Expression of Na+,K+-ATPase in Pichia pastoris: analysis of wild type and D369N mutant proteins by Fe2+-catalyzed oxidative cleavage and molecular modeling.
Na+,K+-ATPase (pig alpha1,beta1) has been expressed in the methylotrophic yeast Pichia pastoris. A protease-deficient strain was used, recombinant clones were screened for multicopy genomic integrants, and protein expression, and time and temperature of methanol induction were optimized. A 3-liter culture provides 300-500 mg of membrane protein with ouabain binding capacity of 30-50 pmol mg-1. Turnover numbers of recombinant and renal Na+,K+-ATPase are similar, as are specific chymotryptic cleavages. Wild type (WT) and a D369N mutant have been analyzed by Fe2+- and ATP-Fe2+-catalyzed oxidative cleavage, described for renal Na+,K+-ATPase. Cleavage of the D369N mutant provides strong evidence for two Fe2+ sites: site 1 composed of residues in P and A cytoplasmic domains, and site 2 near trans-membrane segments M3/M1. The D369N mutation suppresses cleavages at site 1, which appears to be a normal Mg2+ site in E2 conformations. The results suggest a possible role of the charge of Asp369 on the E1 <--> E2 conformational equilibrium. 5'-Adenylyl-beta,gamma-imidodi-phosphate(AMP-PNP)-Fe2+-catalyzed cleavage of the D369N mutant produces fragments in P (712VNDS) and N (near 440VAGDA) domains, described for WT, but only at high AMP-PNP-Fe2+ concentrations, and a new fragment in the P domain (near 367CSDKTGT) resulting from cleavage. Thus, the mutation distorts the active site. A molecular dynamic simulation of ATP-Mg2+ binding to WT and D351N structures of Ca2+-ATPase (analogous to Asp369 of Na+,K+-ATPase) supplies possible explanations for the new cleavage and for a high ATP affinity, which was observed previously for the mutant. The Asn351 structure with bound ATP-Mg2+ may resemble the transition state of the WT poised for phosphorylation.
Strugatsky
David
D
Department of Biological Chemistry, Weizmann Institute of Science, Rehovoth, 76100, Israel.
Gottschalk
Kay-Eberhard
KE
Goldshleger
Rivka
R
Bibi
Eitan
E
Karlish
Steven J D
SJ
D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002918:Chymotrypsin / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D016681:Genome, Fungal; D007501:Iron / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D010766:Phosphorylation; D010843:Pichia / Q000201:enzymology*; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D013552:Swine; D013696:Temperature; D013997:Time Factors
D018076:DNA, Complementary; D011994:Recombinant Proteins; D001224:Aspartic Acid; D007501:Iron; D002918:Chymotrypsin; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D010100:Oxygen