Complete replacement of basic amino acid residues with cysteines in Rickettsia prowazekii ATP/ADP translocase.
The ATP/ADP translocase (Tlc) of Rickettsia prowazekii is a basic protein with isoelectric point (pI)=9.84. It is conceivable, therefore, that basic residues in this protein are involved in electrostatic interactions with negatively charged substrates. We tested this hypothesis by individually mutating all basic residues in Tlc to Cys. Unexpectedly, mutations of only 20 out of 51 basic residues resulted in greater than 80% inhibition of transport activity. Moreover, 12 of 51Cys-substitution mutants exhibited higher than wild-type (WT) activity. At least in one case this up-effect was additive and the double mutant Lys422Cys Lys427Cys transported ATP five-fold better than WT protein. Since in these two single mutants and in the corresponding double mutant K(m)'s were similar to that of WT protein, we conclude that Tlc may have evolved a mechanism that limits the transporter's exchange rate and that at least these two basic residues play a key role in that mechanism. Based on the alignment of 16 Tlc homologs, the loss of activity in the mutants poorly correlates with charge conservation within the Tlc family. Also, despite the presence of three positively charged and one negatively charged intramembrane residues, we have failed to identify potential charge pairs (salt bridges) by either charge reversal or charge neutralization approaches.
Alexeyev
Mikhail F
MF
Laboratory of Molecular Biology, Department of Microbiology and Immunology, University of South Alabama College of Medicine, LMB Building, Mobile, AL 36688-0001, USA.
Winkler
Herbert H
HH
D024361:Amino Acids, Basic / Q000737:chemistry*; D001120:Arginine / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D003593:Cytoplasm / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008698:Mesylates / Q000737:chemistry / Q000494:pharmacology; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019897:Periplasm / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012283:Rickettsia prowazekii / Q000201:enzymology*
D024361:Amino Acids, Basic; D008698:Mesylates; C109153:(2-sulfonatoethyl)methanethiosulfonate; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D000226:Mitochondrial ADP, ATP Translocases; D001120:Arginine; D008239:Lysine; D003545:Cysteine