scholarly journals Conformational changes in the catalytically inactive nucleotide-binding site of CFTR

2013 ◽  
Vol 142 (1) ◽  
pp. 61-73 ◽  
Author(s):  
László Csanády ◽  
Csaba Mihályi ◽  
Andras Szollosi ◽  
Beáta Töröcsik ◽  
Paola Vergani
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Nucleotide Binding ◽  
Structural Rearrangement ◽  
Nucleotide Binding Site ◽  
Binding Domains ◽  
Channel Opening ◽  
Catalytically Active ◽  
Opening And Closing ◽  
Closing Rate

A central step in the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the association of its two cytosolic nucleotide-binding domains (NBDs) into a head-to-tail dimer, with two nucleotides bound at the interface. Channel opening and closing, respectively, are coupled to formation and disruption of this tight NBD dimer. CFTR is an asymmetric adenosine triphosphate (ATP)-binding cassette protein in which the two interfacial-binding sites (composite sites 1 and 2) are functionally different. During gating, the canonical, catalytically active nucleotide-binding site (site 2) cycles between dimerized prehydrolytic (state O1), dimerized post-hydrolytic (state O2), and dissociated (state C) forms in a preferential C→O1→O2→C sequence. In contrast, the catalytically inactive nucleotide-binding site (site 1) is believed to remain associated, ATP-bound, for several gating cycles. Here, we have examined the possibility of conformational changes in site 1 during gating, by studying gating effects of perturbations in site 1. Previous work showed that channel closure is slowed, both under hydrolytic and nonhydrolytic conditions, by occupancy of site 1 by N6-(2-phenylethyl)-ATP (P-ATP) as well as by the site-1 mutation H1348A (NBD2 signature sequence). Here, we found that P-ATP prolongs wild-type (WT) CFTR burst durations by selectively slowing (>2×) transition O1→O2 and decreases the nonhydrolytic closing rate (transition O1→C) of CFTR mutants K1250A (∼4×) and E1371S (∼3×). Mutation H1348A also slowed (∼3×) the O1→O2 transition in the WT background and decreased the nonhydrolytic closing rate of both K1250A (∼3×) and E1371S (∼3×) background mutants. Neither P-ATP nor the H1348A mutation affected the 1:1 stoichiometry between ATP occlusion and channel burst events characteristic to WT CFTR gating in ATP. The marked effect that different structural perturbations at site 1 have on both steps O1→C and O1→O2 suggests that the overall conformational changes that CFTR undergoes upon opening and coincident with hydrolysis at the active site 2 include significant structural rearrangement at site 1.

ATP-binding Cassette Exporters: Structure and Mechanism with a Focus on P-glycoprotein and MRP1

2019 ◽  
Vol 26 (7) ◽  
pp. 1062-1078 ◽  
Author(s):  
Maite Rocío Arana ◽  
Guillermo Alejandro Altenberg
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Structural Information ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Atp Binding ◽  
Binding Domains ◽  
P Glycoprotein ◽  
And Function ◽  
Atp Binding Cassette

Background:Proteins that belong to the ATP-binding cassette superfamily include transporters that mediate the efflux of substrates from cells. Among these exporters, P-glycoprotein and MRP1 are involved in cancer multidrug resistance, protection from endo and xenobiotics, determination of drug pharmacokinetics, and the pathophysiology of a variety of disorders. Objective:To review the information available on ATP-binding cassette exporters, with a focus on Pglycoprotein, MRP1 and related proteins. We describe tissue localization and function of these transporters in health and disease, and discuss the mechanisms of substrate transport. We also correlate recent structural information with the function of the exporters, and discuss details of their molecular mechanism with a focus on the nucleotide-binding domains. Methods: Evaluation of selected publications on the structure and function of ATP-binding cassette proteins. Conclusions:Conformational changes on the nucleotide-binding domains side of the exporters switch the accessibility of the substrate-binding pocket between the inside and outside, which is coupled to substrate efflux. However, there is no agreement on the magnitude and nature of the changes at the nucleotide- binding domains side that drive the alternate-accessibility. Comparison of the structures of Pglycoprotein and MRP1 helps explain differences in substrate selectivity and the bases for polyspecificity. P-glycoprotein substrates are hydrophobic and/or weak bases, and polyspecificity is explained by a flexible hydrophobic multi-binding site that has a few acidic patches. MRP1 substrates are mostly organic acids, and its polyspecificity is due to a single bipartite binding site that is flexible and displays positive charge.

scholarly journals Conformational Changes in the Catalytically Inactive Nucleotide Binding Site of CFTR

2013 ◽  
Vol 104 (2) ◽  
pp. 626a
Author(s):  
Csaba Mihalyi ◽  
Andras Szollosi ◽  
Paola Vergani ◽  
Laszlo Csanady
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site

Conformational changes at the nucleotide pocket of motor proteins monitored by electron paramagnetic resonance spectroscopy

2011 ◽  
Vol 83 (9) ◽  
pp. 1675-1684 ◽  
Author(s):  
Nariman Naber ◽  
Roger Cooke ◽  
Edward Pate
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Binding Site ◽  
Conformational Changes ◽  
Motor Proteins ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Nucleotide Site ◽  
Electron Paramagnetic

A fundamental goal in the field of motor proteins is to identify the conformational changes associated with the hydrolysis of the physiological substrate, ATP, and to define how these conformational changes are modulated by binding to the polymer track and translated into biologically useful movement. We have used electron paramagnetic resonance (EPR) spectroscopy to monitor conformational changes at the nucleotide-binding site of myosin- and kinesin-family motors. A novel set of nucleotide-analog EPR spin probes were synthesized and used to localize a spin moiety at the nucleotide site. This allows a reporter group to be placed with high specificity at the ATP binding site. Our results indicate that the nucleotide-binding site of myosin motors opens when the motor binds to its polymer roadway, actin, while that of kinesin closes on binding to microtubules (MTs). However, the transition is not all-or-none. There is instead an equilibrium between open and closed conformations. The different conformational changes in the two motor families can be correlated with differences in their biochemical cycles. Thus, we can now define the relationship between nucleotide-site structure, biochemistry and polymer binding for the two motors.

scholarly journals Prolonged Nonhydrolytic Interaction of Nucleotide with CFTR's NH2-terminal Nucleotide Binding Domain and its Role in Channel Gating

2003 ◽  
Vol 122 (3) ◽  
pp. 333-348 ◽  
Author(s):  
Claudia Basso ◽  
Paola Vergani ◽  
Angus C. Nairn ◽  
David C. Gadsby
Keyword(s):  
Channel Gating ◽  
Nucleotide Binding ◽  
Nucleoside Triphosphate ◽  
Binding Domains ◽  
Rate Limiting Step ◽  
Channel Opening ◽  
Nucleotide Interactions ◽  
Dependent Protein ◽  
Opening And Closing ◽  
Ion Pore

CFTR, the protein defective in cystic fibrosis, functions as a Cl− channel regulated by cAMP-dependent protein kinase (PKA). CFTR is also an ATPase, comprising two nucleotide-binding domains (NBDs) thought to bind and hydrolyze ATP. In hydrolyzable nucleoside triphosphates, PKA-phosphorylated CFTR channels open into bursts, lasting on the order of a second, from closed (interburst) intervals of a second or more. To investigate nucleotide interactions underlying channel gating, we examined photolabeling by [α32P]8-N3ATP or [γ32P]8-N3ATP of intact CFTR channels expressed in HEK293T cells or Xenopus oocytes. We also exploited split CFTR channels to distinguish photolabeling at NBD1 from that at NBD2. To examine simple binding of nucleotide in the absence of hydrolysis and gating reactions, we photolabeled after incubation at 0°C with no washing. Nucleotide interactions under gating conditions were probed by photolabeling after incubation at 30°C, with extensive washing, also at 30°C. Phosphorylation of CFTR by PKA only slightly influenced photolabeling after either protocol. Strikingly, at 30°C nucleotide remained tightly bound at NBD1 for many minutes, in the form of nonhydrolyzed nucleoside triphosphate. As nucleotide-dependent gating of CFTR channels occurred on the time scale of seconds under comparable conditions, this suggests that the nucleotide interactions, including hydrolysis, that time CFTR channel opening and closing occur predominantly at NBD2. Vanadate also appeared to act at NBD2, presumably interrupting its hydrolytic cycle, and markedly delayed termination of channel open bursts. Vanadate somewhat increased the magnitude, but did not alter the rate, of the slow loss of nucleotide tightly bound at NBD1. Kinetic analysis of channel gating in Mg8-N3ATP or MgATP reveals that the rate-limiting step for CFTR channel opening at saturating [nucleotide] follows nucleotide binding to both NBDs. We propose that ATP remains tightly bound or occluded at CFTR's NBD1 for long periods, that binding of ATP at NBD2 leads to channel opening wherupon its hydrolysis prompts channel closing, and that phosphorylation acts like an automobile clutch that engages the NBD events to drive gating of the transmembrane ion pore.

Characterization of Nucleotide Binding ­Site-Encoding Genes in Sweetpotato, Ipomoea batatas(L.) Lam., and Their Response to Biotic and Abiotic Stresses

2021 ◽  
pp. 1-15
Author(s):  
Zengzhi Si ◽  
Yake Qiao ◽  
Kai Zhang ◽  
Zhixin Ji ◽  
Jinling Han
Keyword(s):  
Binding Site ◽  
Abiotic Stresses ◽  
Ipomoea Batatas ◽  
Expression Profiles ◽  
Nucleotide Binding ◽  
Regulatory Elements ◽  
Nucleotide Binding Site ◽  
Biotic And Abiotic Stresses ◽  
Encoding Genes

Sweetpotato, <i>Ipomoea batatas</i> (L.) Lam., is an important and widely grown crop, yet its production is affected severely by biotic and abiotic stresses. The nucleotide binding site (NBS)-encoding genes have been shown to improve stress tolerance in several plant species. However, the characterization of NBS-encoding genes in sweetpotato is not well-documented to date. In this study, a comprehensive analysis of NBS-encoding genes has been conducted on this species by using bioinformatics and molecular biology methods. A total of 315 NBS-encoding genes were identified, and 260 of them contained all essential conserved domains while 55 genes were truncated. Based on domain architectures, the 260 NBS-encoding genes were grouped into 6 distinct categories. Phylogenetic analysis grouped these genes into 3 classes: TIR, CC (I), and CC (II). Chromosome location analysis revealed that the distribution of NBS-encoding genes in chromosomes was uneven, with a number ranging from 1 to 34. Multiple stress-related regulatory elements were detected in the promoters, and the NBS-encoding genes’ expression profiles under biotic and abiotic stresses were obtained. According to the bioinformatics analysis, 9 genes were selected for RT-qPCR analysis. The results revealed that <i>IbNBS75</i>, <i>IbNBS219</i>, and <i>IbNBS256</i> respond to stem nematode infection; <i>Ib­NBS240</i>, <i>IbNBS90</i>, and <i>IbNBS80</i> respond to cold stress, while <i>IbNBS208</i>, <i>IbNBS71</i>, and <i>IbNBS159</i> respond to 30% PEG treatment. We hope these results will provide new insights into the evolution of NBS-encoding genes in the sweetpotato genome and contribute to the molecular breeding of sweetpotato in the future.

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