scholarly journals Nucleotide-binding Domains of Cystic Fibrosis Transmembrane Conductance Regulator, an ABC Transporter, Catalyze Adenylate Kinase Activity but Not ATP Hydrolysis

2006 ◽  
Vol 281 (7) ◽  
pp. 4058-4068 ◽  
Author(s):  
Christian H. Gross ◽  
Norzehan Abdul-Manan ◽  
John Fulghum ◽  
Judith Lippke ◽  
Xun Liu ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Abc Transporter ◽  
Adenylate Kinase ◽  
Kinase Activity ◽  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Cystic Fibrosis Transmembrane

scholarly journals Inhibition of ATPase, GTPase and adenylate kinase activities of the second nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator by genistein

1999 ◽  
Vol 340 (1) ◽  
pp. 227-235 ◽  
Author(s):  
Christoph RANDAK ◽  
Ennes A. AUERSWALD ◽  
Irmgard ASSFALG-MACHLEIDT ◽  
William W. REENSTRA ◽  
Werner MACHLEIDT
Keyword(s):  
Cystic Fibrosis ◽  
Adenylate Kinase ◽  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Enhanced Fluorescence ◽  
Chloride Currents ◽  
Cystic Fibrosis Transmembrane ◽  
Ordered State

In the presence of ATP, genistein, like the ATP analogue adenosine 5ʹ-[β,γ-imido]triphosphate (pp[NH]pA), increases cystic fibrosis transmembrane conductance regulator (CFTR) chloride currents by prolonging open times. As pp[NH]pA is thought to increase CFTR currents by interfering with ATP hydrolysis at the second nucleotide-binding fold (NBF-2), the present study was undertaken to investigate the effects of genistein on a fusion protein comprising maltose-binding protein (MBP) and NBF-2 (MBP-NBF-2). MBP-NBF-2 exhibited ATPase, GTPase and adenylate kinase activities that were inhibited by genistein in a partial non-competitive manner with respect to ATP or GTP. Ki values for competitive and uncompetitive inhibition were respectively 20 μM and 63 μM for ATPase, 15 μM and 54 μM for GTPase, and 46 μM and 142 μM for adenylate kinase. For ATPase activity, genistein reduced Vmax by 29% and Vmax/Km by 77%. Additional evidence for complex-formation between genistein and MBP-NBF-2 was obtained by the detection of genistein-dependent alterations in the CD spectrum of MBP-NBF-2 that were consistent with the formation of a higher-ordered state. Addition of MBP-NBF-2 increased the fluorescence intensity of genistein, consistent with a change to a less polar environment. pp[NH]pA partially eliminated this enhanced fluorescence of genistein. These observations provide the first direct biochemical evidence that genistein interacts with CFTR, thus inhibiting NBF-2 activity, and suggest a similar mechanism for genistein-dependent stimulation of CFTR chloride currents.

Mg2+-dependent ATP occlusion at the first nucleotide-binding domain (NBD1) of CFTR does not require the second (NBD2)

2008 ◽  
Vol 416 (1) ◽  
pp. 129-136 ◽  
Author(s):  
Luba Aleksandrov ◽  
Andrei Aleksandrov ◽  
John R. Riordan
Keyword(s):  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Bivalent Cation ◽  
Binding Domains ◽  
Rapid Hydrolysis ◽  
Cystic Fibrosis Transmembrane

ATP binding to the first and second NBDs (nucleotide-binding domains) of CFTR (cystic fibrosis transmembrane conductance regulator) are bivalent-cation-independent and -dependent steps respectively [Aleksandrov, Aleksandrov, Chang and Riordan (2002) J. Biol. Chem. 277, 15419–15425]. Subsequent to the initial binding, Mg2+ drives rapid hydrolysis at the second site, while promoting non-exchangeable trapping of the nucleotide at the first site. This occlusion at the first site of functional wild-type CFTR is somewhat similar to that which occurs when the catalytic glutamate residues in both of the hydrolytic sites of P-glycoprotein are mutated, which has been proposed to be the result of dimerization of the two NBDs and represents a transient intermediate formed during ATP hydrolysis [Tombline and Senior (2005) J. Bioenerg. Biomembr. 37, 497–500]. To test the possible relevance of this interpretation to CFTR, we have now characterized the process by which NBD1 occludes [32P]N3ATP (8-azido-ATP) and [32P]N3ADP (8-azido-ADP). Only N3ATP, but not N3ADP, can be bound initially at NBD1 in the absence of Mg2+. Despite the lack of a requirement for Mg2+ for ATP binding, retention of the NTP at 37 °C was dependent on the cation. However, at reduced temperature (4 °C), N3ATP remains locked in the binding pocket with virtually no reduction over a 1 h period, even in the absence of Mg2+. Occlusion occurred identically in a ΔNBD2 construct, but not in purified recombinant NBD1, indicating that the process is dependent on the influence of regions of CFTR in addition to NBD1, but not NBD2.

Sign in / Sign up

Export Citation Format

Share Document