ABCMdb

PMID: 25533467

Kluth M, Stindt J, Droge C, Linnemann D, Kubitz R, Schmitt L
A mutation within the extended X loop abolished substrate-induced ATPase activity of the human liver ATP-binding cassette (ABC) transporter MDR3.
J Biol Chem. 2015 Feb 20;290(8):4896-907. doi: 10.1074/jbc.M114.588566. Epub 2014 Dec 22., [PubMed]

Sentences

No. Mutations Sentence Comment

64 ABCB4 p.Glu558Gln Fermentation of MDR3 Transformed P. pastoris Cells-For large-scale expression, P. pastoris cells containing the chromosomally integrated wild type MDR3, the E558Q/E1702Q double mutant, or the Q1174E mutant gene were fermented in a 15-liter table-top glass fermenter (Applikon Biotechnology) according to the P. pastoris fermentation guidelines from Invitrogen. Login to comment 65 ABCB4 p.Glu558Gln Fermentation of MDR3 Transformed P. pastoris Cells-For large-scale expression, P. pastoris cells containing the chromosomally integrated wild type MDR3, the E558Q/E1702Q double mutant, or the Q1174E mutant gene were fermented in a 15-liter table-top glass fermenter (Applikon Biotechnology) according to the P. pastoris fermentation guidelines from Invitrogen. Login to comment 118 ABCB4 p.Glu558Gln ABCB4 p.Glu1207Gln RESULTS Expression and Purification of the Human ABC Transporter MDR3 in P. pastoris-Previously, we described the expression of wild type MDR3 and the ATP hydrolysis-deficient mutant (E558Q/E1207Q, later called the EQ/EQ mutant) in the methylotrophic yeast P. pastoris. Login to comment 136 ABCB4 p.Glu558Gln Groen et al. (7) reported an important cytotoxicity caused by expression of wild type MDR3 in HEK293T cells, which was counteracted by the single mutation E558Q of the Walker B motif of the first NBD resulting in an inactive floppase. Login to comment 156 ABCB4 p.Glu558Gln ABCB4 p.Glu1207Gln A, human wild type MDR3, the E558Q/E1207Q double mutant, and the Q1174E mutant purified from P. pastoris. Login to comment 182 ABCB4 p.Glu558Gln ABCB4 p.Glu1207Gln of purifications Km (MgATP) vmax kcat mg/100 g wet cell weight mM nmol minafa;1 mgafa;1 safa;1 Wild type 6.3 afe; 1.2 8 2.17 afe; 0.20 354 afe; 13 0.83 afe; 0.03 1.78 afe; 0.10a 536 afe; 11a 1.26 afe; 0.03a Wild type-BODIPY 1.26 afe; 0.10 186 afe; 6 0.44 afe; 0.01 1.43 afe; 0.19a 175 afe; 10a 0.41 afe; 0.02a E558Q/E1207Q 3.4 afe; 0.6 5 b b b Q1174E 2.0 afe; 0.2 5 1.04 afe; 0.15 286 afe; 16 0.64 afe; 0.04 Q1174E-BODIPY 0.74 afe; 0.10 198 afe; 5 0.46 afe; 0.01 a ATPase activity was in the presence of 300 òe;M DOPC lipids. Login to comment 220 ABCB10 p.Glu602Arg ABCB10 p.Glu602Asp Oancea et al. (24) substituted the conserved glutamate (Glu-602) of the X loop of the transporter associated with antigen processing (TAP1/2) and demonstrated that peptide binding was not affected; however, transport activity was reduced from 20% for the E602D mutant to complete disruption for the E602R mutant, suggesting a pivotal role in transmitting conformational changes generated by ATP hydrolysis and substrate translocation. Login to comment 227 ABCB4 p.Gln1181Glu Thus, we used this structural model of MDR3 to generate the structure of the MDR3 Q1181E mutant (Q1174E isoform A). Login to comment 269 ABCB4 p.Glu558Gln ABCB4 p.Glu1207Gln ATPase Activity of Human MDR3 4904 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290ߦNUMBER 8ߦFEBRUARY 20, 2015 at SEMMELWEIS UNIV OF MEDICINE on December , Cross-linking of wild type MDR3 with maleimide-BODIPY blocks basal and PC-induced ATPase activity as demonstrated for MDR1 (33-35), whereas the ATP hydrolysis-deficient EQ double mutant (E558Q/E1207Q) showed no PC stimulation, and ATPase activity of the labeled EQ/EQ mutant was only marginally reduced (Table 1 and Fig. 1). Login to comment 270 ABCB4 p.Glu558Gln ABCB4 p.Glu1207Gln ATPase Activity of Human MDR3 4904 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290ߦNUMBER 8ߦFEBRUARY 20, 2015 at SEMMELWEIS UNIV OF MEDICINE on December , Cross-linking of wild type MDR3 with maleimide-BODIPY blocks basal and PC-induced ATPase activity as demonstrated for MDR1 (33-35), whereas the ATP hydrolysis-deficient EQ double mutant (E558Q/E1207Q) showed no PC stimulation, and ATPase activity of the labeled EQ/EQ mutant was only marginally reduced (Table 1 and Fig. 1). Login to comment 309 ABCB1 p.Asp164Gln They demonstrated that the amino acid residue Gln-1175 of NBD2, which is identical to Gln-1174 in MDR3, hydrogen bonds with Asp-164 (Asp-166 in MDR3) of the ICL1 and identified this residue pair Asp-164-Gln-1175 as key residue pair in the transmission interface. Login to comment 310 ABCB1 p.Asp164Gln They demonstrated that the amino acid residue Gln-1175 of NBD2, which is identical to Gln-1174 in MDR3, hydrogen bonds with Asp-164 (Asp-166 in MDR3) of the ICL1 and identified this residue pair Asp-164-Gln-1175 as key residue pair in the transmission interface. Login to comment