scholarly journals CryoEM reveals the stochastic nature of individual ATP binding events in a group II chaperonin

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yanyan Zhao ◽  
Michael F. Schmid ◽  
Judith Frydman ◽  
Wah Chiu
Keyword(s):  
Atp Hydrolysis ◽  
Biochemical Property ◽  
Atp Binding ◽  
Methanococcus Maripaludis ◽  
Particle Level ◽  
Random Manner ◽  
Quaternary Structures ◽  
The Individual ◽  
Atp Binding And Hydrolysis ◽  
Group Ii Chaperonin

AbstractChaperonins are homo- or hetero-oligomeric complexes that use ATP binding and hydrolysis to facilitate protein folding. ATP hydrolysis exhibits both positive and negative cooperativity. The mechanism by which chaperonins coordinate ATP utilization in their multiple subunits remains unclear. Here we use cryoEM to study ATP binding in the homo-oligomeric archaeal chaperonin from Methanococcus maripaludis (MmCpn), consisting of two stacked rings composed of eight identical subunits each. Using a series of image classification steps, we obtained different structural snapshots of individual chaperonins undergoing the nucleotide binding process. We identified nucleotide-bound and free states of individual subunits in each chaperonin, allowing us to determine the ATP occupancy state of each MmCpn particle. We observe distinctive tertiary and quaternary structures reflecting variations in nucleotide occupancy and subunit conformations in each chaperonin complex. Detailed analysis of the nucleotide distribution in each MmCpn complex indicates that individual ATP binding events occur in a statistically random manner for MmCpn, both within and across the rings. Our findings illustrate the power of cryoEM to characterize a biochemical property of multi-subunit ligand binding cooperativity at the individual particle level.

scholarly journals 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

2006 ◽  
Vol 401 (2) ◽  
pp. 581-586 ◽  
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.

scholarly journals In vivo FRET analyses reveal a role of ATP hydrolysis–associated conformational changes in human P-glycoprotein

2020 ◽  
Vol 295 (15) ◽  
pp. 5002-5011 ◽  
Author(s):  
Ryota Futamata ◽  
Fumihiko Ogasawara ◽  
Takafumi Ichikawa ◽  
Atsushi Kodan ◽  
Yasuhisa Kimura ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Hek293 Cells ◽  
Atp Binding ◽  
Binding Domains ◽  
P Glycoprotein ◽  
Atp Binding And Hydrolysis

P-glycoprotein (P-gp; also known as MDR1 or ABCB1) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. At least two P-gp states are required for transport. In the inward-facing (pre-drug transport) conformation, the two NBDs are separated, and the two TMDs are open to the intracellular side; in the outward-facing (post-drug transport) conformation, the NBDs are dimerized, and the TMDs are slightly open to the extracellular side. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations, and these changes help translocate substrates across the membrane. However, how ATP hydrolysis is coupled to these conformational changes remains unclear. In this study, we used a new FRET sensor that detects conformational changes in P-gp to investigate the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living HEK293 cells. We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of γ-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state. The findings of our study underscore the utility of using FRET analysis in living cells to elucidate the function of membrane proteins such as multidrug transporters.

Learning the ABCs one at a time: structure and mechanism of ABC transporters

2019 ◽  
Vol 47 (1) ◽  
pp. 23-36 ◽  
Author(s):  
Robert C. Ford ◽  
Konstantinos Beis
Keyword(s):  
Abc Transporters ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Bacterial Cells ◽  
The Novel ◽  
Mechanism Model ◽  
Binding Domains ◽  
Atp Binding And Hydrolysis ◽  
Alternating Access

Abstract ATP-binding cassette (ABC) transporters are essential proteins that are found across all kingdoms of life. ABC transporters harness the energy of ATP hydrolysis to drive the import of nutrients inside bacterial cells or the export of toxic compounds or essential lipids across bacteria and eukaryotic membranes. Typically, ABC transporters consist of transmembrane domains (TMDs) and nucleotide-binding domains (NBDs) to bind their substrate and ATP, respectively. The TMDs dictate what ligands can be recognised, whereas the NBDs are the power engine of the ABC transporter, carrying out ATP binding and hydrolysis. It has been proposed that they utilise the alternating access mechanism, inward- to outward-facing conformation, to transport their substrates. Here, we will review the recent progress on the structure determination of eukaryotic and bacterial ABC transporters as well as the novel mechanisms that have also been proposed, that fall out of the alternating access mechanism model.

scholarly journals Cryo-EM structures reveal how ATP and DNA binding in MutS coordinate the sequential steps of DNA mismatch repair

2021 ◽  
Author(s):  
Alessandro Borsellini ◽  
Vladislav Kunetsky ◽  
Peter Friedhoff ◽  
Meindert H. Lamers
Keyword(s):  
Dna Binding ◽  
Mismatch Repair ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Dna Mismatch Repair ◽  
Atp Binding ◽  
Dna Mismatch ◽  
Sliding Clamp ◽  
Adp Release ◽  
Atp Binding And Hydrolysis

DNA mismatch repair detects and removes mismatches from DNA reducing the error rate of DNA replication a 100-1000 fold. The MutS protein is one of the key players that scans for mismatches and coordinates the repair cascade. During this, MutS undergoes multiple conformational changes that initiate the subsequent steps, in response to ATP binding, hydrolysis, and release. How ATP induces the different conformations in MutS is not well understood. Here we present four cryo-EM structures of Escherichia coli MutS at sequential stages of the ATP hydrolysis cycle. These structures reveal how ATP binding and hydrolysis induces a closing and opening of the MutS dimer, respectively. Additional biophysical analysis furthermore explains how DNA binding modulates the ATPase cycle by preventing hydrolysis during scanning and mismatch binding, while preventing ADP release in the sliding clamp state. Nucleotide release is achieved when MutS encounters single stranded DNA that is produced during the removal of the daughter strand. This way, the combination of the ATP binding and hydrolysis and its modulation by DNA enable MutS to adopt different conformations needed to coordinate the sequential steps of the mismatch repair cascade.

scholarly journals Mutations in Sensor 1 and Walker B in the Bovine Papillomavirus E1 Initiator Protein Mimic the Nucleotide-Bound State

2009 ◽  
Vol 84 (4) ◽  
pp. 1912-1919 ◽  
Author(s):  
Xiaofei Liu ◽  
Arne Stenlund
Keyword(s):  
Atp Hydrolysis ◽  
Mutational Analysis ◽  
Structural Data ◽  
Bound State ◽  
Atp Binding ◽  
Bovine Papillomavirus ◽  
Viral Genomes ◽  
Initiator Protein ◽  
Close Proximity ◽  
Atp Binding And Hydrolysis

ABSTRACT Viral replication initiator proteins are multifunctional proteins that utilize ATP binding and hydrolysis by their AAA+ modules for multiple functions in the replication of their viral genomes. These proteins are therefore of particular interest for understanding how AAA+ proteins carry out multiple ATP driven functions. We have performed a comprehensive mutational analysis of the residues involved in ATP binding and hydrolysis in the papillomavirus E1 initiator protein based on the recent structural data. Ten of the eleven residues that were targeted were defective for ATP hydrolysis, and seven of these were also defective for ATP binding. The three mutants that could still bind nucleotide represent the Walker B motif (D478 and D479) and Sensor 1 (N523), three residues that are in close proximity to each other and generally are considered to be involved in ATP hydrolysis. Surprisingly, however, two of these mutants, D478A and N523A, mimicked the nucleotide bound state and were capable of binding DNA in the absence of nucleotide. However, these mutants could not form the E1 double trimer in the absence of nucleotide, demonstrating that there are two qualitatively different consequences of ATP binding by E1, one that can be mimicked by D478A and N523A and one which cannot.

scholarly journals Establishing the role of ATP for the function of the RIG-I innate immune sensor

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
David C Rawling ◽  
Megan E Fitzgerald ◽  
Anna Marie Pyle
Keyword(s):  
Immune Response ◽  
Atp Hydrolysis ◽  
Innate Immune ◽  
Atp Binding ◽  
Rna Molecules ◽  
Proof Reading ◽  
Atp Binding And Hydrolysis ◽  
Signal Activation ◽  
Inducible Gene

Retinoic acid-inducible gene I (RIG-I) initiates a rapid innate immune response upon detection and binding to viral ribonucleic acid (RNA). This signal activation occurs only when pathogenic RNA is identified, despite the ability of RIG-I to bind endogenous RNA while surveying the cytoplasm. Here we show that ATP binding and hydrolysis by RIG-I play a key role in the identification of viral targets and the activation of signaling. Using biochemical and cell-based assays together with mutagenesis, we show that ATP binding, and not hydrolysis, is required for RIG-I signaling on viral RNA. However, we show that ATP hydrolysis does provide an important function by recycling RIG-I and promoting its dissociation from non-pathogenic RNA. This activity provides a valuable proof-reading mechanism that enhances specificity and prevents an antiviral response upon encounter with host RNA molecules.

The intact CFTR protein mediates ATPase rather than adenylate kinase activity

2008 ◽  
Vol 412 (2) ◽  
pp. 315-321 ◽  
Author(s):  
Mohabir Ramjeesingh ◽  
Francisca Ugwu ◽  
Fiona L. L. Stratford ◽  
Ling-Jun Huan ◽  
Canhui Li ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Atpase Activity ◽  
Mechanism Of Action ◽  
Adenylate Kinase ◽  
Kinase Activity ◽  
Residual Activity ◽  
Atp Binding ◽  
Cftr Protein ◽  
The Individual ◽  
Atp Binding And Hydrolysis

The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain–domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.

scholarly journals Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel

2010 ◽  
Vol 135 (5) ◽  
pp. 399-414 ◽  
Author(s):  
Ming-Feng Tsai ◽  
Min Li ◽  
Tzyh-Chang Hwang
Keyword(s):  
Chloride Channel ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Abc Proteins ◽  
Atp Binding Site ◽  
Functional Studies ◽  
Binding Domains ◽  
Gating Model ◽  
Atp Binding And Hydrolysis ◽  
Opening And Closing

Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR’s opening–closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N3-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N3-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1–NBD2 interface. The open state of CFTR has been shown to represent a two-ATP–bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a “partial NBD dimer” state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and “partial” separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR’s NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed.

scholarly journals The role of ATP hydrolysis in conformational changes of human P-glycoprotein in living cells

2019 ◽  
Author(s):  
Ryota Futamata ◽  
Fumihiko Ogasawara ◽  
Takafumi Ichikawa ◽  
Atsushi Kodan ◽  
Yasuhisa Kimura ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Atp Binding ◽  
P Glycoprotein ◽  
Atp Binding And Hydrolysis

AbstractP-glycoprotein (P-gp; also known as MDR1 or ABCB1) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. P-gp takes at least two states during transport; the inward-facing (pre-drug transport) conformation, in which the two NBDs are separated and the two TMDs are open to the intracellular side, and the outward-facing (post-drug transport) conformation, in which the NBDs are dimerized and the TMDs are slightly open to the extracellular side. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations to translocate substrates across the membrane. However, it remains unclear how ATP is used during these conformational changes in living cells. In this study, we investigated the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living cells by using fluorescence resonance energy transfer (FRET). We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of γ-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state.

scholarly journals Dynein activation in vivo is regulated by the nucleotide states of its AAA3 domain

2021 ◽  
Author(s):  
Rongde Qiu ◽  
Jun Zhang ◽  
Jeremy D. Rotty ◽  
Xin Xiang
Keyword(s):  
Atp Hydrolysis ◽  
Cytoplasmic Dynein ◽  
The Other ◽  
Atp Binding ◽  
Motor Activation ◽  
Early Endosomes ◽  
Arginine Finger ◽  
Atp Binding And Hydrolysis

SummaryCytoplasmic dynein is activated by dynactin and cargo adapters in vitro, and the activation also needs LIS1 (Lissencephaly 1) in vivo. How this process is regulated remains unclear. Here we found in Aspergillus nidulans that a dynein AAA4 arginine-finger mutation bypasses the requirement of LIS1 for dynein activation driven by the early endosomal adapter HookA. As the AAA4 arginine-finger is implicated in AAA3 ATP hydrolysis, we examined AAA3 mutants defective in ATP binding and hydrolysis respectively. Astonishingly, blocking AAA3 ATP hydrolysis allows dynein activation by dynactin in the absence of LIS1 or HookA. As a consequence, dynein accumulates at microtubule minus ends while early endosomes stay near the plus ends. On the other hand, blocking AAA3 ATP binding abnormally prevents LIS1 from being dissociated from dynein upon motor activation. Thus, the AAA3 ATPase cycle regulates the coordination between dynein activation and cargo binding as well as the dynamic dynein-LIS1 interaction.

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