In VitroDissociation of BiP-Peptide Complexes Requires a Conformational Change in BiP after ATP Binding but Does Not Require ATP Hydrolysis

ABSTRACT P143 is a DNA helicase that tightly binds both double-stranded and single-stranded DNA. DNA-protein complexes rapidly dissociated in the presence of ATP and Mg2+. This finding suggests that ATP hydrolysis causes a conformational change in P143 which decreases affinity for DNA. This supports the model of an inchworm mechanism of DNA unwinding.

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Involvement of F1296 and N1303 of CFTR in induced-fit conformational change in response to ATP binding at NBD2

The Journal of General Physiology ◽

10.1085/jgp.201010434 ◽

2010 ◽

Vol 136 (4) ◽

pp. 407-423 ◽

Cited By ~ 17

Author(s):

Andras Szollosi ◽

Paola Vergani ◽

László Csanády

Keyword(s):

Conformational Change ◽

Bound States ◽

Atp Hydrolysis ◽

Chloride Ion ◽

Atp Binding ◽

Toggle Switch ◽

Regulatory Domain ◽

Induced Fit ◽

Subsequent Formation ◽

Abc Protein

The chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR) displays a typical adenosine trisphosphate (ATP)-binding cassette (ABC) protein architecture comprising two transmembrane domains, two intracellular nucleotide-binding domains (NBDs), and a unique intracellular regulatory domain. Once phosphorylated in the regulatory domain, CFTR channels can open and close when supplied with cytosolic ATP. Despite the general agreement that formation of a head-to-tail NBD dimer drives the opening of the chloride ion pore, little is known about how ATP binding to individual NBDs promotes subsequent formation of this stable dimer. Structural studies on isolated NBDs suggest that ATP binding induces an intra-domain conformational change termed “induced fit,” which is required for subsequent dimerization. We investigated the allosteric interaction between three residues within NBD2 of CFTR, F1296, N1303, and R1358, because statistical coupling analysis suggests coevolution of these positions, and because in crystal structures of ABC domains, interactions between these positions appear to be modulated by ATP binding. We expressed wild-type as well as F1296S, N1303Q, and R1358A mutant CFTR in Xenopus oocytes and studied these channels using macroscopic inside-out patch recordings. Thermodynamic mutant cycles were built on several kinetic parameters that characterize individual steps in the gating cycle, such as apparent affinities for ATP, open probabilities in the absence of ATP, open probabilities in saturating ATP in a mutant background (K1250R), which precludes ATP hydrolysis, as well as the rates of nonhydrolytic closure. Our results suggest state-dependent changes in coupling between two of the three positions (1296 and 1303) and are consistent with a model that assumes a toggle switch–like interaction pattern during the intra-NBD2 induced fit in response to ATP binding. Stabilizing interactions of F1296 and N1303 present before ATP binding are replaced by a single F1296-N1303 contact in ATP-bound states, with similar interaction partner toggling occurring during the much rarer ATP-independent spontaneous openings.

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Coupling between ATP hydrolysis and protein conformational change in maltose transporter

Proteins Structure Function and Bioinformatics ◽

10.1002/prot.25160 ◽

2016 ◽

Vol 85 (2) ◽

pp. 207-220

Author(s):

Xiaoying Lv ◽

Hao Liu ◽

Haifeng Chen ◽

Haipeng Gong

Keyword(s):

Conformational Change ◽

Atp Hydrolysis ◽

Protein Conformational Change ◽

Maltose Transporter

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Roles of the AX4GKS and Arginine-Rich Motifs of Hepatitis C Virus RNA Helicase in ATP- and Viral RNA-Binding Activity

Journal of Virology ◽

10.1128/jvi.74.20.9732-9737.2000 ◽

2000 ◽

Vol 74 (20) ◽

pp. 9732-9737 ◽

Cited By ~ 17

Author(s):

Shin C. Chang ◽

Ju-Chien Cheng ◽

Yi-Hen Kou ◽

Chuan-Hong Kao ◽

Chiung-Hui Chiu ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Binding ◽

Atp Hydrolysis ◽

Nonstructural Protein ◽

Binding Activity ◽

Viral Rna ◽

Atp Binding ◽

Rna Unwinding ◽

Arginine Rich Motif

ABSTRACT The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses protease, nucleoside triphosphatase, and helicase activities. Although the enzymatic activities have been extensively studied, the ATP- and RNA-binding domains of the NS3 helicase are not well-characterized. In this study, NS3 proteins with point mutations in the conserved helicase motifs were expressed inEscherichia coli, purified, and analyzed for their effects on ATP binding, RNA binding, ATP hydrolysis, and RNA unwinding. UV cross-linking experiments indicate that the lysine residue in the AX4GKS motif is directly involved in ATP binding, whereas the NS3(GR1490DT) mutant in which the arginine-rich motif (1486-QRRGRTGR-1493) was changed to QRRDTTGR bound ATP as well as the wild type. The binding activity of HCV NS3 helicase to the viral RNA was drastically reduced with the mutation at Arg1488 (R1488A) and was also affected by the K1236E substitution in the AX4GKS motif and the R1490A and GR1490DT mutations in the arginine-rich motif. Previously, Arg1490 was suggested, based on the crystal structure of an NS3-deoxyuridine octamer complex, to directly interact with the γ-phosphate group of ATP. Nevertheless, our functional analysis demonstrated the critical roles of Arg1490 in binding to the viral RNA, ATP hydrolysis, and RNA unwinding, but not in ATP binding.

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

Biochemical Journal ◽

10.1042/bj20081068 ◽

2008 ◽

Vol 416 (1) ◽

pp. 129-136 ◽

Cited By ~ 14

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.

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ATP-dependent thermostabilization of human P-glycoprotein (ABCB1) is blocked by modulators

Biochemical Journal ◽

10.1042/bcj20190736 ◽

2019 ◽

Vol 476 (24) ◽

pp. 3737-3750 ◽

Cited By ~ 5

Author(s):

Sabrina Lusvarghi ◽

Suresh V. Ambudkar

Keyword(s):

Conformational Changes ◽

Drug Transport ◽

Atp Hydrolysis ◽

Atp Binding ◽

Dependent Manner ◽

Deficient Mutant ◽

Concentration Dependent Manner ◽

Glycoprotein P ◽

Binding Domains ◽

P Glycoprotein

P-glycoprotein (P-gp), an ATP-binding cassette transporter associated with multidrug resistance in cancer cells, is capable of effluxing a number of xenobiotics as well as anticancer drugs. The transport of molecules through the transmembrane (TM) region of P-gp involves orchestrated conformational changes between inward-open and inward-closed forms, the details of which are still being worked out. Here, we assessed how the binding of transport substrates or modulators in the TM region and the binding of ATP to the nucleotide-binding domains (NBDs) affect the thermostability of P-gp in a membrane environment. P-gp stability after exposure at high temperatures (37–80°C) was assessed by measuring ATPase activity and loss of monomeric P-gp. Our results show that P-gp is significantly thermostabilized (>22°C higher IT50) by the binding of ATP under non-hydrolyzing conditions (in the absence of Mg2+). By using an ATP-binding-deficient mutant (Y401A) and a hydrolysis-deficient mutant (E556Q/E1201Q), we show that thermostabilization of P-gp requires binding of ATP to both NBDs and their dimerization. Additionally, we found that transport substrates do not affect the thermal stability of P-gp either in the absence or presence of ATP; in contrast, inhibitors of P-gp including tariquidar and zosuquidar prevent ATP-dependent thermostabilization in a concentration-dependent manner, by stabilizing the inward-open conformation. Altogether, our data suggest that modulators, which bind in the TM regions, inhibit ATP hydrolysis and drug transport by preventing the ATP-dependent dimerization of the NBDs of P-gp.

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Yeast Chd1p Unwraps the Exit Side DNA upon ATP Binding to Facilitate the Nucleosome Translocation Occurring upon ATP Hydrolysis

Biochemistry ◽

10.1021/acs.biochem.0c00747 ◽

2020 ◽

Vol 59 (47) ◽

pp. 4481-4487

Author(s):

Jaewon Kirk ◽

Ju Yeon Lee ◽

Yejin Lee ◽

Chanshin Kang ◽

Soochul Shin ◽

...

Keyword(s):

Atp Hydrolysis ◽

Atp Binding ◽

Exit Side

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Ligand-induced Conformational Shift in the N-terminal Domain of GRP94, an Hsp90 Chaperone

Journal of Biological Chemistry ◽

10.1074/jbc.m405253200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 46162-46171 ◽

Cited By ~ 74

Author(s):

Robert M. Immormino ◽

D. Eric Dollins ◽

Paul L. Shaffer ◽

Karen L. Soldano ◽

Melissa A. Walker ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Crystal Structures ◽

Conformational Change ◽

Atp Hydrolysis ◽

Dna Gyrase ◽

Regulatory Domain ◽

Conformational Switch ◽

Terminal Ligand ◽

Conformational Rearrangement ◽

Hsp90 Chaperone

GRP94 is the endoplasmic reticulum paralog of cytoplasmic Hsp90. Models of Hsp90 action posit an ATP-dependent conformational switch in the N-terminal ligand regulatory domain of the chaperone. However, crystal structures of the isolated N-domain of Hsp90 in complex with a variety of ligands have yet to demonstrate such a conformational change. We have determined the structure of the N-domain of GRP94 in complex with ATP, ADP, and AMP. Compared with theN-ethylcarboxamidoadenosine and radicicol-bound forms, these structures reveal a large conformational rearrangement in the protein. The nucleotide-bound form exposes new surfaces that interact to form a biochemically plausible dimer that is reminiscent of those seen in structures of MutL and DNA gyrase. Weak ATP binding and a conformational change in response to ligand identity are distinctive mechanistic features of GRP94 and suggest a model for how GRP94 functions in the absence of co-chaperones and ATP hydrolysis.

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Theoretical Study on the ATP Hydrolysis Mechanism of HisP Protein, the ATP-Binding Subunit of ABC Transporter

MATERIALS TRANSACTIONS ◽

10.2320/matertrans.48.735 ◽

2007 ◽

Vol 48 (4) ◽

pp. 735-739 ◽

Cited By ~ 1

Author(s):

Qiang Pei ◽

Carlos A. Del Carpio ◽

Hideyuki Tsuboi ◽

Michihisa Koyama ◽

Akira Endou ◽

...

Keyword(s):

Abc Transporter ◽

Theoretical Study ◽

Atp Hydrolysis ◽

Atp Binding ◽

Hydrolysis Mechanism ◽

Binding Subunit

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The Walker B motif of the second nucleotide-binding domain (NBD2) of CFTR plays a key role in ATPase activity by the NBD1–NBD2 heterodimer

Biochemical Journal ◽

10.1042/bj20060968 ◽

2006 ◽

Vol 401 (2) ◽

pp. 581-586 ◽

Cited By ~ 29

Author(s):

Fiona L. L. Stratford ◽

Mohabir Ramjeesingh ◽

Joanne C. Cheung ◽

Ling-JUN Huan ◽

Christine E. Bear

Keyword(s):

Atpase Activity ◽

Atp Hydrolysis ◽

Nucleotide Binding ◽

Vital Role ◽

Atp Binding ◽

Primary Role ◽

Binding Domains ◽

Membrane Spanning ◽

Atp Binding And Hydrolysis

CFTR (cystic fibrosis transmembrane conductance regulator), a member of the ABC (ATP-binding cassette) superfamily of membrane proteins, possesses two NBDs (nucleotide-binding domains) in addition to two MSDs (membrane spanning domains) and the regulatory ‘R’ domain. The two NBDs of CFTR have been modelled as a heterodimer, stabilized by ATP binding at two sites in the NBD interface. It has been suggested that ATP hydrolysis occurs at only one of these sites as the putative catalytic base is only conserved in NBD2 of CFTR (Glu1371), but not in NBD1 where the corresponding residue is a serine, Ser573. Previously, we showed that fragments of CFTR corresponding to NBD1 and NBD2 can be purified and co-reconstituted to form a heterodimer capable of ATPase activity. In the present study, we show that the two NBD fragments form a complex in vivo, supporting the utility of this model system to evaluate the role of Glu1371 in ATP binding and hydrolysis. The present studies revealed that a mutant NBD2 (E1371Q) retains wild-type nucleotide binding affinity of NBD2. On the other hand, this substitution abolished the ATPase activity formed by the co-purified complex. Interestingly, introduction of a glutamate residue in place of the non-conserved Ser573 in NBD1 did not confer additional ATPase activity by the heterodimer, implicating a vital role for multiple residues in formation of the catalytic site. These findings provide the first biochemical evidence suggesting that the Walker B residue: Glu1371, plays a primary role in the ATPase activity conferred by the NBD1–NBD2 heterodimer.

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