Large scale purification of detergent-soluble P-glycoprotein from Pichia pastoris cells and characterization of nucleotide binding properties of wild-type, Walker A, and Walker B mutant proteins

P-glycoprotein (Pgp; mouse MDR3) was expressed in Pichia pastoris, grown in fermentor culture, and purified. The final pure product is of high specific ATPase activity and is soluble at low detergent concentration. 120 g of cells yielded 6 mg of pure Pgp; >4 kg of cells were obtained from a single fermentor run. Properties of the pure protein were similar to those of previous preparations, except there was significant ATPase activity in absence of added lipid. Mutant mouse MDR3 P-glycoproteins were purified by the same procedure after growth of cells in flask culture, with similar yields and purity. This procedure should open up new avenues of structural, biophysical, and biochemical studies of Pgp. Equilibrium nucleotide-binding parameters of wild-type mouse MDR3 Pgp were studied using 2'-(3')-O-(2,4,6-trinitrophenyl)adenosine tri- and diphosphate. Both analogs were found to bind with K(d) in the low micromolar range, to a single class of site, with no evidence of cooperativity. ATP displacement of the analogs was seen. Similar binding was seen with K429R/K1072R and D551N/D1196N mutant mouse MDR3 Pgp, showing that these Walker A and B mutations had no significant effect on affinity or stoichiometry of nucleotide binding. These residues, known to be critical for catalysis, are concluded to be involved primarily in stabilization of the catalytic transition state in Pgp.     

Properties of P-glycoprotein with mutations in the "catalytic carboxylate" glutamate residues

It is known from earlier work that two conserved Glu residues, designated "catalytic carboxylates," are critical for function in P-glycoprotein (Pgp). Here the role of these residues (Glu-552 and Glu-1197 in mouse MDR3 Pgp) was studied further. Mutation E552Q or E1197Q reduced Pgp-ATPase to low but still measurable rates. Two explanations previously offered for effects of these mutations, namely that ADP release is slowed or that a second (drug site-resetting) round of ATP hydrolysis is blocked, were evaluated and appeared unsatisfactory. Thus the study was extended to include E552A, -D, and -K and E1197A, -D, and -K mutants. All reduced ATPase to similar low but measurable rates. Orthovanadate-trapping experiments showed that mutation to Gln, Ala, Asp, or Lys altered characteristics of the transition state but did not eliminate its formation in contrast e.g. with mutation of the analogous catalytic Glu in F1-ATPase. Retention of ATP as well as ADP was seen in Ala, Asp, and Lys mutants. Mutation E552A in nucleotide binding domain 1 (NBD1) was combined with mutation S528A or S1173A in the LSGGQ sequence of NBD1 or NBD2, respectively. Synergistic effects were seen. E552A/S1173A had extremely low turnover rate for ATPase, while E552A/S528A showed zero or close to zero ATPase. Both showed orthovanadate-independent retention of ATP and ADP. We propose that mutations of the catalytic Glu residues interfere with formation and characteristics of a closed conformation, involving an interdigitated NBD dimer interface, which normally occurs immediately following ATP binding and progresses to the transition state.     

Quantitative determination of direct binding of b subunit to F1 in Escherichia coli F1F0-ATP synthase

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 the alpha(3)beta(3) hexagon of F(1). To quantitatively characterize binding of b subunit to the F(1) alpha(3)beta(3) hexagon, we developed fluorimetric assays in which wild-type F(1), or F(1) enzymes containing introduced Trp residues, were titrated with a soluble portion of the b subunit (b(ST34-156)). With five different F(1) enzymes, K(d)(b(ST34-156)) ranged from 91 to 157 nm. Binding was strongly Mg(2+)-dependent; in EDTA buffer, K(d)(b(ST34-156)) was increased to 1.25 microm. The addition of the cytoplasmic portion of the b subunit increases the affinity of binding of delta subunit to delta-depleted F(1). The apparent K(d)(b(ST34-156)) for this effect was increased from 150 nm in Mg(2+) buffer to 1.36 microm in EDTA buffer. This work demonstrates quantitatively how binding of the cytoplasmic portion of the b subunit directly to F(1) contributes to stator resistance and emphasizes the importance of Mg(2+) in stator interactions.     

Atrazine catabolism by a combined bacterial association (KRA30) under carbon- and nitrogen-limitations in a retentostat

AIMS: Nutrient-limited atrazine catabolism study in continuous cultures with biomass retention to mimic in situ environmental conditions and thus gain insight of the efficacy of biosupplementation/biostimulation to eliminate reduced herbicide bioavailability. METHODS AND RESULTS: Carbon- and nitrogen-limited retentostat (1 and 5 l) cultivation of a combined atrazine (100 mg l-1)-catabolizing association KRA30 was made. As a nitrogen source, through citrate supplementation, increased herbicide catabolism resulted and was complete in the absence of NH4-N. Co-metabolism of the molecule in the presence of succinate was identified. Population characterization by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) indicated component species numerical dominance shifts in response to changes in nutrient limitation, mineral salts composition and biofilm formation, although the total species complement and catabolic potential were retained. CONCLUSIONS: Biomass and catabolic capacity maintenance, through cost-effective biosupplementation/biostimulation, should promote atrazine bioavailability and so ensure successful amelioration. SIGNIFICANCE AND IMPACT OF THE STUDY: All planning, implementation and monitoring of bioremediation programmes should be underpinned by a combination of molecular and (continuous) culture-based methods.     

The Three-Dimensional Solution Structure of the Src Homology Domain-2 of the Growth Factor Receptor-Bound Protein-2

A set of high-resolution three-dimensional solution structures of the Src homology region-2 (SH2) domain of the growth factor receptor-bound protein-2 was determined using heteronuclear NMR spectroscopy. The NMR data used in this study were collected on a stable monomeric protein solution that was free of protein aggregates and proteolysis. The solution structure was determined based upon a total of 1439 constraints, which included 1326 nuclear Overhauser effect distance constraints, 70 hydrogen bond constraints, and 43 dihedral angle constraints. Distance geometry-simulated annealing calculations followed by energy minimization yielded a family of 18 structures that converged to a root-mean-square deviation of 1.09 Å for all backbone atoms and 0.40 Å for the backbone atoms of the central -sheet. The core structure of the SH2 domain contains an antiparallel -sheet flanked by two parallel -helices displaying an overall architecture that is similar to other known SH2 domain structures. This family of NMR structures is compared to the X-ray structure and to another family of NMR solution structures determined under different solution conditions.     

Solubilization of adenosine triphosphatase from membranes of Escherichia coli: effect of p-aminobenzamidine.

The five subunits of the membrane-bound adenosine triphosphatase (F1) from Escherichia coli were identified on electrophoretograms of membranes which had been washed with a low-ionic-strength buffer containing the protease inhibitor p-aminobenzamidine. All of the subunits of the membrane-bound F1 appeared to have the same molecular weights and isoelectric points as those of the soluble F1, as judged by two-dimensional electrophoresis. p-Aminobenzamidine inhibited the solubilization of F1 rebound to F1-depleted membranes, and was found to inhibit the membrane-bound adenosine triphosphatase activity to a much greater extent than the solubilized activity. It is therefore unlikely that p-aminobenzamidine inhibits the solubilization of F1 by inhibiting a protease, as suggested previously by Cox et al. (G.B. Cox, J.A. Downie, D.R.H. Fayle, F. Gibson, and J. Radik, J. Bacteriol. 133:287--292, 1978).     

The uncA gene codes for the alpha-subunit of the adenosine triphosphatase of Escherichia coli. Electrophoretic analysis of uncA mutant strains.

Four mutant strains of Escherichia coli which lack membrane-bound adenosine triphosphatase activity were shown by genetic-complementation tests to carry mutations in the uncA gene. A soluble inactive F1-ATPase aggregate was released from the membranes of three of the uncA mutant strains by low-ionic-strength washing, and purified by procedures developed for the purification of F1-ATPase from normal strains. Analysis of the subunit structure by two-dimensional gel electrophoresis indicated that the F1-ATPase in strains carrying the uncA401 or uncA453 alleles had a subunit structure indistinguishable from normal F1-ATPase. In contrast, the F1-ATPase from the strain carrying the uncA447 allele contained an alpha-subunit of normal molecular weight, but abnormal net charge. Membranes from strains carrying the uncA450 allele did not have F1-ATPase aggregates that could be solubilized by low-ionic-strength washing. However, a partial dipolid strain carrying both the uncA+ and uncA450 alleles formed an active F1-ATPase aggregate which could be solubilized by low-ionic-strength washing of the membranes and which contained two types of alpha-subunit, one of which was normal and the other had abnormal net charge. It is concluded that the uncA gene codes for the alpha-subunit of the adenosine triphosphatase.     

Removal of "tightly bound" nucleotides from soluble mitochondrial adenosine triphosphatase (F1).

Soluble mitochondrial ATPase (F1) from beef heart prepared in this laboratory contained approximately 1.8 mol of ADP and 0 mol of ATP/mol of F1 which were not removed by repeated precipitation of the enzyme with ammonium sulfate solution or by gel filtration in low ionic strength buffer containing EDTA. This enzyme had full coupling activity. Treatment of the enzyme with trypsin (5 mug/mg of F1 for 3 min) reduced the "tightly bound" ADP to zero, abolished coupling activity, but had no effect on the ATPase activity, stability, or membrane-binding capability of the F1. When the trypsin concentration was varied between 0 and 5 mug/mg of F1, tightly bound ADP was removed to varying degrees, and a correlation was seen between amount of residual tightly bound ADP and residual coupling activity. Gel filtration of the native F1 in high ionic strength buffer containing EDTA also caused complete loss of tightly bound ADP and coupling ability, whereas ATPase activity, stability, and membrane-binding capability were retained. The ADP-depleted F1 preparations were unable to rebind normal amounts of ADP or any ATP in simple reloading experiments. The results strongly suggest that tightly bound ADP is required for ATP synthesis and for energy-coupled ATP hydrolysis on F1. The results also suggest that ATP synthesis and energy-linked ATP hydrolysis rather than involving one nucleotide binding site on F1, involve a series or "cluster" of sites. The ATP hydrolysis site may represent one component of this cluster. The results show that nonenergy-coupled ATP hydrolysis on F1 can occur in the absence of tightly bound ADP or ATP.     

Removal of "tightly bound" nucleotides from phosphorylating submitochondrial particles.

Phosphorylating submitochondrial particles from beef heart (ETPH) prepared here contained about 2.4 nmol of ATP and 1.9 nmol of ADP/mg of protein after repeated washing of the particles. Essentially all of the "tightly bound " ATP and ADP was removed by trypsin treatment. The trypsin-treated ETPH had increased ATPase activity, undiminished NADH oxidase and succinate oxidase activity, but energy-coupling activity (ATP-driven reversed electron transfer) was abolished. Removal of half the ATP and ADP occurred at low levels of trypsin and was associated with loss of half of the coupling activity. Gel filtration of ETPH in high ionic strength buffer also removed ADP and ATP from the particles, resulting in loss of energy-coupling activity, while ATPase activity was increased. The results support the contention that the tightly bound ADP is essential in energy coupling in mitochondria. Tightly bound ATP may also play an essential role.