scholarly journals Access of Extracellular Cations to their Binding Sites in Na,K-ATPase: Role of the Second Extracellular Loop of the α Subunit

2006 ◽  
Vol 127 (3) ◽  
pp. 341-352 ◽  
Author(s):  
Oihana Capendeguy ◽  
Pierre Chodanowski ◽  
Olivier Michielin ◽  
Jean-Daniel Horisberger
Keyword(s):  
Binding Sites ◽  
Structural Model ◽  
Maximum Activity ◽  
Extracellular Loop ◽  
Animal Cells ◽  
Α Subunit ◽  
Transmembrane Segments ◽  
Gating Mechanism ◽  
Extracellular Side ◽  
Active Transport System

Na,K-ATPase, the main active transport system for monovalent cations in animal cells, is responsible for maintaining Na+ and K+ gradients across the plasma membrane. During its transport cycle it binds three cytoplasmic Na+ ions and releases them on the extracellular side of the membrane, and then binds two extracellular K+ ions and releases them into the cytoplasm. The fourth, fifth, and sixth transmembrane helices of the α subunit of Na,K-ATPase are known to be involved in Na+ and K+ binding sites, but the gating mechanisms that control the access of these ions to their binding sites are not yet fully understood. We have focused on the second extracellular loop linking transmembrane segments 3 and 4 and attempted to determine its role in gating. We replaced 13 residues of this loop in the rat α1 subunit, from E314 to G326, by cysteine, and then studied the function of these mutants using electrophysiological techniques. We analyzed the results using a structural model obtained by homology with SERCA, and ab initio calculations for the second extracellular loop. Four mutants were markedly modified by the sulfhydryl reagent MTSET, and we investigated them in detail. The substituted cysteines were more readily accessible to MTSET in the E1 conformation for the Y315C, W317C, and I322C mutants. Mutations or derivatization of the substituted cysteines in the second extracellular loop resulted in major increases in the apparent affinity for extracellular K+, and this was associated with a reduction in the maximum activity. The changes produced by the E314C mutation were reversed by MTSET treatment. In the W317C and I322C mutants, MTSET also induced a moderate shift of the E1/E2 equilibrium towards the E1(Na) conformation under Na/Na exchange conditions. These findings indicate that the second extracellular loop must be functionally linked to the gating mechanism that controls the access of K+ to its binding site.

3D Reconstruction of Na+, K+-ATpase from Tubular Crystals

2000 ◽  
Vol 6 (S2) ◽  
pp. 238-239
Author(s):  
W. J. Rice ◽  
H.S. Young ◽  
D.W. Martin ◽  
J. R. Sachs ◽  
D.L. Stokes
Keyword(s):  
Amino Acids ◽  
Transmembrane Protein ◽  
Phosphorylation Site ◽  
Animal Cells ◽  
Α Subunit ◽  
A Subunit ◽  
Extracellular Side ◽  
Membrane Spanning ◽  
P Type ◽  
Tubular Crystals

The Na+,K+-ATPase is a transmembrane protein, located in the plasma membrane of virtually all animal cells, which controls Na+ and K+ gradients. It is a member of the P-type ATPase family of ion pumps, a group of enzymes which pump ions against a concentration gradient, forming a phosphorylated intermediate during the pumping cycle. For each mole of ATP hydrolysed, 3 Na + ions are moved out of the cell and 2 K+ ions are moved into the cell. Unlike most other members of this family, which have one subunit, Na+, K+-ATPase is a heterodimer of α and β subunits. The a subunit consists of 1020 amino acids and has been predicted to have 10 membrane-spanning a-helices as well as a large cytoplasmic headpiece which forms the ATP binding and phosphorylation site. The α subunit, 300 amino acids in length, has one membrane spanning helix and has most of its mass located on the extracellular side of the membrane.

scholarly journals Secondary Structural Model of MALAT1 Becomes Unstructured in Chronic Myeloid Leukemia and Undergoes Structural Rearrangement in Cervical Cancer

2021 ◽  
Vol 7 (1) ◽  
pp. 6
Author(s):  
Matthew C. Wang ◽  
Phillip J. McCown ◽  
Grace E. Schiefelbein ◽  
Jessica A. Brown
Keyword(s):  
Cervical Cancer ◽  
Chronic Myeloid Leukemia ◽  
Hela Cells ◽  
Binding Sites ◽  
Myeloid Leukemia ◽  
Structural Changes ◽  
Structural Model ◽  
Dimethyl Sulfate ◽  
K562 Cells ◽  
Cancer Types

Long noncoding RNAs (lncRNAs) influence cellular function through binding events that often depend on the lncRNA secondary structure. One such lncRNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), is upregulated in many cancer types and has a myriad of protein- and miRNA-binding sites. Recently, a secondary structural model of MALAT1 in noncancerous cells was proposed to form 194 hairpins and 13 pseudoknots. That study postulated that, in cancer cells, the MALAT1 structure likely varies, thereby influencing cancer progression. This work analyzes how that structural model is expected to change in K562 cells, which originated from a patient with chronic myeloid leukemia (CML), and in HeLa cells, which originated from a patient with cervical cancer. Dimethyl sulfate-sequencing (DMS-Seq) data from K562 cells and psoralen analysis of RNA interactions and structure (PARIS) data from HeLa cells were compared to the working structural model of MALAT1 in noncancerous cells to identify sites that likely undergo structural alterations. MALAT1 in K562 cells is predicted to become more unstructured, with almost 60% of examined hairpins in noncancerous cells losing at least half of their base pairings. Conversely, MALAT1 in HeLa cells is predicted to largely maintain its structure, undergoing 18 novel structural rearrangements. Moreover, 50 validated miRNA-binding sites are affected by putative secondary structural changes in both cancer types, such as miR-217 in K562 cells and miR-20a in HeLa cells. Structural changes unique to K562 cells and HeLa cells provide new mechanistic leads into how the structure of MALAT1 may mediate cancer in a cell-type specific manner.

scholarly journals Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel

2016 ◽  
Vol 113 (5) ◽  
pp. E644-E653 ◽  
Author(s):  
Nurit Degani-Katzav ◽  
Revital Gortler ◽  
Lilach Gorodetzki ◽  
Yoav Paas
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Parasitic Diseases ◽  
Β Subunit ◽  
River Blindness ◽  
Subunit Stoichiometry ◽  
Binding Pockets ◽  
Extracellular Side ◽  
Electrophysiological Characterization

The invertebrate glutamate-gated chloride-selective receptors (GluClRs) are ion channels serving as targets for ivermectin (IVM), a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. The native GluClR is a heteropentamer consisting of α and β subunit types, with yet unknown subunit stoichiometry and arrangement. Based on the recent crystal structure of a homomeric GluClαR, we introduced mutations at the intersubunit interfaces where Glu (the neurotransmitter) binds. By electrophysiological characterization of these mutants, we found heteromeric assemblies with two equivalent Glu-binding sites at β/α intersubunit interfaces, where the GluClβ and GluClα subunits, respectively, contribute the “principal” and “complementary” components of the putative Glu-binding pockets. We identified a mutation in the IVM-binding site (far away from the Glu-binding sites), which significantly increased the sensitivity of the heteromeric mutant receptor to both Glu and IVM, and improved the receptor subunits’ cooperativity. We further characterized this heteromeric GluClR mutant as a receptor having a third Glu-binding site at an α/α intersubunit interface. Altogether, our data unveil heteromeric GluClR assemblies having three α and two β subunits arranged in a counterclockwise β-α-β-α-α fashion, as viewed from the extracellular side, with either two or three Glu-binding site interfaces.

Binding Investigation of Integrin αvβ3 With Its Inhibitors by SPR Technology and Molecular Docking Simulation

2010 ◽  
Vol 15 (2) ◽  
pp. 131-137 ◽  
Author(s):  
Yaqin Liu ◽  
Yuanjiang Pan ◽  
Yuhong Xu
Keyword(s):  
Molecular Docking ◽  
Binding Sites ◽  
Structural Model ◽  
Docking Simulation ◽  
Integrin Αvβ3 ◽  
Equilibrium Dissociation Constant ◽  
Molecular Docking Simulation ◽  
Spr Biosensor ◽  
Inhibitor Discovery ◽  
Equilibrium Dissociation

Integrins play critical roles in the process of angiogenesis and are attractive targets for anticancer therapies. It is desirable to develop new types of small-molecule inhibitors of integrin. Herein, the binding features of several inhibitors to integrin αvβ3 have been studied by surface plasmon resonance (SPR) biosensor technology and molecular docking analyses. The SPR results indicated that the equilibrium dissociation constant (KD) values are evaluated for the inhibitors and showed that the KD value of cyclopeptide c-Lys is much lower than the reference molecule. In addition, the 3D structural model of integrin αvβ3 was generated according to the crystal structure of the integrin αvβ3 complex, and the molecular docking simulation analyses revealed that the predicted binding sites for the most active cyclopeptide c-Lys were consistent with the reported structure. These results thus implied that cyclopeptide c-Lys could be developed as a novel inhibitor for integrin αvβ3. The current work has potential for application in structure-based integrin αvβ3 inhibitor discovery.

scholarly journals The extracellular loop of the membrane permease VraG interacts with GraS to sense cationic antimicrobial peptides in Staphylococcus aureus

2021 ◽  
Vol 17 (3) ◽  
pp. e1009338
Author(s):  
Junho Cho ◽  
Stephen K. Costa ◽  
Rachel M. Wierzbicki ◽  
William F. C. Rigby ◽  
Ambrose L. Cheung
Keyword(s):  
Positive Charge ◽  
Efflux Pump ◽  
Survival Strategies ◽  
Extracellular Loop ◽  
Major Question ◽  
Defense Proteins ◽  
Bacterial Membranes ◽  
Transmembrane Segments ◽  
Charge Interaction ◽  
Sense And Respond

Host defense proteins (HDPs), aka defensins, are a key part of the innate immune system that functions by inserting into the bacterial membranes to form pores to kill invading and colonizing microorganisms. To ensure survival, microorganism such as S. aureus has developed survival strategies to sense and respond to HDPs. One key strategy in S. aureus is a two-component system (TCS) called GraRS coupled to an efflux pump that consists of a membrane permease VraG and an ATPase VraF, analogous to the BceRS-BceAB system of Bacillus subtilis but with distinct differences. While the 9 negatively charged amino acid extracellular loop of the membrane sensor GraS has been shown to be involved in sensing, the major question is how such a small loop can sense diverse HDPs. Mutation analysis in this study divulged that the vraG mutant phenocopied the graS mutant with respect to reduced activation of downstream effector mprF, reduction in surface positive charge and enhanced 2 hr. killing with LL-37 as compared with the parental MRSA strain JE2. In silico analysis revealed VraG contains a single 200-residue extracellular loop (EL) situated between the 7th and 8th transmembrane segments (out of 10). Remarkably, deletion of EL in VraG enhanced mprF expression, augmented surface positive charge and improved survival in LL-37 vs. parent JE2. As the EL of VraG is rich in lysine residues (16%), in contrast to a preponderance of negatively charged aspartic acid residues (3 out of 9) in the EL of GraS, we divulged the role of charge interaction by showing that K380 in the EL of VraG is an important residue that likely interacts with GraS to interfere with GraS-mediated signaling. Bacterial two-hybrid analysis also supported the interaction of EL of VraG with the EL of GraS. Collectively, we demonstrated an interesting facet of efflux pumps whereby the membrane permease disrupts HDP signaling by inhibiting GraS sensing that involves charged residues in the EL of VraG.

scholarly journals Cryo-EM structure of the complete and ligand-saturated insulin receptor ectodomain

2019 ◽  
Author(s):  
Theresia Gutmann ◽  
Ingmar Schäfer ◽  
Chetan Poojari ◽  
Beate Brankatschk ◽  
Ilpo Vattulainen ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Binding Sites ◽  
Structural Model ◽  
Receptor Site ◽  
Insulin Binding ◽  
Proximal Region ◽  
Dynamics Simulation ◽  
Structural Basis ◽  
Structure Based Drug Design ◽  
Receptor Interactions

AbstractGlucose homeostasis and growth essentially depend on the peptide hormone insulin engaging its receptor. Despite biochemical and structural advances, a fundamental contradiction has persisted in the current understanding of insulin ligand–receptor interactions. While biochemistry predicts two distinct insulin binding sites, 1 and 2, recent structural analyses have only resolved site 1. Using a combined approach of cryo-EM and atomistic molecular dynamics simulation, we determined the structure of the entire dimeric insulin receptor ectodomain saturated with four insulin molecules. Complementing the previously described insulin–site 1 interaction, we present the first view of insulin bound to the discrete insulin receptor site 2. Insulin binding stabilizes the receptor ectodomain in a T-shaped conformation wherein the membrane-proximal domains converge and contact each other. These findings expand the current models of insulin binding to its receptor and of its regulation. In summary, we provide the structural basis enabling a comprehensive description of ligand–receptor interactions that ultimately will inform new approaches to structure-based drug design.In briefA cryo-EM structure of the complete insulin receptor ectodomain saturated with four insulin ligands is reported. The structural model of the insulin–insulin receptor complex adopts a T-shaped conformation, reveals two additional insulin-binding sites potentially involved in the initial interaction of insulin with its receptor, and resolves the membrane proximal region.

scholarly journals Crystal structure of MICU2 and comparison with MICU1 reveal insights into the uniporter gating mechanism

2019 ◽  
Vol 116 (9) ◽  
pp. 3546-3555 ◽  
Author(s):  
Kimberli J. Kamer ◽  
Wei Jiang ◽  
Virendar K. Kaushik ◽  
Vamsi K. Mootha ◽  
Zenon Grabarek
Keyword(s):  
Binding Sites ◽  
Mammalian Cells ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Functional Coupling ◽  
Gating Mechanism ◽  
Ef Hand ◽  
Negative Effect ◽  
Oligomeric Complex

The mitochondrial uniporter is a Ca2+-channel complex resident within the organelle’s inner membrane. In mammalian cells the uniporter’s activity is regulated by Ca2+ due to concerted action of MICU1 and MICU2, two paralogous, but functionally distinct, EF-hand Ca2+-binding proteins. Here we present the X-ray structure of the apo form of Mus musculus MICU2 at 2.5-Å resolution. The core structure of MICU2 is very similar to that of MICU1. It consists of two lobes, each containing one canonical Ca2+-binding EF-hand (EF1, EF4) and one structural EF-hand (EF2, EF3). Two molecules of MICU2 form a symmetrical dimer stabilized by highly conserved hydrophobic contacts between exposed residues of EF1 of one monomer and EF3 of another. Similar interactions stabilize MICU1 dimers, allowing exchange between homo- and heterodimers. The tight EF1–EF3 interface likely accounts for the structural and functional coupling between the Ca2+-binding sites in MICU1, MICU2, and their complex that leads to the previously reported Ca2+-binding cooperativity and dominant negative effect of mutation of the Ca2+-binding sites in either protein. The N- and C-terminal segments of the two proteins are distinctly different. In MICU2 the C-terminal helix is significantly longer than in MICU1, and it adopts a more rigid structure. MICU2’s C-terminal helix is dispensable in vitro for its interaction with MICU1 but required for MICU2’s function in cells. We propose that in the MICU1–MICU2 oligomeric complex the C-terminal helices of both proteins form a central semiautonomous assembly which contributes to the gating mechanism of the uniporter.

scholarly journals Rotational symmetry of the structured Chip/LDB-SSDP core module of the Wnt enhanceosome

2019 ◽  
Vol 116 (42) ◽  
pp. 20977-20983 ◽  
Author(s):  
Miha Renko ◽  
Marc Fiedler ◽  
Trevor J. Rutherford ◽  
Jonas V. Schaefer ◽  
Andreas Plückthun ◽  
...  
Keyword(s):  
Cell Fate ◽  
Structural Model ◽  
Mutational Analysis ◽  
Binding Protein ◽  
Rotational Symmetry ◽  
Lim Domain ◽  
Animal Cells ◽  
Developmental Control ◽  
Cell Fate Decisions ◽  
Core Module

The Chip/LIM-domain binding protein (LDB)–single-stranded DNA-binding protein (SSDP) (ChiLS) complex controls numerous cell-fate decisions in animal cells, by mediating transcription of developmental control genes via remote enhancers. ChiLS is recruited to these enhancers by lineage-specific LIM-domain proteins that bind to its Chip/LDB subunit. ChiLS recently emerged as the core module of the Wnt enhanceosome, a multiprotein complex that primes developmental control genes for timely Wnt responses. ChiLS binds to NPFxD motifs within Pygopus (Pygo) and the Osa/ARID1A subunit of the BAF chromatin remodeling complex, which could synergize with LIM proteins in tethering ChiLS to enhancers. Chip/LDB and SSDP both contain N-terminal dimerization domains that constitute the bulk of their structured cores. Here, we report the crystal structures of these dimerization domains, in part aided by DARPin chaperones. We conducted systematic surface scanning by structure-designed mutations, followed by in vitro and in vivo binding assays, to determine conserved surface residues required for binding between Chip/LDB, SSDP, and Pygo-NPFxD. Based on this, and on the 4:2 (SSDP-Chip/LDB) stoichiometry of ChiLS, we derive a highly constrained structural model for this complex, which adopts a rotationally symmetrical SSDP2-LDB2-SSDP2 architecture. Integrity of ChiLS is essential for Pygo binding, and our mutational analysis places the NPFxD pockets on either side of the Chip/LDB dimer, each flanked by an SSDP dimer. The symmetry and multivalency of ChiLS underpin its function as an enhancer module integrating Wnt signals with lineage-specific factors to operate context-dependent transcriptional switches that are pivotal for normal development and cancer.

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