Characterization of the Reaction Cycle of MJ0796: A Model Archaeal Adenosine Triphosphate-Binding Cassette Transporter Nucleotide Binding Domain

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2006-12-20

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Moody, Jonathan Edward

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Abstract

Adenosine-triphosphate binding cassette (ABC) transporters couple nucleotide hydrolysis to vectorial transport of solutes across lipid bilayers. These proteins, found in all kingdoms of life, have been implicated in a variety of human genetic disorders and engender drug resistance in cancer cells and infectious prokaryotes. Despite the widely varying solutes transported by these protein machines, a conserved functional mechanism is suggested by the high degree of amino acid conservation found in the nucleotide binding domains of all ABC transporters. Using two model archaeal ABC transporter nucleotide binding domains, MJ0796 and MJ1267, from Methanocaldococcus jannaschii the highly conserved Walker A, Walker B, and LSGGQ motifs were probed using site-directed mutagenesis. Catalytic carboxylate mutants, MJ0796-E171Q and MJ1267-E179Q, exhibited nucleotide-dependent dimerization upon analytical gel filtration and equilibrium centrifugation experiments. This self-association was negatively affected by changes in the electrostatic environment, as shown using alanine substitutions at these loci as well as altering the ionic conditions of the experiments. The MJ0796-E171Q protein was crystallized, and its structure solved to 1.9 angstrom resolution. The structure reveals an ATP sandwich dimer with two nucleotides bound at the dimeric interface, with each binding site composed of Walker A and B residues from one monomer and LSGGQ residues from the opposing monomer.
A proposed reaction cycle based upon the MJ0796-E171Q dimer structure was probed using Walker A, Walker B, and LSGGQ point mutants. Mixtures of the Walker A mutant MJ0796-K44A with LSGGQ mutant MJ0796-S147F, both hydrolytically deficient in isolation, did not exhibit activity. In stark contrast, mixtures of MJ0796-S147F and MJ0796-E171Q did exhibit 25% wild type activity, suggesting a mechanism whereby two nucleotide binding events and a single hydrolysis event complete the minimal reaction cycle. This also suggests that during wild type hydrolysis, two nucleotides are hydrolyzed per cycle. These heterodimeric mutant mixtures were further analyzed using tryptophan fluorescence emission and anisotropy. Mixing experiments were performed using a full transporter system, the lipoprotein release machinery from Escherichia coli. A modified ABC transporter reaction cycle is presented.

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