Binding of the fluorescent substrate analog 2',3'-O-(2,4,6-trinitrophenylcyclohexadienylidene)adenosine 5'-triphosphate to the gastric hydrogen ion-potassium-ATPase: evidence for cofactor-induced conformational changes in the enzyme

Faller, Larry D.

doi:10.1021/bi00465a004

Cited by 31 publications

(28 citation statements)

References 27 publications

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“…Thus, ATP inhibition of K ATP channel activity is thought to involve direct interaction with Kir subunits despite the lack of identifiable nucleotide-binding motifs. The recent demonstration of the photoaffinity labeling of Kir6.2 channel by 8-azido-[␥- 32 P]ATP (17,18) also supports the direct binding of ATP with the pore-forming subunit of K ATP channels. In addition, mutations in both the NH 2 -and COOH-terminal regions of the Kir6.2 (13, 19 -23) and Kir1.1 (24) subunits alter the EC 50 for ATP-mediated channel gating.…”

mentioning

confidence: 76%

“…TNP-ATP Binding-To assess the binding of ATP to these recombinant fusion proteins, we used fluorescent TNP-ATP (Molecular Probes, Inc.) (29,30), which has been widely employed to study nucleotide binding to enzymes and other proteins (31)(32)(33)(34). The binding of TNP-ATP to recombinant proteins was performed generally as described by Faller (32).…”

Section: Dna Constructs and Mutagenesis-dnas Encoding The Nhmentioning

confidence: 99%

“…Excitation wavelength (403 nm) and emission wavelength (546 nm) were determined for the Kir1.1 COOH terminus fusion protein and utilized for all recombinant proteins (slit widths, 5 nm) because they did not vary significantly among proteins examined. A typical 10-nm blue shift in emission wavelength was detected upon binding of TNP-ATP to proteins (32). The temperature was maintained at 22 Ϯ 0.1°C by a circulating water bath (Neslab, Newington, NH).…”

Section: Dna Constructs and Mutagenesis-dnas Encoding The Nhmentioning

confidence: 99%

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The Carboxyl Termini of KATP Channels Bind Nucleotides

Vanoye¹,

MacGregor²,

Dong³

et al. 2002

Journal of Biological Chemistry

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Glutaraldehyde cross-linking demonstrated the multimerization potential of these COOH termini, suggesting that these cytosolic segments may directly interact in intact tetrameric channels. Thus, the COOH termini of K ATP tetrameric channels contain the nucleotide-binding pockets of these metabolically regulated channels with four potential nucleotide-binding sites/channel tetramer. ATP-sensitive or ATP-regulated potassium (K ATP )1 channels couple metabolism to either cell excitability (Kir6.x) (1-6) or potassium secretion (Kir1.1 in kidney) (7,8) and provide therapeutic targets for diseases including tissue ischemia, diabetes, hypertension, and disorders of potassium homeostasis. K ATP channels are formed by an octameric complex of four pore-forming subunits (Kir6.x or Kir1.1) and four sulfonylurea receptors, SUR1 or SUR2 for Kir6.x (2) or the cystic fibrosis transmembrane conductance regulator or SUR2b for Kir1.1 (9, 10).Although the SUR/fibrosis transmembrane conductance regulator subunits contain nucleotide-binding folds (11, 12), this subunit is not required for ATP-mediated inhibition of K ϩ channel activity. For example, deletion of the last 36 amino acids from the COOH terminus of Kir6.2 (Kir6.2⌬C36) produces functional K ϩ channels in the absence of coexpressed SURs that are sensitive to ATP (13). Nevertheless, SUR subunits are required for ADP-mediated activation of K ATP channels (14 -16). Thus, ATP inhibition of K ATP channel activity is thought to involve direct interaction with Kir subunits despite the lack of identifiable nucleotide-binding motifs. The recent demonstration of the photoaffinity labeling of Kir6.2 channel by 8-azido-[␥-32 P]ATP (17, 18) also supports the direct binding of ATP with the pore-forming subunit of K ATP channels. In addition, mutations in both the NH 2 -and COOH-terminal regions of the Kir6.2 (13, 19 -23) and Kir1.1 (24) subunits alter the EC 50 for ATP-mediated channel gating. Because ATP-mediated inhibition of channel activity must be a complex process involving residues that form an ATP-binding pocket and others that may be required for linking ATP binding to channel closure, those mutational studies of channel gating by nucleotides do not provide unequivocal evidence for direct involvement of those residues in ATP binding.In the present study, we assessed the direct binding of fluorescent 2Ј,3Ј-O-(2,4,6-trinitrophenylcyclo-hexadienylidene) adenosine triphosphate (TNP-ATP) to purified maltose-binding fusion proteins of the cytosolic NH 2 and COOH termini of the three known K ATP channels and the COOH terminus of a ATP-insensitive inward rectifier K ϩ channel, Kir2.1 (25). We provide herein what we believe to be the first evidence of direct binding of ATP to cytosolic domains of the pore-forming subunits of K ATP channels and show that the COOH termini, but not the NH 2 termini, of Kir subunits of K ATP channels bind TNP-ATP. The kinetic analyses of TNP-ATP binding suggest that the COOH termini have a single nucleotide-binding site. Based on glutaraldehyde cross-linking...

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mentioning

confidence: 76%

Section: Dna Constructs and Mutagenesis-dnas Encoding The Nhmentioning

confidence: 99%

Section: Dna Constructs and Mutagenesis-dnas Encoding The Nhmentioning

confidence: 99%

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The Carboxyl Termini of KATP Channels Bind Nucleotides

Vanoye¹,

MacGregor²,

Dong³

et al. 2002

Journal of Biological Chemistry

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“…The absolute magnitude depends on the specific protein environment within the nucleotide-binding pocket and was introduced as a fitting parameter (see below). The fluorescence intensity of TNP-AXP in buffer is low in the absence of TAP1-NBD and can be described by Equation 1, taking inner filter effects into account (48,49). The fluorescence constants Q 1 and Q 2 in buffer B (with and without 5 mM MgCl 2 ) were determined in the absence of protein by stepwise addition of TNP-AXP from a 1 mM stock solution to assay buffer.…”

Section: Heterologous Expression Of Tap1-nbd and Mutants In Ementioning

confidence: 99%

Engineering ATPase Activity in the Isolated ABC Cassette of Human TAP1

Ernst

Koch

Horn

et al. 2006

Journal of Biological Chemistry

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The human transporter associated with antigen processing (TAP) translocates antigenic peptides from the cytosol into the endoplasmic reticulum lumen. The functional unit of TAP is a heterodimer composed of the TAP1 and TAP2 subunits, both of which are members of the ABC-transporter family. ABC-transporters are ATP-dependent pumps, channels, or receptors that are composed of four modules: two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Although the TMDs are rather divergent in sequence, the NBDs are conserved with respect to structure and function. Interestingly, the NBD of TAP1 contains mutations at amino acid positions that have been proposed to be essential for catalytic activity. Instead of a glutamate, proposed to act as a general base, TAP1 contains an aspartate and a glutamine instead of the conserved histidine, which has been suggested to act as the linchpin. We used this degeneration to evaluate the individual contribution of these two amino acids to the ATPase activity of the engineered TAP1-NBD mutants. Based on our results a catalytic hierarchy of these two fundamental amino acids in ATP hydrolysis of the mutated TAP1 motor domain was deduced. ATP-binding cassette (ABC)3 -transporters form a large superfamily of primary transmembrane proteins (1), which ultimately use the energy of ATP hydrolysis to translocate their substrate or allocrite (2), the term we prefer to distinguish the transported substrate from ATP. Found in all three kingdoms of life, ABC-transporters contain certain sequence motifs such as the Walker A and B motifs, the C-loop (ABC signature motif), and the D-loop (3, 4). These sequences serve as diagnostic tools to identify ABC-transporters. Furthermore, all members of this family show a modular architecture composed of two nucleotide-binding domains (NBDs), which contain all the conserved sequence motifs, and two transmembrane domains (TMDs). In archea and eubacteria, these four modules are generally encoded on separate genes, whereas they are fused on a single polypeptide chain ("full-size" transporter) in eukarya. Additionally, fusions of one NBD and one TMD, which are called "half-size" transporters, are found in all organisms. The functional unit of these systems would be a homo-or heterodimer of two half-size transporters. Examples of the latter are hemolysin B (HlyB) from Escherichia coli (5) and the human transporter associated with antigen processing (TAP) (6).Human TAP, located in the membrane of the endoplasmic reticulum (ER), is a key component of major histocompatibility complex class I-mediated adaptive immunity (7). The functional TAP complex is a heterodimer composed of TAP1 and TAP2, both of which comprise a TMD-NBD fusion. It is now firmly established that the allocrites of TAP, antigenic peptides, which are generated in the cytosol by proteasomal degradation (8, 9), are transported in an ATP-dependent manner across the ER membrane for subsequent loading of major histocompatibility complex class I molecules (10). After passing the ER quali...

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“…Q and Q 2 are constants (fluorescence intensity per M or M 2 of free TNP-ATP, respectively) derived independently from the concentration dependence of TNP-ATP fluorescence intensity in buffer alone (F Buffer ) and account for the inner filter effect (25):…”

Section: Dnamentioning

confidence: 99%