermost glucose residues from the Glc3Man9GlcNAc2 transferred to nascent proteins and the glucose added by UDP-Glc:glycoprotein glucosyltransferase. GII is composed of a catalytic GIIα subunit and a regulatory GIIβ subunit. GIIβ participates in the endoplasmic reticulum localization of GIIα and mediates in vivo enhancement of N-glycan trimming by GII through its C-terminal MRH domain. We determined the structure of a functional GIIβ MRH domain by NMR spectroscopy. It adopts a β-barrel fold similar to that of other MRH domains, but its binding pocket is the most shallow known to date as it accommodates a single mannose residue. In addition, we identified a conserved residue outside the binding pocket (Trp-409) present in GIIβ but not in other MRHs that influences GII glucose trimming activity.

Characterization of the Acyl Substrate Binding Pocket of Acetyl-CoA Synthetase†

Biochemistry ◽  
2006 ◽  
Vol 45 (38) ◽  
pp. 11482-11490 ◽  
Author(s):  
Cheryl Ingram-Smith ◽  
Barrett I. Woods ◽  
Kerry S. Smith
Keyword(s):  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Acetyl Coa
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