Characterization of the Reaction Cycle of Mj0796: A Model Archaeal Adenosine Triphosphate- Binding Cassette Transporter Nucleotide Binding Domain

Show full item record

Title: Characterization of the Reaction Cycle of Mj0796: A Model Archaeal Adenosine Triphosphate- Binding Cassette Transporter Nucleotide Binding Domain
Author: Moody, Jonathan Edward
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 .
URI: http : / /hdl .handle .net /2152 .5 /658
Date: 2006-12-20

Citation

Characterization of the Reaction Cycle of Mj0796: A Model Archaeal Adenosine Triphosphate- Binding Cassette Transporter Nucleotide Binding Domain. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /658 .

Files in this item

Files Size Format View
moodyjonathan.pdf 7.945Mb application/pdf View/Open

This item appears in the following Collection(s)

Show full item record

Search DSpace

Advanced Search

Browse