Expression and Characterization of the NBD1-R Domain Region of CFTR: Evidence for Subunit-Subunit Interactions

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• 作者：David C. A. Neville ; Christine R. Rozanas ; Barry M. Tulk ; R. Reid Townsend ; and A. S. Verkman
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：February 24, 1998
• 年：1998
• 卷：37
• 期：8
• 页码：2401 - 2409
• 全文大小：187K
• 年卷期：v.37,no.8(February 24, 1998)
• ISSN：1520-4995

文摘

To study interactions between the contiguous NBD1 and Rdomains of CFTR, wild-type andmages/gifchars/Delta.gif" BORDER=0 >F508 NBD1-R (amino acids 404-830, in fusion with His₆tag) were expressed as single proteins inEscherichia coli. NBD1-R (10-25 mg/L culture) waspurified from inclusion bodies in 8 M urea byNi-affinity chromatography, and renatured by rapid dilution at pH 5.In vitro phosphorylation by proteinkinase A increased the apparent size of NBD1-R from ~52 to ~56 kDaby SDS-PAGE. The fluorescentATP analogue TNP-ATP bound to renatured NBD1-R withof 0.81 ± 0.1 mages/entities/mgr.gif">M (wild-type), 0.93 ±0.1 mages/entities/mgr.gif">M (wild-type, phosphorylated), 0.75 ± 0.1 mages/entities/mgr.gif">M (mages/gifchars/Delta.gif" BORDER=0 >F508NBD1-R), and 0.72 ± 0.1 mages/entities/mgr.gif">M (mages/gifchars/Delta.gif" BORDER=0 >F508NBD1-R, phosphorylated) with a stoichiometry of ~1 TNP-ATP site perNBD1-R molecule; TNP-ATPbinding was reversed by ATP, AMP-PCP, and AMP-PNP withK_Is of ~3.2, 4.2, and 4.6 mM,respectively.Secondary structure analysis by circular dichroism gave 19%mages/gifchars/alpha.gif" BORDER=0>-helix, 43% mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-sheet and turn, and 38%"other" structure. To determine if nucleotide binding to NBD1influenced R domain phosphorylation,NBD1-R was in vitro phosphorylated with protein kinase A and[mages/gifchars/gamma.gif" BORDER=0 >-³²P]ATP in the presence of AMP-PCP, AMP-PNP, or TNP-ATP. Whereas the nucleotide analogues did notaffect ³²P-incorporation incontrol proteins (Kemptide, GST-R domain), phosphorylation of NBD1-Rwas reduced >75% by AMP-PNP or AMP-PCP (0.25 mM) and >50% by TNP-ATP (0.25 mages/entities/mgr.gif">M).Analysis of phosphorylation sitesindicated that inhibition involved multiple sites in NBD1-R, includingserines 660, 712, 737, 795, and813. These results establish the conditions for NBD1-R expression,purification, and renaturation. Theinhibition of R domain phosphorylation by nucleotide binding to theNBD1 domain indicates significantdomain-domain interactions and suggests a novel mechanism forregulation of CFTR phosphorylation.