Molecular dynamics analysis of the wild type and dF508 mutant structures of the human CFTR–nucleotide binding domain 1

The kinase activity of human p34cdc2 is negatively regulated by phosphorylation at Thr-14 and Tyr-15. These residues lie within the putative nucleotide binding domain of p34cdc2. It has been proposed that phosphorylation within this motif ablates the binding of ATP to the active site of p34cdc2, thereby inhibiting p34cdc2 kinase activity (K. Gould and P. Nurse, Nature [London] 342:39-44, 1989). To understand the mechanism of this inactivation, various forms of p34cdc2 were tested for the ability to bind nucleotide. The active site of p34cdc2 was specifically modified by the MgATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The apparent Km for modification of wild-type, monomeric p34cdc2 was 148 microM FSBA and was not significantly affected by association with cyclin B. Tyrosine-phosphorylated p34cdc2 was modified by FSBA with a slightly higher Km (241 microM FSBA). FSBA modification of both tyrosine-phosphorylated and unphosphorylated p34cdc2 was competitively inhibited by ATP, and half-maximal inhibition in each case occurred at approximately 250 microM ATP. In addition to being negatively regulated by phosphorylation, the kinase activity of p34cdc2 was positively regulated by the cyclin-dependent phosphorylation of Thr-161. Mutation of p34cdc2 at Thr-161 resulted in the formation of an enzymatically inactive p34cdc2/cyclin B complex both in vivo and in vitro. However, mutation of Thr-161 did not significantly affect the ability of p34cdc2 to bind nucleotide (FSBA). Taken together, these results indicate that inhibition of p34cdc2 kinase activity by phosphorylation of Tyr-15 (within the putative ATP binding domain) or by mutation of Thr-161 involves a mechanism other than inhibition of nucleotide binding. We propose instead that the defect resides at the level of catalysis.

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Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease-associated ATP Binding Cassette (ABC) Transporter, ABCA4

Journal of Biological Chemistry ◽

10.1074/jbc.m112.409623 ◽

2012 ◽

Vol 287 (53) ◽

pp. 44097-44107 ◽

Cited By ~ 12

Author(s):

Esther E. Biswas-Fiss ◽

Stephanie Affet ◽

Malissa Ha ◽

Subhasis B. Biswas

Keyword(s):

Abc Transporter ◽

Nucleotide Binding ◽

Binding Domain ◽

Atp Binding ◽

Nucleotide Binding Domain ◽

Stargardt Disease ◽

Binding Properties ◽

Nucleotide Binding Domain 1 ◽

Atp Binding Cassette

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Glucose-regulated protein 78 substrate-binding domain alters its conformation upon EGCG inhibitor binding to nucleotide-binding domain: Molecular dynamics studies

Scientific Reports ◽

10.1038/s41598-018-22905-6 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 8

Author(s):

K. R. D. Sagara N. S. Gurusinghe ◽

Aanchal Mishra ◽

Seema Mishra

Keyword(s):

Molecular Dynamics ◽

Substrate Binding ◽

Nucleotide Binding ◽

Binding Domain ◽

Nucleotide Binding Domain ◽

Inhibitor Binding ◽

Substrate Binding Domain ◽

Glucose Regulated Protein

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ΔF508-CFTR Modulator Screen Based on Cell Surface Targeting of a Chimeric Nucleotide Binding Domain 1 Reporter

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555218763310 ◽

2018 ◽

Vol 23 (8) ◽

pp. 823-831

Author(s):

Puay-Wah Phuan ◽

Guido Veit ◽

Joseph-Anthony Tan ◽

Ariel Roldan ◽

Walter E. Finkbeiner ◽

...

Keyword(s):

Cystic Fibrosis ◽

Cell Surface ◽

Nucleotide Binding ◽

Surface Expression ◽

Cell Surface Expression ◽

Wild Type ◽

Nucleotide Binding Domain ◽

Δf508 Cftr ◽

Nucleotide Binding Domain 1 ◽

Cftr Modulators

The most common cystic fibrosis–causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine at residue 508 (∆F508). The ∆F508 mutation impairs folding of nucleotide binding domain 1 (NBD1) and interfacial interactions of NBD1 and the membrane spanning domains. Here, we report a domain-targeted screen to identify ∆F508-CFTR modulators that act on NBD1. A biochemical screen for ΔF508-NBD1 cell surface expression was done in Madin–Darby canine kidney cells expressing a chimeric reporter consisting of ΔF508-NBD1, the CD4 transmembrane domain, and an extracellular horseradish peroxidase (HRP) reporter. Using a luminescence readout of HRP activity, the screen was robust with a Z′ factor of 0.7. The screening of ~20,000 synthetic small molecules allowed the identification of compounds from four chemical classes that increased ∆F508-NBD1 cell surface expression by up to 4-fold; for comparison, a 12-fold increased cell surface expression was found for a wild-type NBD1 chimera. While the compounds were inactive as correctors of full-length ΔF508-CFTR, several carboxamide-benzothiophenes had potentiator activity with low micromolar EC50. Interestingly, the potentiators did not activate G551D or wild-type CFTR. Our results provide a proof of concept for a cell-based NBD1 domain screen to identify ∆F508-CFTR modulators that target the NBD1 domain.

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Mechanisms of p34cdc2 regulation

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1675-1685.1993 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1675-1685

Author(s):

S Atherton-Fessler ◽

L L Parker ◽

R L Geahlen ◽

H Piwnica-Worms

Keyword(s):

Active Site ◽

Kinase Activity ◽

Nucleotide Binding ◽

Binding Domain ◽

Cyclin B ◽

Wild Type ◽

Nucleotide Binding Domain ◽

Maximal Inhibition

The kinase activity of human p34cdc2 is negatively regulated by phosphorylation at Thr-14 and Tyr-15. These residues lie within the putative nucleotide binding domain of p34cdc2. It has been proposed that phosphorylation within this motif ablates the binding of ATP to the active site of p34cdc2, thereby inhibiting p34cdc2 kinase activity (K. Gould and P. Nurse, Nature [London] 342:39-44, 1989). To understand the mechanism of this inactivation, various forms of p34cdc2 were tested for the ability to bind nucleotide. The active site of p34cdc2 was specifically modified by the MgATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The apparent Km for modification of wild-type, monomeric p34cdc2 was 148 microM FSBA and was not significantly affected by association with cyclin B. Tyrosine-phosphorylated p34cdc2 was modified by FSBA with a slightly higher Km (241 microM FSBA). FSBA modification of both tyrosine-phosphorylated and unphosphorylated p34cdc2 was competitively inhibited by ATP, and half-maximal inhibition in each case occurred at approximately 250 microM ATP. In addition to being negatively regulated by phosphorylation, the kinase activity of p34cdc2 was positively regulated by the cyclin-dependent phosphorylation of Thr-161. Mutation of p34cdc2 at Thr-161 resulted in the formation of an enzymatically inactive p34cdc2/cyclin B complex both in vivo and in vitro. However, mutation of Thr-161 did not significantly affect the ability of p34cdc2 to bind nucleotide (FSBA). Taken together, these results indicate that inhibition of p34cdc2 kinase activity by phosphorylation of Tyr-15 (within the putative ATP binding domain) or by mutation of Thr-161 involves a mechanism other than inhibition of nucleotide binding. We propose instead that the defect resides at the level of catalysis.

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