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ABSTRACT: Cysteine-free mouse MDR3 P-glycoprotein (Pgp) was constructed by mutagenesis of the nine natural Cys to Ala. The Cys-free protein was expressed in Pichia pastoris and purified. Yield, purity, ATPase activity, K(m)(MgATP), and stimulation of ATPase by verapamil, were similar to wild-type mouse Ppg. Mouse Cys-free Pgp was superior in yield and stability to Cys-free human MDR1 Pgp. Mutants Y1040A and Y1040C were constructed in mouse Cys-free Pgp background. Both showed extremely low ATPase activity, strongly-impaired vanadate-trapping of ADP, and reduced photolabeling by 8-azido-ATP. The results are consistent with the conclusion that Tyr-1040 is located in the MgATP-binding site in NBD2 and is required for correct binding and/or orientation of bound MgATP substrate in Pgp as previously suggested by X-ray structures of other ABC transporters and by sequencing of photolabeled Pgp. The results also support our previous conclusion that both catalytic sites must be intact for normal function in Pgp.
Archives of Biochemistry and Biophysics 02/2006; 445(1):124-8. · 2.93 Impact Factor
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ABSTRACT: The stator in F(1)F(o)-ATP synthase resists strain generated by rotor torque. In Escherichia coli, the b(2)delta subunit complex comprises the stator, bound to subunit a in F(o) and to the alpha(3)beta(3) hexagon of F(1). Previous work has shown that N-terminal residues of alpha subunit are involved in binding delta. A synthetic peptide consisting of the first 22 residues of alpha (alphaN1-22) binds specifically to isolated wild-type delta subunit with 1:1 stoichiometry and high affinity, accounting for a major portion of the binding energy between delta and F(1). Residues alpha6-18 are predicted by secondary structure algorithms and helical wheels to be alpha-helical and amphipathic, and a potential helix capping box occurs at residues alpha3-8. We introduced truncations, deletions, and mutations into alphaN1-22 peptide and examined their effects on binding to the delta subunit. The deletions and mutations were introduced also into the N-terminal region of the uncA (alpha subunit) gene to determine effects on cell growth in vivo and membrane ATP synthase activity in vitro. Effects seen in the peptides were well correlated with those seen in the uncA gene. The results show that, with the possible exception of residues close to the initial Met, all of the alphaN1-22 sequence is required for binding of delta to alpha. Within this sequence, an amphipathic helix seems important. Hydrophobic residues on the predicted nonpolar surface are important for delta binding, namely alphaIle-8, alphaLeu-11, alphaIle-12, alphaIle-16, and alphaPhe-19. Several or all of these residues probably make direct interaction with helices 1 and 5 of delta. The potential capping box sequence per se appeared less important. Impairment of alpha/delta binding brings about functional impairment due to reduced level of assembly of ATP synthase in cells.
Journal of Biological Chemistry 07/2004; 279(24):25673-9. · 4.77 Impact Factor
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ABSTRACT: The stator in F(1)F(0)-ATP synthase resists strain generated by rotor torque. In Escherichia coli the b(2)delta subunit complex comprises the stator, bound to subunit a in F(0) and to alpha(3)beta(3) hexagon of F(1). Proteolysis and cross-linking had suggested that N-terminal residues of alpha subunit are involved in binding delta. Here we demonstrate that a synthetic peptide consisting of the first 22 residues of alpha ("alpha N1-22") binds specifically to isolated wild-type delta subunit with high affinity (K(d) = 130 nm), accounting for a major portion of the binding energy when delta-depleted F(1) and isolated delta bind together (K(d) = 1.4 nm). Stoichiometry of binding of alpha N1-22 to delta at saturation was 1/1, showing that in intact F(1)F(0)-ATP synthase only one of the three alpha subunits is involved in delta binding. When alpha N1-22 was incubated with delta subunits containing mutations in helices 1 or 5 on the F(1)-binding face of delta, peptide binding was impaired as was binding of delta-depleted F(1). Residues alpha 6-18 are predicted to be helical, and a potential helix capping box occurs at residues alpha 3-8. Circular dichroism measurements showed that alpha N1-22 had significant helical content. Hypothetically a helical region of residues alpha N1-22 packs with helices 1 and 5 on the F(1)-binding face of delta, forming the alpha/delta interface.
Journal of Biological Chemistry 05/2003; 278(16):13623-6. · 4.77 Impact Factor
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ABSTRACT: The stator in F1F0-ATP synthase resists strain generated by rotor torque. In Escherichia coli theb
2δ subunit complex comprises the stator, bound to subunit a in F0 and to α3β3 hexagon of F1. Proteolysis and cross-linking had suggested that N-terminal residues of α subunit are involved in binding δ. Here we demonstrate
that a synthetic peptide consisting of the first 22 residues of α (“αN1–22”) binds specifically to isolated wild-type δ subunit
with high affinity (K
d = 130 nm), accounting for a major portion of the binding energy when δ-depleted F1 and isolated δ bind together (K
d = 1.4 nm). Stoichiometry of binding of αN1–22 to δ at saturation was 1/1, showing that in intact F1F0-ATP synthase only one of the three α subunits is involved in δ binding. When αN1–22 was incubated with δ subunits containing
mutations in helices 1 or 5 on the F1-binding face of δ, peptide binding was impaired as was binding of δ-depleted F1. Residues α6–18 are predicted to be helical, and a potential helix capping box occurs at residues α3–8. Circular dichroism
measurements showed that αN1–22 had significant helical content. Hypothetically a helical region of residues αN1–22 packs
with helices 1 and 5 on the F1-binding face of δ, forming the α/δ interface.
Journal of Biological Chemistry 04/2003; 278(16):13623-13626. · 4.77 Impact Factor
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ABSTRACT: The presence of the soluble intracellular heat shock protein gp96 (an endoplasmic reticulum resident protein) at the surface of certain cell types is an intriguing phenomenon whose physiological significance has been unclear. We have shown that the active surface expression of gp96 by some immune cells is found throughout the vertebrate phylum including the Agnatha, the only vertebrate taxon whose members (lamprey, hagfish) lack an adaptive immune system. To determine whether gp96 surface expression can be modulated by pathogens, we investigated the effects of in vitro stimulation by purified lipopolysaccharide (LPS) and the heat-killed gram-negative bacteria, Escherichia coli and Aeromonas hydrophilia. Purified Xenopus B cells are readily activated and markedly proliferate in vitro in response to the heat-killed bacteria but not to purified LPS. Furthermore, messenger ribonucleic acid, and intracellular and surface protein expressions of both gp96 and immunoglobulin were upregulated only after activation of B cells by heat-killed bacteria. These data are consistent with an ancestral immunological role of gp96 as an antigen-presenting or danger-signaling molecule, or both, interacting directly with antigen-presenting cells, T cells, or natural killer cells, (or all), to trigger or amplify immune responses.
Cell Stress and Chaperones 02/2003; 8(3):265-71. · 3.01 Impact Factor
