Exchange, efflux and substrate binding by cysteine mutants of the lactose permease of Escherichia coli

Iwaarden, Pierre R. van; Driessen, Arnold J.M.; Lolkema, Juke S.; Kaback, H. Ronald; Konings, Wn

doi:10.1021/bi00071a017

Cited by 38 publications

(52 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Mutation C154G results in a phenotype that binds ligand but does little or no translocation of lactose across the membrane (42,43,44), and the protein appears to be locked in an outwardly facing conformation (44). When this mutation is combined with the A122C/C148A mutations, LacY binds ligand with about 10 times greater affinity than mutant A122C/ C148A but catalyzes essentially no lactose translocation.…”

Section: Discussionmentioning

confidence: 99%

“…However, the marked increase in the rate of reaction in the presence of ⌬ H ϩ suggests more interesting possibilities. To investigate the problem further, the A122C/C148A mutations were combined with C154G, a mutation that leads to increased binding affinity but almost complete loss of transport activity (42,43,44). RSO vesicles with mutant A122C/C148A/C154G exhibit a K d of about 20 M, about 10-fold lower than mutant A122C/C148A, as determined by TDG protection against alkylation with NEM (Table I).…”

Section: Inactivation By Mts-galactoside Occurs At Amentioning

confidence: 99%

See 1 more Smart Citation

Probing the Mechanism of a Membrane Transport Protein with Affinity Inactivators

Guan

Sahin–Tóth

Kálai

et al. 2003

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Affinity inactivators are useful for probing catalytic mechanisms. Here we describe the synthesis and properties of methanethiosulfonyl (MTS) galactose or glucose derivatives with respect to a well studied membrane transport protein, the lactose permease of Escherichia coli. The MTS-galactose derivatives behave as affinity inactivators of a functional mutant with Ala 122 3 Cys in a background otherwise devoid of Cys residues. A proton electrochemical gradient (⌬ H ؉) markedly increases the rate of reaction between Cys 122 and MTS-galactose derivatives; nonspecific labeling with the corresponding MTS-glucose derivatives is unaffected. When the Ala 122 3 Cys mutation is combined with a mutation (Cys 154 3 Gly) that blocks transport but increases binding affinity, discrimination between the MTS-galactose and -glucose derivatives is abolished, and ⌬ H ؉ has no effect. The results provide strong confirmation that the non-galactosyl moiety of permease substrates abuts Ala 122 in helix IV. In addition, the findings demonstrate that the MTS-galactose derivatives do not react with the Cys residue at position 122 upon binding per se but at a subsequent step in the overall transport mechanism. Thus, these inactivators behave as unique suicide substrates.The lactose permease (LacY) 1 of Escherichia coli transduces the free energy stored in an electrochemical H ϩ gradient into a concentration gradient of D-galactopyranosides and vice versa (galactoside/H ϩ symport) (1-4). LacY is a 12-transmembranehelix bundle with the N and C termini on the cytoplasmic face of the membrane (5-7). Several lines of evidence indicate that it is both functionally (8) and structurally a monomer (9 -11).Analysis of an extensive library of mutants, particularly Cys replacement mutants (12), with a battery of site-directed biophysical and biochemical techniques has led to the formulation of a teriary structure model (49) that includes tilts as well as a working model for the transport mechanism (13).LacY is in close proximity to the nongalactosyl portion of ligand (24). Affinity labeling is based upon specific recognition of an inactivating ligand at the binding site. Because Cys 148 (helix V), which is in close proximity to Ala 122 , makes direct contact with the galactopyranosyl moiety of the substrate, numerous attempts to develop an affinity reagent specific for this side chain have failed. On the other hand, Ala 122 abuts the nongalactosyl moiety of disaccharide substrates, and alkyation of a Cys residue at this position blocks binding of disaccharides with no effect on galactose transport or binding. Therefore, methanethiosulfonyl (MTS) galactoside with an ethylene linkage between the galactopyranoside ring and MTS and MTS-5-galactose with a carbamide linkage were synthesized as potential affinity reagents for the single-Cys A122C mutant in LacY with the corresponding glucosides as controls. EXPERIMENTAL PROCEDURES ReagentsMTS-galactoside and MTS-glucoside-MTS-galactoside and MTSglucoside were synthesized as shown in the synthetic scheme (Sch...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Inactivation By Mts-galactoside Occurs At Amentioning

confidence: 99%

Probing the Mechanism of a Membrane Transport Protein with Affinity Inactivators

Guan

Sahin–Tóth

Kálai

et al. 2003

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Interestingly, the LacY(C148S) mutant displays monophasic (non-equilibrium exchange) kinetics with only the high affinity state for lactose, whereas the wild-type protein exhibits biphasic kinetics with both a high and low icapp [279]. The maximal rates of efflux ~tx M and exchange of the C148S mutant are about 5-fold reduced relative to the wild-type LacY protein, and high affinity binding of NPG is no longer detected in LacY(C148S).…”

Section: Vii-c Cysteine Residuesmentioning

confidence: 97%

“…Specifically, it has been suggested that in LacY sulfhydryl-disulfide interchange occurs either as intermediate of the respiratory chain [272] or as proton carrier that is affected by Ap-mediated redox changes [273,274]. However, characterization of LacY mutated at each of the 8 cysteinyl residues in the molecule has demonstrated that none of the cysteines is essential for transport [275]. Even the cysteine-less LacY protein displays significant lactose-H + symport activity [276].…”

Section: Vii-c Cysteine Residuesmentioning

confidence: 99%

See 1 more Smart Citation

Secondary solute transport in bacteria

Poolman

Konings

1993

Biochimica et Biophysica Acta (BBA) - Bioenergetics

Self Cite

202

160

View full text Add to dashboard Cite

Ligand‐Induced conformational changes in the lactose permease of escherichia coli: Evidence for two binding sites

Frillingos

Voss

et al. 1994

Protein Science

View full text Add to dashboard Cite

By using a lactose permease mutant containing a single Cys residue in place of Val 331 (helix X), conformational changes induced by ligand binding were studied. With right-side-out membrane vesicles containing Val 331 + Cys permease, lactose transport is inactivated by either N-ethylmaleimide (NEM) or 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). Remarkably, 0,D-galactopyranosyl I-thio-0,D-galactopyranoside (TDG) enhances the rate of inactivation by CPM, a hydrophobic sulfhydryl reagent, whereas NEM inactivation is attenuated by the ligand. Val 33 1 + Cys permease was then purified and studied in dodecyl-0,D-maltoside by site-directed fluorescence spectroscopy. The reactivity of Val 331 "+ Cys permease with 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS) is not changed over a low range of TDG concentrations (<0.8 mM), but the fluorescence of the MIANS-labeled protein is quenched in a saturable manner (apparent Kd 0.12 mM) without a change in emission maximum. In contrast, over a higher range of TDG concentrations (1-10 mM), the reactivity of Val 331 + Cys permease with MIANS is enhanced and the emission maximum of MIANS-labeled permease is blue shifted by 3-7 nm. Furthermore, the fluorescence of MIANS-labeled Val 331 --t Cys permease is quenched by both acrylamide and iodide, but the former is considerably more effective. A low concentration of TDG (0.2 mM) does not alter quenching by either compound, whereas a higher concentration of ligand (10mM) decreases the quenching constant for iodide by about 50% and for acrylamide by about 20%. Finally, the EPR spectrum of nitroxide spin-labeled Val 331 + Cys permease exhibits 2 components with different mobilities, and TDG causes the immobilized component to increase. The results provide evidence for the argument that lac permease has more than a single binding site. TDG binding to a higher affinity site quenches the fluorescence of MIANS-labeled Val 33 1 + Cys permease, and occupation of a second lower affinity site causes position 33 1 to become more accessible from a hydrophobic environment.Keywords: C-less permease; ligand-protein interaction; MIANS; site-directed fluorescence; site-directed spin labeling Lactose permease of Escherichia coli is a hydrophobic, polytopic, plasma membrane protein that catalyzes the coupled stoichiometric translocation of 0-galactosides and H + (reviewed in Kaback, , 1989Kaback, , 1992. The permease is encoded by the lacy Reprint requests to: H. Ronald Kaback, HHMI/UCLA, 6-720 MacDonald Building, 10833 Le Conte Avenue, Los Angeles, California 90024-1662; e-mail: ronaldk@hhmi.ucla.edu.Abbreviurions: lac, lactose; C-less permease, functional lac permease devoid of Cys residues; NEM, N-ethylmaleimide; CPM, 7-diethylamino-3-(4"maleimidylphenyl)-4-methylcoumarin; MIANS, 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid; A H + , the H + electrochemical gradient across the membrane; dodecylmaltoside, dodecyl-0,D-maltoside; KPi, potassium phosphate; TDG, 6,D-galctopyranosyl 1-thio-0,D-galactopyranoside;...

show abstract

Exchange, efflux and substrate binding by cysteine mutants of the lactose permease of Escherichia coli

Abstract: Citation for published version (APA): Iwaarden, P. R. V., Driessen, A.

Cited by 38 publications

References 34 publications

Probing the Mechanism of a Membrane Transport Protein with Affinity Inactivators

Probing the Mechanism of a Membrane Transport Protein with Affinity Inactivators

Secondary solute transport in bacteria

Ligand‐Induced conformational changes in the lactose permease of escherichia coli: Evidence for two binding sites

Contact Info

Product

Resources

About