title: ATP binding to two sites is necessary for dimerization of nucleotide-binding domains of ABC proteins.
abstract: ATP binding cassette (ABC) transporters have a functional unit formed by two transmembrane domains and two nucleotide binding domains (NBDs). ATP-bound NBDs dimerize in a head-to-tail arrangement, with two nucleotides sandwiched at the dimer interface. Both NBDs contribute residues to each of the two nucleotide-binding sites (NBSs) in the dimer. In previous studies, we showed that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii forms ATP-bound dimers that dissociate completely following hydrolysis of one of the two bound ATP molecules. Since hydrolysis of ATP at one NBS is sufficient to drive dimer dissociation, it is unclear why all ABC proteins contain two NBSs. Here, we used luminescence resonance energy transfer (LRET) to study ATP-induced formation of NBD homodimers containing two NBSs competent for ATP binding, and NBD heterodimers with one active NBS and one binding-defective NBS. The results showed that binding of two ATP molecules is necessary for NBD dimerization. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dissociation, but two binding sites are required to form the ATP-sandwich NBD dimer necessary for hydrolysis.
authors.affiliation: Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, TX 79430-6551, USA; Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX 79430-6551, USA. Electronic address: maria.zoghbi@ttuhsc.edu.
authors.forename: Maria E
authors.initials: ME
authors.lastname: Zoghbi
authors.affiliation: Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, TX 79430-6551, USA; Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX 79430-6551, USA. Electronic address: g.altenberg@ttuhsc.edu.
authors.forename: Guillermo A
authors.initials: GA
authors.lastname: Altenberg
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics; D000255:Adenosine Triphosphate / Q000737:chemistry*; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer; D006868:Hydrolysis; D063747:Methanocaldococcus; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary
chemical_list: D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate
keywords: 8-N(3)ATP-2′,3′-biotin-long chain-hydrazide; 8-azido-ATP-biotin; ABC; ABC transporter; ATP-binding cassette; Cys-less single-Trp mutant MJ0796-C53G-C128I-G174W; Dimerization; Fluorescence; Kinetics; LRET; Luminescence resonance energy transfer; MJ; MJ-CL; MJ-K44A; MJ-K44E; MJ-S42F; MJ-Y11A; MJI; NBD; NBS; luminescence (or lanthanide-based) resonance energy transfer; mutant based on MJ in which Lys44 was replaced with Ala; mutant based on MJ in which Lys44 was replaced with Glu; mutant based on MJ in which Ser42 was replaced with Phe; mutant based on MJ in which Tyr11 was replaced with Ala; nucleotide-binding domain; nucleotide-binding site; single-Cys mutant E171Q based on MJ; single-Cys mutant G14C based on MJ-CL