Intermolecular cross-linking of monomers in Helicobacter pylori Na+/H+ antiporter NhaA at the dimer interface inhibits antiporter activity

2010 ◽  
Vol 426 (1) ◽  
pp. 99-108 ◽  
Author(s):  
Akira Karasawa ◽  
Keiji Mitsui ◽  
Masafumi Matsushita ◽  
Hiroshi Kanazawa
Keyword(s):  
Helicobacter Pylori ◽  
Conformational Changes ◽  
Cross Linking ◽  
Flanking Sequence ◽  
Native Page ◽  
Dimer Interface ◽  
Reducing Conditions ◽  
Cysteine Scanning Mutagenesis ◽  
Functional Analyses ◽  
Β Sheet

We have previously shown that HPNhaA (Helicobacter pylori Na+/H+ antiporter) forms an oligomer in a native membrane of Escherichia coli, and conformational changes of oligomer occur between monomers of the oligomer during ion transport. In the present study, we use Blue-native PAGE to show that HPNhaA forms a dimer. Cysteine-scanning mutagenesis of residues 55–61 in a putative β-sheet region of loop1 and subsequent functional analyses revealed that the Q58C mutation resulted in an intermolecular disulfide bond. G56C, I59C and G60C were found to be cross-linked by bifunctional cross-linkers. Furthermore, the Q58E mutant did not form a dimer, possibly due to electrostatic repulsion between monomers. These results imply that Gln-58 and the flanking sequence in the putative β-sheet of the monomer are located close to the identical residues in the dimer. The Q58C mutant of NhaA was almost inactive under non-reducing conditions, and activity was restored under reducing conditions. This result showed that cross-linking at the dimer interface reduces transporter activity by interfering with the flexible association between the monomers. A mutant HPNhaA protein with three amino acid substitutions at residues 57–59 did not form a dimer, and yet was active, indicating that the monomer is functional.

scholarly journals TTYH family members form tetrameric complexes within the cell membrane

2021 ◽  
Author(s):  
Emelia Melvin ◽  
Elia Shlush ◽  
Moshe Giladi ◽  
Yoni Haitin
Keyword(s):  
Single Molecule ◽  
Spatial Organization ◽  
Size Exclusion ◽  
Cross Linking ◽  
Native Page ◽  
Intact Mouse ◽  
Detergent Solubilization ◽  
Blue Native Page ◽  
Exclusion Chromatography ◽  
Functional Analyses

The conserved Tweety homolog (TTYH) family consists of three paralogs in vertebrates, displaying a ubiquitous expression pattern. Although considered as ion channels for almost two decades, recent structural and functional analyses refuted this role. Intriguingly, while all paralogs, studied following detergent solubilization, shared a dimeric stoichiometry, their spatial organization differed. Here, we determined the stoichiometry of intact mouse TTYH (mTTYH) complexes in cells. Using cross-linking and single-molecule fluorescence microscopy, we demonstrated that mTTYH1 and mTTYH3 form tetramers at the plasma membrane. Blue-native PAGE and fluorescence-detection size-exclusion chromatography analyses revealed that detergent solubilization results in the dissolution of tetramers into dimers, suggesting a dimer-of-dimers assembly mode. As cross-linking analysis of the soluble extracellular domains also showed tetrameric stoichiometry, we explored the effect of membrane solubilization and disulfide bridges integrity and established their contribution to tetramer stability. Future studies of the native tetrameric TTYH characterized here may illuminate their long-sought cellular function.

Influence of Calcium Ions on the Plant Cell Surface: Membrane Fusions and Conformational Changes

1974 ◽  
Vol 32 ◽  
pp. 154-155
Author(s):  
D. James Morré ◽  
Charles E. Bracker ◽  
William J. VanDerWoude
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Conformational Changes ◽  
Calcium Ions ◽  
Surface Membrane ◽  
Carboxyl Groups ◽  
Cross Linking ◽  
Ultrastructural Evidence ◽  
Glycine Max L ◽  
Wall Extensibility

Calcium ions in the concentration range 5-100 mM inhibit auxin-induced cell elongation and wall extensibility of plant stems. Inhibition of wall extensibility requires that the tissue be living; growth inhibition cannot be explained on the basis of cross-linking of carboxyl groups of cell wall uronides by calcium ions. In this study, ultrastructural evidence was sought for an interaction of calcium ions with some component other than the wall at the cell surface of soybean (Glycine max (L.) Merr.) hypocotyls.

scholarly journals Structural control of fibrin bioactivity by mechanical deformation

2020 ◽  
Author(s):  
Sachin Kumar ◽  
Yujen Wang ◽  
Manuel K. Rausch ◽  
Sapun H. Parekh
Keyword(s):  
Biological Activity ◽  
Conformational Changes ◽  
Structural Control ◽  
Structural Changes ◽  
Tensile Deformation ◽  
Mechanical Strain ◽  
Fibrous Protein ◽  
Binding Partners ◽  
Blood Clots ◽  
Β Sheet

AbstractFibrin is a fibrous protein network that entraps blood cells and platelets to form blood clots following vascular injury. As a biomaterial, fibrin acts a biochemical scaffold as well as a viscoelastic patch that resists mechanical insults. The biomechanics and biochemistry of fibrin have been well characterized independently, showing that fibrin is a hierarchical material with numerous binding partners. However, comparatively little is known about how fibrin biomechanics and biochemistry are coupled: how does fibrin deformation influence its biochemistry at the molecular level? In this study, we show how mechanically-induced molecular structural changes in fibrin affect fibrin biochemistry and fibrin-platelet interaction. We found that tensile deformation of fibrin lead to molecular structural transitions of α-helices to β-sheets, which reduced binding of tissue plasminogen activator (tPA), an enzyme that initiates fibrinolysis, at the network and single fiber level. Moreover, binding of tPA and Thioflavin T (ThT), a commonly used β-sheet marker, was primarily mutually exclusive such that tPA bound to native (helical) fibrin whereas ThT bound to strained fibrin. Finally, we demonstrate that conformational changes in fibrin suppressed the biological activity of platelets on mechanically strained fibrin due to attenuated αIIbβ3 integrin binding. Our work shows that mechanical strain regulates fibrin molecular structure and fibrin biological activity in an elegant mechano-chemical feedback loop, which likely influences fibrinolysis and wound healing kinetics.

scholarly journals OX40L/OX40 Signal Promotes IL-9 Production by Mucosal MAIT Cells During Helicobacter pylori Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Siqi Ming ◽  
Mei Zhang ◽  
Zibin Liang ◽  
Chunna Li ◽  
Jianzhong He ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Potential Role ◽  
Critical Role ◽  
Underlying Mechanism ◽  
Mucosal Inflammation ◽  
Cross Linking ◽  
Mait Cells ◽  
H Pylori ◽  
Mait Cell

Mucosal associated invariant T (MAIT) cells play a critical role in Helicobacter pylori (H. pylori)-induced gastritis by promoting mucosal inflammation and aggravating mucosal injuries (1, 2). However, the underlying mechanism and key molecules involved are still uncertain. Here we identified OX40, a co-stimulatory molecule mainly expressed on T cells, as a critical regulator to promote proliferation and IL-9 production by MAIT cells and facilitate mucosal inflammation in H. pylori-positive gastritis patients. Serum examination revealed an increased level of IL-9 in gastritis patients. Meanwhile, OX40 expression was increased in mucosal MAIT cells, and its ligand OX40L was also up-regulated in mucosal dendritic cells (DCs) of gastritis patients, compared with healthy controls. Further results demonstrated that activation of the OX40/OX40L pathway promoted IL-9 production by MAIT cells, and MAIT cells displayed a highly-activated phenotype after the cross-linking of OX40 and OX40L. Moreover, the level of IL-9 produced by MAIT cells was positively correlated with inflammatory indexes in the gastric mucosa, suggesting the potential role of IL-9-producing MAIT cells in mucosal inflammation. Taken together, we elucidated that OX40/OX40L axis promoted mucosal MAIT cell proliferation and IL-9 production in H. pylori-induced gastritis, which may provide potential targeting strategies for gastritis treatment.

scholarly journals Characterization of the stilbenedisulphonate binding site on band 3 Memphis variant II (Pro-854→Leu)

1996 ◽  
Vol 317 (2) ◽  
pp. 509-514 ◽  
Author(s):  
James M. SALHANY ◽  
Renee L. SLOAN ◽  
Lawrence M. SCHOPFER
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Covalent Binding ◽  
Band 3 ◽  
Blood Group Antigen ◽  
Cross Linking ◽  
Anion Exchange Protein ◽  
Membrane Bound ◽  
And Control

Band 3 Memphis variant II is a mutant anion-exchange protein associated with the Diego a+ blood group antigen. There are two mutations in this transporter: Lys-56 → Glu within the cytoplasmic domain, and Pro-854 → Leu within the membrane-bound domain. The Pro-854 mutation, which is thought to give rise to the antigenicity, is located within the C-terminal subdomain of the membrane-bound domain. Yet, there is an apparent enhancement in the rate of covalent binding of H2DIDS (4,4´-di-isothiocyanatodihydro-2,2´-stilbenedisulphonate) to ‘lysine A’ (Lys-539) in the N-terminal subdomain, suggesting widespread conformational changes. In this report, we have used various kinetic assays which differentiate between conformational changes in the two subdomains, to characterize the stilbenedisulphonate site on band 3 Memphis variant II. We have found a significantly higher H2DIDS (a C-terminal-sensitive inhibitor) affinity for band 3 Memphis variant II, due to a lower H2DIDS ‘off’ rate constant, but no difference was found between mutant and control when DBDS (4,4´-dibenzamido-2,2´-stilbenedisulphonate) (a C-terminal-insensitive inhibitor) ‘off’ rates were measured. Furthermore, there were no differences in the rates of covalent binding to lysine A, for either DIDS (4,4´-di-isothiocyanato-2,2´-stilbenedisulphonate) or H2DIDS. However, the rate of covalent intrasubunit cross-linking of Lys-539 and Lys-851 by H2DIDS was abnormally low for band 3 Memphis variant II. These results suggest that the Pro-854 → Leu mutation causes a localized conformational change in the C-terminal subdomain of band 3.

scholarly journals Inter-Active Site Communication Mediated by the Dimer Interface β-Sheet in the Half-the-Sites Enzyme, Thymidylate Synthase

Biochemistry ◽  
2019 ◽  
Vol 58 (30) ◽  
pp. 3302-3313 ◽  
Author(s):  
Paul J. Sapienza ◽  
Konstantin I. Popov ◽  
David D. Mowrey ◽  
Bradley T. Falk ◽  
Nikolay V. Dokholyan ◽  
...  
Keyword(s):  
Thymidylate Synthase ◽  
Active Site ◽  
Dimer Interface ◽  
Β Sheet

scholarly journals Conformational Changes in the Capsid of a Calicivirus upon Interaction with Its Functional Receptor

2010 ◽  
Vol 84 (11) ◽  
pp. 5550-5564 ◽  
Author(s):  
Robert J. Ossiboff ◽  
Yi Zhou ◽  
Patrick J. Lightfoot ◽  
B. V. Venkataram Prasad ◽  
John S. L. Parker
Keyword(s):  
Conformational Changes ◽  
Viral Entry ◽  
Structural Changes ◽  
Feline Calicivirus ◽  
Soluble Receptor ◽  
Viral Capsids ◽  
Dimer Interface ◽  
Metastable Structures ◽  
Growth Properties ◽  
Functional Receptor

ABSTRACT Nonenveloped viral capsids are metastable structures that undergo conformational changes during virus entry that lead to interactions of the capsid or capsid fragments with the cell membrane. For members of the Caliciviridae, neither the nature of these structural changes in the capsid nor the factor(s) responsible for inducing these changes is known. Feline junctional adhesion molecule A (fJAM-A) mediates the attachment and infectious viral entry of feline calicivirus (FCV). Here, we show that the infectivity of some FCV isolates is neutralized following incubation with the soluble receptor at 37°C. We used this property to select mutants resistant to preincubation with the soluble receptor. We isolated and sequenced 24 soluble receptor-resistant (srr) mutants and characterized the growth properties and receptor-binding activities of eight mutants. The location of the mutations within the capsid structure of FCV was mapped using a new 3.6-Å structure of native FCV. The srr mutations mapped to the surface of the P2 domain were buried at the protruding domain dimer interface or were present in inaccessible regions of the capsid protein. Coupled with data showing that both the parental FCV and the srr mutants underwent increases in hydrophobicity upon incubation with the soluble receptor at 37°C, these findings indicate that FCV likely undergoes conformational change upon interaction with its receptor. Changes in FCV capsid conformation following its interaction with fJAM-A may be important for subsequent interactions of the capsid with cellular membranes, membrane penetration, and genome delivery.

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