The intact CFTR protein mediates ATPase rather than adenylate kinase activity.
The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain-domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.
Ramjeesingh
Mohabir
M
Programme of Structural Biology and Biochemistry, Research Institute of the Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8.
Ugwu
Francisca
F
Stratford
Fiona L L
FL
Huan
Ling-Jun
LJ
Li
Canhui
C
Bear
Christine E
CE
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D015226:Dinucleoside Phosphates / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D002712:Chlorides; D015226:Dinucleoside Phosphates; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D000263:Adenylate Kinase; D000251:Adenosine Triphosphatases