Premium
ATP‐driven dimerization of nucleotide binding domains, asymmetric occlusion of one nucleotide and ADP‐driven disassembly of the occluded state are important reaction intermediates of the P‐glycoprotein transport cycle
Author(s) -
Sauna Zuben Erach,
Kim InWha,
Nandigama Krishnamachary,
Ambudkar Suresh V
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a242-b
Subject(s) - nucleotide , dimer , atp binding cassette transporter , atp hydrolysis , biochemistry , adenosine triphosphate , chemistry , walker motifs , biophysics , stereochemistry , atpase , transporter , biology , enzyme , gene , organic chemistry
The ATP‐binding cassette (ABC) transport proteins have a central role in biology and are important clinical targets. Early mechanistic studies used P‐glycoprotein (Pgp) an ABC protein that confers multidrug resistance to cancer cells. Recent structural studies of nucleotide binding domains of bacterial ABC proteins have led to several new concepts. (i) Binding of ATP at each of the two ATP sites results in the formation of a symmetric dimer. (ii) Formation of ADP during hydrolysis disassembles this dimer. (iii) The closed dimer induces conformational changes in the transmembrane domains (TMDs), converting a high‐affinity transport‐substrate site to a low‐affinity site. To test these hypotheses we used a mutant of Pgp (E556Q/E1201Q), where ATP hydrolysis is severely impaired or ATP‐γ‐S a non‐hydrolyzable ATP analog. Both strategies result in occlusion of the nucleoside triphosphate (but not the diphosphate) in a non‐exchangeable form and the stoichiometry of nucleotide:Pgp is 1 mol/mol. Thus the ATP‐driven dimer is an important step in the transport cycle, however, though two ATP molecules may initially bind to form a symmetric dimer only one forms an asymmetric occluded transition state. This occlusion of nucleotide results in the high‐affinity to a low‐affinity switch at the TMDs. The asymmetric occlusion of ATP is consistent with the structures of intact ABC proteins and recent molecular dynamic simulations.