scholarly journals Glutamic acid 327 in the sheep α 1 isoform of Na+,K+-ATPase is a pivotal residue for cation-induced conformational changes

1995 ◽  
Vol 309 (1) ◽  
pp. 187-194 ◽  
Author(s):  
C L Johnson ◽  
T A Kuntzweiler ◽  
J B Lingrel ◽  
C G Johnson ◽  
E T Wallick
Keyword(s):  
Glutamic Acid ◽  
Conformational Changes ◽  
Conformational Transition ◽  
Cation Binding ◽  
Monovalent Cations ◽  
Wild Type ◽  
Binding Studies ◽  
Ouabain Binding ◽  
Na Ions ◽  
Inhibition Curve

The cation binding characteristics of the mutant E327A formed in the sheep alpha 1 isoform of the Na+,K(+)-ATPase were examined using [3H]ouabain binding as a function of monovalent cation concentrations. Equilibrium competition binding assays in the presence of Mg2+, inorganic phosphate and various amounts of unlabelled ouabain indicated that both wild-type sheep alpha 1 protein and the E327A mutant expressed in 3T3 cells had similar affinities for ouabain (KD = 1.53 and 1.31 nM respectively). Sodium inhibition of ouabain binding appeared competitive in both enzymes. However, binding of three Na+ ions was required to explain the steep character of the Na+ inhibition curve for the wild-type Na+,K(+)-ATPase (Ki = 12.8 +/- 1.6 mM), whereas the binding of two Na+ ions was detected for the mutant E327A (Ki = 19.2 +/- 2.5 mM). Potassium binding of [3H]ouabain binding displayed a partially competitive nature with Hill coefficients of 2 for both wild-type sheep alpha 1 (Ki = 0.743 +/- 0.044 mM) and E327A (Ki = 0.875 +/- 0.067 mM). At concentrations of K+ above 10 mM, the sheep alpha 1 competition curve levelled off whereas the inhibition curve for E327A displayed a stimulation in ouabain binding. This stimulation in [3H]ouabain binding also occurred with Rb+, Cs+ and Li+, but was never observed with choline or Na+, suggesting that this effect was not due to ionic strength. From these [3H]ouabain-binding studies, it is obvious that the mutant enzyme E327A in the presence of Mg2+, Pi and ouabain, interacts with monovalent cations in a unique fashion. One interpretation of these data is that the glutamic acid residue at position 327 is involved in a conformational transition induced by the binding of monovalent cations to the Na+,K+-ATPase and that this transition is inhibited by the mutation of E327A.

scholarly journals Partial reactions of the Na,K-ATPase: determination of rate constants.

1994 ◽  
Vol 104 (2) ◽  
pp. 197-240 ◽  
Author(s):  
S Heyse ◽  
I Wuddel ◽  
H J Apell ◽  
W Stürmer
Keyword(s):  
Electric Current ◽  
Rate Constant ◽  
Conformational Changes ◽  
Rate Constants ◽  
Conformational Transition ◽  
Divalent Cations ◽  
Quantitative Description ◽  
Reaction Model ◽  
Na Ions

Experiments were designed to characterize several partial reactions of the Na,K-ATPase and to demonstrate that a model can be defined that reproduces most of the transport features of the pump with a single set of kientic parameters. We used the fluorescence label 5-iodoacetamidofluorescein, which is thought to be sensitive to conformational changes, and the styryl dye RH 421, which can be applied to detect ion-binding and -release reactions. In addition transient electric currents were measured, which are associated mainly with the E1-->E2 conformational transition. Numerical simulations were performed on the basis of a reaction model, that has been developed from the Post-Albers cycle. Analysis of the experimental data allows the determination of several rate constants of the pump cycle. Our conclusions may be summarized as follows: (a) binding of one Na+ ion at the cytoplasmic face is electrogenic. This Na+ ion is specifically bound to a neutral binding site with an affinity of 8 mM in the presence of 10 mM Mg2+. In the absence of divalent cations, the intrinsic binding affinity was found to be 0.7 mM. (b) The analysis of fluorescence experiments with the cardiotonic steroid strophanthidin indicates that the 5-iodoacetamidofluorescein label monitors the conformational transition (Na3)E1-P-->P-E2(Na2), which is accompanied by the release of one Na+ ion. 5-IAF does not respond to the release of the subsequent two Na+ ions, which can be monitored by the RH 421 dye. These experiments indicate further that the conformational transition E1P-->P-E2 is the rate limiting process of the Na+ translocation. The corresponding rate constant was determined to be 22 s-1 at 20 degrees C. From competition experiments with cardiotonic steroids, we estimated that the remaining 2 Na+ ions are released subsequently with a rate constant of at least 5,000 s-1 from their negatively charged binding sites. (c) Comparing the fluorescence experiments with electric current transients, which were performed at various Na concentrations in the absence and presence of strophanthidin, we found that the transition (Na3).E1-P-->P-E2.(Na2) is the major charge translocating step in the reaction sequence Na3.E1-->(Na3).E1-P-->P-E2.(Na2)-->P-E2. The subsequent release of 2 Na+ ions contributed less than 25% to the total electric current transient. (d) The well known antagonism between cardiotonic steroids and K+ binding can be explained by a kinetic model. A quantitative description has been obtained under the assumption that these inhibitors bind only to the states P-E2(Na2) and P-E2(K2).(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals The Role of Loop 6/7 in Folding and Functional Performance of Na,K-ATPase

2004 ◽  
Vol 279 (44) ◽  
pp. 45594-45602 ◽  
Author(s):  
Guiyan Xu ◽  
David J. Kane ◽  
Larry D. Faller ◽  
Robert A. Farley
Keyword(s):  
Atpase Activity ◽  
Functional Performance ◽  
Intrinsic Rate ◽  
Cation Binding ◽  
Wild Type ◽  
Ouabain Binding ◽  
Cation Binding Site ◽  
Charged Residues ◽  
Ion Binding Sites ◽  
Phosphorylation Domain

Alanine substitutions were made for 15 amino acids in the cytoplasmic loop between transmembrane helices 6 and 7 (L6/7) of the human α1-subunit of Na,K-ATPase. Most mutations reduced Na,K-ATPase activity by less than 50%; however, the mutations R834A, R837A, and R848A reduced Na,K-ATPase activity by 75, 89, and 66%, respectively. Steady-state phosphoenzyme formation from ATP was reduced in mutants R834A, R837A, and R848A, and R837A also had a faster E2P → E2dephosphorylation rate compared with the wild-type enzyme. Effects of L6/7 mutations on the phosphorylation domain of the protein were also demonstrated by18O exchange, which showed that intrinsic rate constants for Pibinding and/or reaction with the protein were altered. Although most L6/7 mutations had no effect on the interaction of Na+or K+with Na,K-ATPase, the E825A, E828A, R834A, and R837A mutations reduced the apparent affinity of the enzyme for both Na+and K+by 1.5–3-fold. 1-Bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU3+), a competitive antagonist of Rb+and Na+occlusion (Hoving, S., Bar-Shimon, M., Tijmes, J. J., Goldschleger, R., Tal, D. M., and Karlish, S. J. D. (1995)J. Biol. Chem. 270, 29788–29793), was used to test whether charged residues in L6/7 are involved in binding monovalent cations and cation antagonists. Br-TITU3+inhibited ouabain binding to wild type Na,K-ATPase with an IC50of 30 μm. Ouabain binding to the E825A, E828A, R834A, or R837A mutants was still inhibited by Br-TITU3+, indicating that Br-TITU3+does not bind to charged residues in L6/7. This observation makes it unlikely that L6/7 functions as a cytoplasmic cation binding site in Na,K-ATPase, and together with the effects of L6/7 mutations on phosphate interactions with the enzyme suggests that L6/7 is important in stabilizing the phosphorylation domain and its relationship to the ion binding sites of the protein.

Constant pH Molecular Dynamics Reveals How Proton Release Drives the Conformational Transition of a Transmembrane Efflux Pump

2017 ◽  
Author(s):  
Jana Shen ◽  
Zhi Yue ◽  
Helen Zgurskaya ◽  
Wei Chen
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Conformational Transition ◽  
Conformational Dynamics ◽  
Proton Release ◽  
Intrinsic Resistance ◽  
Constant Ph ◽  
Wide Range ◽  
Constant Ph Molecular Dynamics

AcrB is the inner-membrane transporter of E. coli AcrAB-TolC tripartite efflux complex, which plays a major role in the intrinsic resistance to clinically important antibiotics. AcrB pumps a wide range of toxic substrates by utilizing the proton gradient between periplasm and cytoplasm. Crystal structures of AcrB revealed three distinct conformational states of the transport cycle, substrate access, binding and extrusion, or loose (L), tight (T) and open (O) states. However, the specific residue(s) responsible for proton binding/release and the mechanism of proton-coupled conformational cycling remain controversial. Here we use the newly developed membrane hybrid-solvent continuous constant pH molecular dynamics technique to explore the protonation states and conformational dynamics of the transmembrane domain of AcrB. Simulations show that both Asp407 and Asp408 are deprotonated in the L/T states, while only Asp408 is protonated in the O state. Remarkably, release of a proton from Asp408 in the O state results in large conformational changes, such as the lateral and vertical movement of transmembrane helices as well as the salt-bridge formation between Asp408 and Lys940 and other sidechain rearrangements among essential residues.Consistent with the crystallographic differences between the O and L protomers, simulations offer dynamic details of how proton release drives the O-to-L transition in AcrB and address the controversy regarding the proton/drug stoichiometry. This work offers a significant step towards characterizing the complete cycle of proton-coupled drug transport in AcrB and further validates the membrane hybrid-solvent CpHMD technique for studies of proton-coupled transmembrane proteins which are currently poorly understood. <p><br></p>

scholarly journals Monoclonal antibodies identify residues 199–216 of the integrin α2 vWFA domain as a functionally important region within α2β1

2000 ◽  
Vol 350 (2) ◽  
pp. 485-493 ◽  
Author(s):  
Danny S. TUCKWELL ◽  
Lyndsay SMITH ◽  
Michelle KORDA ◽  
Janet A. ASKARI ◽  
Sentot SANTOSO ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Conformational Changes ◽  
Cation Binding ◽  
Inhibitory Antibody ◽  
Collagen Binding ◽  
Collagen Peptide ◽  
Binding Residue ◽  
Important Region ◽  
Domain Mapping

Integrin α2β1 is the major receptor for collagens in the human body, and the collagen-binding site on the α2 subunit von Willebrand factor A-type domain (vWFA domain) is now well defined. However, the biologically important conformational changes that are associated with collagen binding, and the means by which the vWFA domain is integrated into the whole integrin are not completely understood. We have raised monoclonal antibodies against recombinant α2 vWFA domain for use as probes of function. Three antibodies, JA202, JA215 and JA218, inhibited binding to collagen, collagen I C-propeptide and E-cadherin, demonstrating that their function is important for structurally diverse α2β1 ligands. Cross-blocking studies grouped the epitopes into two clusters: (I) JA202, the inhibitory antibody, Gi9, and a non-inhibitory antibody, JA208; (II) JA215 and JA218. Both clusters were sensitive to events at the collagen binding site, as binding of Gi9, JA202, JA215 and JA218 were inhibited by collagen peptide, JA208 binding was enhanced by collagen peptide, and binding of JA202 was decreased after mutagenesis of the cation-binding residue Thr221 to alanine. Binding of cluster I antibodies was inhibited by the anti-functional anti-β1 antibody Mab13, and binding of Gi9 and JA218 to α2β1 was inhibited by substituting Mn2+ for Mg2+, demonstrating that these antibodies were sensitive to changes initiated outside the vWFA domain. Mapping of epitopes showed that JA202 and Gi9 bound between residues 212–216, while JA208 bound between residues 199–216. We have therefore identified two epitope clusters with novel properties; i.e. they are intimately associated with the collagen-binding site, responsive to conformational changes at the collagen-binding site and sensitive to events initiated outside the vWFA domain.

scholarly journals Temperature effects on sodium pump phosphoenzyme distribution in human red blood cells.

1985 ◽  
Vol 85 (1) ◽  
pp. 123-136 ◽  
Author(s):  
J H Kaplan ◽  
L J Kenney
Keyword(s):  
Atpase Activity ◽  
Cell Membranes ◽  
Conformational Transition ◽  
Fold Increase ◽  
Inhibitory Effect ◽  
Red Cell ◽  
Na Pump ◽  
Na Ions ◽  
Red Cell Membranes ◽  
Increasing Temperature

Phosphorylation of red cell membranes at ambient temperatures with micromolar [32P]ATP in the presence of Na ions produced phosphoenzyme that was dephosphorylated rapidly upon the addition of ADP or K ions. However, as first observed by Blostein (1968, J. Biol. Chem., 243:1957), the phosphoenzyme formed at 0 degrees C under otherwise identical conditions was insensitive to the addition of K ions but was dephosphorylated rapidly by ADP. This suggested that the conformational transition from ADP-sensitive, K-insensitive Na pump phosphoenzyme (E1 approximately P) to K-sensitive, ADP-insensitive phosphoenzyme (E2P) is blocked at 0 degrees C. Since the ATP:ADP exchange reaction is a partial reaction of the overall enzyme cycle dependent upon the steady state level of E1 approximately P that is regulated by [Na], we examined the effects of temperature on the curve relating [Na] to ouabain-sensitive ATP:ADP exchange. The characteristic triphasic curve seen at higher temperatures when [Na] was between 0.5 and 100 mM was not obtained at 0 degrees C. Simple saturation was observed instead with a K0.5 for Na of approximately 1 mM. The effect of increasing temperature on the ATP:ADP exchange at fixed (150 mM) Na was compared with the effect of increasing temperature on (Na + K)-ATPase activity of the same membrane preparation. It was observed that (a) at 0 degrees C, there was significant ouabain-sensitive ATP:ADP exchange activity, (b) at 0 degrees C, ouabain-sensitive (Na + K)-ATPase activity was virtually absent, and (c) in the temperature range 5-37 degrees C, there was an approximately 300-fold increase in (Na + K)-ATPase activity with only a 9-fold increase in the ATP:ADP exchange. These observations are in keeping with the suggestion that the E1 approximately P----E2P transition of the Na pump in human red cell membranes is blocked at 0 degrees C. Previous work has shown that the inhibitory effect of Na ions and the low-affinity stimulation by Na of the rate of ATP:ADP exchange occur at the extracellular surface of the Na pump. The absence of both of these effects at 0 degrees C, where E1 approximately P is maximal, supports the idea that external Na acts through sites on the E2P form of the phosphoenzyme.

Estrogen Receptor-Independent Catechol Estrogen Binding Activity:  Protein Binding Studies in Wild-Type, Estrogen Receptor-α KO, and Aromatase KO Mice Tissues†

Biochemistry ◽  
2004 ◽  
Vol 43 (21) ◽  
pp. 6698-6708 ◽  
Author(s):  
Brian J. Philips ◽  
Pete J. Ansell ◽  
Leslie G. Newton ◽  
Nobuhiro Harada ◽  
Shin-Ichiro Honda ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Protein Binding ◽  
Estrogen Receptor Α ◽  
Binding Activity ◽  
Wild Type ◽  
Binding Studies ◽  
Catechol Estrogen ◽  
Estrogen Binding

scholarly journals New Insights into the Spring-Loaded Conformational Change of Influenza Virus Hemagglutinin

2002 ◽  
Vol 76 (9) ◽  
pp. 4456-4466 ◽  
Author(s):  
Jennifer A. Gruenke ◽  
R. Todd Armstrong ◽  
William W. Newcomb ◽  
Jay C. Brown ◽  
Judith M. White
Keyword(s):  
Influenza Virus ◽  
Conformational Change ◽  
Conformational Changes ◽  
Limited Proteolysis ◽  
Coiled Coil ◽  
Fusion Peptide ◽  
Wild Type ◽  
Influenza Virus Hemagglutinin ◽  
Target Membrane ◽  
Mutant Forms

ABSTRACT Influenza virus hemagglutinin undergoes a conformational change in which a loop-to-helix “spring-loaded” conformational change forms a coiled coil that positions the fusion peptide for interaction with the target bilayer. Previous work has shown that two proline mutations designed to disrupt this change disrupt fusion but did not determine the basis for the fusion defect. In this work, we made six additional mutants with single proline substitutions in the region that undergoes the spring-loaded conformational change and two additional mutants with double proline substitutions in this region. All double mutants were fusion inactive. We analyzed one double mutant, F63P/F70P, as an example. We observed that F63P/F70P undergoes key low-pH-induced conformational changes and binds tightly to target membranes. However, limited proteolysis and electron microscopy observations showed that the mutant forms a coiled coil that is only ∼50% the length of the wild type, suggesting that it is splayed in its N-terminal half. This work further supports the hypothesis that the spring-loaded conformational change is necessary for fusion. Our data also indicate that the spring-loaded conformational change has another role beyond presenting the fusion peptide to the target membrane.

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