The conserved dipole in transmembrane helix 5 of KdpB in the Escherichia coli KdpFABC P-type ATPase is crucial for coupling and the electrogenic K+-Translocation step

DC FieldValueLanguage
dc.contributor.authorBecker, Doris
dc.contributor.authorFendler, Klaus
dc.contributor.authorAltendorf, Karlheinz
dc.contributor.authorGreie, Joerg-Christian
dc.date.accessioned2021-12-23T16:07:08Z-
dc.date.available2021-12-23T16:07:08Z-
dc.date.issued2007
dc.identifier.issn00062960
dc.identifier.urihttps://osnascholar.ub.uni-osnabrueck.de/handle/unios/7720-
dc.description.abstractThe KdpFABC complex of Escherichia coli, a high-affinity K+-uptake system, belongs to the group of P-type ATPases and is responsible for ATP-driven K+ uptake in the case of K+ limitation. Sequence alignments identified two conserved charged residues, D583 and K586, which are located at the center of transmembrane helix 5 (TM 5) of the catalytic KdpB subunit, and which are supposed to establish a dipole involved in energy coupling. Cells in which the two charges were eliminated or inverted by mutagenesis displayed a clearly slower growth rate with respect to wild-type cells under K+-limiting conditions. Purified KdpFABC complexes from several K586 mutants and a D583K:K586D double mutant showed a reduced K+-stimulated ATPase activity together with an increased resistance to orthovanadate. Upon reconstitution into liposomes, only the conservative K586R mutant was able to facilitate K+ transport, whereas the elimination of the positive charge at position 586 as well as inverting the charges at positions 583 and 586 (D583K:K586D) led to an uncoupling of ATP hydrolysis and K+ transport. Electrophysiological measurements with KdpFABC-containing proteoliposomes adsorbed to planar lipid bilayers revealed that in case of the D583K:K586D double mutant the characteristic K+-independent electrogenic step within the reaction cycle is lacking, thereby clearly arguing for an exact positioning of the dipole for coupling within the functional enzyme complex. In addition, these findings strongly suggest that the dipole residues in KdpB are not directly responsible for the characteristic electrogenic reaction step of KdpFABC, which most likely occurs within the K+-translocating KdpA subunit.
dc.language.isoen
dc.publisherAMER CHEMICAL SOC
dc.relation.ispartofBIOCHEMISTRY
dc.subjectAMINO-ACID SUBSTITUTIONS
dc.subjectBINDING
dc.subjectBiochemistry & Molecular Biology
dc.subjectCA2+-ATPASE
dc.subjectCALCIUM-PUMP
dc.subjectCOMPLEX
dc.subjectCRYSTAL-STRUCTURE
dc.subjectPURIFICATION
dc.subjectSARCOPLASMIC-RETICULUM
dc.subjectSUBUNIT
dc.subjectTRANSPORT ATPASE
dc.titleThe conserved dipole in transmembrane helix 5 of KdpB in the Escherichia coli KdpFABC P-type ATPase is crucial for coupling and the electrogenic K+-Translocation step
dc.typejournal article
dc.identifier.doi10.1021/bi701394h
dc.identifier.isiISI:000251241500025
dc.description.volume46
dc.description.issue48
dc.description.startpage13920
dc.description.endpage13928
dc.contributor.orcid0000-0002-5699-7060
dc.publisher.place1155 16TH ST, NW, WASHINGTON, DC 20036 USA
dcterms.isPartOf.abbreviationBiochemistry
crisitem.author.deptFB 05 - Biologie/Chemie-
crisitem.author.deptidfb05-
crisitem.author.parentorgUniversität Osnabrück-
crisitem.author.netidAlKa770-
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