scholarly journals ATP-binding sites in brain p97/VCP (valosin-containing protein), a multifunctional AAA ATPase

2003 ◽  
Vol 374 (2) ◽  
pp. 473-480 ◽  
Author(s):  
Ran ZALK ◽  
Varda SHOSHAN-BARMATZ
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Covalent Binding ◽  
Protein A ◽  
Atp Binding ◽  
Amino Acid Sequencing ◽  
Protein Determination ◽  
Atp Binding Site ◽  
Aaa Atpase ◽  
Nucleotide Specificity

VCP (valosin-containing protein) or p97 is a member of the AAA family (ATPases associated with a variety of cellular activities family), a diverse group of proteins sharing a key conserved AAA module containing duplicate putative ATP-binding sites. Although the functions of the AAA family are related to their putative ATP-binding sites, the binding of ATP to these sites has not yet been demonstrated. In the present study, the ATP-binding site(s) of brain VCP was characterized using the photoreactive ATP analogue, BzATP [3′-O-(4-benzoylbenzoyl)ATP]. Photo-activation of Bz-[α-32P]ATP resulted in its covalent binding to a 97-kDa purified soluble or membrane-associated protein, identified by amino acid sequencing as VCP. Bz-[α-32P]ATP covalently bound to the purified homo-hexameric VCP with an apparent high affinity (74–111 nM). A molar stoichiometry of 2.23±0.14 BzATP bound per homo-hexameric VCP (n=6) was determined using different methods for analysis of radiolabelling and protein determination. Nucleotides inhibited the binding of Bz-[α-32P]ATP to VCP with the following efficiency: BzATP>ATP>ADP≫adenosine 5′-[β,γ-imido]triphosphate≥adenosine 5′-[β,γ-methylene]triphosphate, whereas AMP, GTP and CTP were ineffective. VCP was observed to possess very low ATPase activity, with nucleotide specificity similar to that for BzATP binding. Conformational changes induced by an alternating site mechanism for ATP binding are suggested as a molecular mechanism for coupling ATP binding to the diverse activities of the AAA family.

Fluorometric study on conformational changes in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase

1995 ◽  
Vol 15 (5) ◽  
pp. 317-326 ◽  
Author(s):  
Tohru Kanazawa ◽  
Hiroshi Suzuki ◽  
Takashi Daiho ◽  
Kazuo Yamasaki
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Binding Site ◽  
Conformational Changes ◽  
Binding Sites ◽  
Transmembrane Domain ◽  
Tryptophan Fluorescence ◽  
Cytoplasmic Domain ◽  
Catalytic Cycle ◽  
Atp Binding ◽  
Atp Binding Site

Changes in the fluoresence of N-acetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine (EDANS), being attached to Cys-674 of sarcoplasmic reticulum Ca2+-ATPase without affecting the catalytic activity, as well as changes in the intrinsic tryptophan fluorescence were followed throughout the catalytic cycle by the steady-state measurements and the stopped-flow spectrofluorometry. EDANS-fluorescence changes reflect conformational changes near the ATP binding site in the cytoplasmic domain, while tryptophan-fluorescence changes most probably reflect conformational changes in or near the transmembrane domain in which the Ca2+ binding sites are located. Formation of the phosphoenzyme intermediates (EP) was also followed by the continuous flow-rapid quenching method. The kinetic analysis of EDANS-fluorescence changes and EP formation revealed that, when ATP is added to the calcium-activated enzyme, conformational changes in the ATP binding site occur in three successive reaction steps; conformational change in the calcium enzyme substrate complex, formation of ADP-sensitive EP, and transition of ADP-sensitive EP to ADP-insensitive EP. In contrast, the ATP-induced tryptophan-fluorescence changes occur only in the latter two steps. Thus, we conclude that conformational changes in the ATP binding site in the cytoplasmic domain are transmitted to the Ca2+-binding sites in the transmembrane domain in these latter two steps.

scholarly journals A Variable Domain near the ATP-Binding Site in Drosophila Muscle Myosin Is Part of the Communication Pathway between the Nucleotide and Actin-binding Sites

2007 ◽  
Vol 368 (4) ◽  
pp. 1051-1066 ◽  
Author(s):  
Becky M. Miller ◽  
Marieke J. Bloemink ◽  
Miklós Nyitrai ◽  
Sanford I. Bernstein ◽  
Michael A. Geeves
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Variable Domain ◽  
Actin Binding ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Communication Pathway ◽  
Muscle Myosin

Abstract 898: Probing accessibility and conformational changes in ATP-binding sites during the catalytic cycle of human P-glycoprotein (ABCB1).

2013 ◽  
Author(s):  
Hong-May Sim ◽  
Jaya Bhatnagar ◽  
Samantha A. Green ◽  
Adam Gonzalez ◽  
Eduardo E. Chufan ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Catalytic Cycle ◽  
Atp Binding ◽  
P Glycoprotein

scholarly journals The flexible N-terminus of BchL autoinhibits activity through interaction with its [4Fe-4S] cluster and relieved upon ATP binding

2020 ◽  
pp. jbc.RA120.016278
Author(s):  
Elliot I Corless ◽  
Syed Muhammad Saad Imran ◽  
Maxwell B Watkins ◽  
John-Paul Bacik ◽  
Jenna Mattice ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Local Environment ◽  
Binding Pocket ◽  
Free Form ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Exchange Mass Spectrometry ◽  
Protochlorophyllide Reduction ◽  
N Terminus ◽  
Poor Tolerance

A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide to chlorophyllide, catalyzed by dark-operative protochlorophyllide oxidoreductase (DPOR). DPOR contains two [4Fe-4S]-containing component proteins (BchL and BchNB) that assemble upon ATP binding to BchL to coordinate electron transfer and protochlorophyllide reduction. But the precise nature of the ATP-induced conformational changes are poorly understood. We present a crystal structure of BchL in the nucleotide-free form where a conserved, flexible region in the N-terminus masks the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions in this region produce a hyper-active enzyme complex, suggesting a role for the N-terminus in auto-inhibition. Hydrogen deuterium exchange mass spectrometry shows that ATP-binding to BchL produces specific conformational changes leading to release of the flexible N-terminus from the docking interface. The release also promotes changes within the local environment surrounding the [4Fe-4S] cluster and promotes BchL complex formation with BchNB. A key patch of amino acids, Asp-Phe-Asp (the ‘DFD patch’), situated at the mouth of the BchL ATP-binding pocket promotes inter-subunit cross stabilization of the two subunits. A linked BchL dimer with one defective ATP-binding site does not support protochlorophyllide reduction, illustrating nucleotide binding to both subunits as a prerequisite for the inter-subunit cross stabilization. The masking of the [4Fe-4S] cluster by the flexible N-terminal region and the associated inhibition of activity is a novel mechanism of regulation in metalloproteins. Such mechanisms are possibly an adaptation to the anaerobic nature of eubacterial cells with poor tolerance for oxygen.

scholarly journals Adenosine nucleotide binding sites on Complex V from diverse organisms

2021 ◽  
Author(s):  
Abhinav Parashar ◽  
Kelath Murali Manoj
Keyword(s):  
Binding Sites ◽  
In Silico ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Atp Binding Site ◽  
In Silico Docking ◽  
Epsilon Subunit ◽  
Nucleotide Binding Sites ◽  
Alpha Beta ◽  
Adenosine Nucleotide

Using in silico docking approaches, we scan the various subunits of Complex V (FoF1ATPase) for putative adenosine nucleotide binding sites. We find that multiple generic ADP/ATP binding sites are present on the alpha-beta binding sites and a conserved ATP binding site is present on the epsilon subunit. These findings support the murburn model of Complex V.

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