scholarly journals The 3 × 120° rotary mechanism ofParacoccus denitrificansF1-ATPase is different from that of the bacterial and mitochondrial F1-ATPases

2020 ◽  
Vol 117 (47) ◽  
pp. 29647-29657 ◽  
Author(s):  
Mariel Zarco-Zavala ◽  
Ryo Watanabe ◽  
Duncan G. G. McMillan ◽  
Toshiharu Suzuki ◽  
Hiroshi Ueno ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Single Molecule ◽  
Paracoccus Denitrificans ◽  
Thermus Thermophilus ◽  
Angular Position ◽  
Adenosine Diphosphate ◽  
Atp Binding ◽  
Clear Trend ◽  
Inhibition Experiment ◽  
Atp Binding And Hydrolysis

The rotation ofParacoccus denitrificansF1-ATPase (PdF1) was studied using single-molecule microscopy. At all concentrations of adenosine triphosphate (ATP) or a slowly hydrolyzable ATP analog (ATPγS), above or belowKm, PdF1showed three dwells per turn, each separated by 120°. Analysis of dwell time between steps showed that PdF1executes binding, hydrolysis, and probably product release at the same dwell. The comparison of ATP binding and catalytic pauses in single PdF1molecules suggested that PdF1executes both elementary events at the same rotary position. This point was confirmed in an inhibition experiment with a nonhydrolyzable ATP analog (AMP-PNP). Rotation assays in the presence of adenosine diphosphate (ADP) or inorganic phosphate at physiological concentrations did not reveal any obvious substeps. Although the possibility of the existence of substeps remains, all of the datasets show that PdF1is principally a three-stepping motor similar to bacterial vacuolar (V1)-ATPase fromThermus thermophilus. This contrasts with all other known F1-ATPases that show six or nine dwells per turn, conducting ATP binding and hydrolysis at different dwells. Pauses by persistent Mg-ADP inhibition or the inhibitory ζ-subunit were also found at the same angular position of the rotation dwell, supporting the simplified chemomechanical scheme of PdF1. Comprehensive analysis of rotary catalysis of F1from different species, including PdF1, suggests a clear trend in the correlation between the numbers of rotary steps of F1and Fodomains of F-ATP synthase. F1motors with more distinctive steps are coupled with proton-conducting Forings with fewer proteolipid subunits, giving insight into the design principle the F1Foof ATP synthase.

scholarly journals Rotary catalysis of bovine mitochondrial F1-ATPase studied by single-molecule experiments

2020 ◽  
Vol 117 (3) ◽  
pp. 1447-1456 ◽  
Author(s):  
Ryohei Kobayashi ◽  
Hiroshi Ueno ◽  
Chun-Biu Li ◽  
Hiroyuki Noji
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Atp Hydrolysis ◽  
Angular Position ◽  
Magnetic Tweezers ◽  
Reaction Scheme ◽  
Atp Binding ◽  
High Speed Imaging ◽  
Hydrolysis Process ◽  
Rotary Catalysis

The reaction scheme of rotary catalysis and the torque generation mechanism of bovine mitochondrial F1 (bMF1) were studied in single-molecule experiments. Under ATP-saturated concentrations, high-speed imaging of a single 40-nm gold bead attached to the γ subunit of bMF1 showed 2 types of intervening pauses during the rotation that were discriminated by short dwell and long dwell. Using ATPγS as a slowly hydrolyzing ATP derivative as well as using a functional mutant βE188D with slowed ATP hydrolysis, the 2 pausing events were distinctively identified. Buffer-exchange experiments with a nonhydrolyzable analog (AMP-PNP) revealed that the long dwell corresponds to the catalytic dwell, that is, the waiting state for hydrolysis, while it remains elusive which catalytic state short pause represents. The angular position of catalytic dwell was determined to be at +80° from the ATP-binding angle, mostly consistent with other F1s. The position of short dwell was found at 50 to 60° from catalytic dwell, that is, +10 to 20° from the ATP-binding angle. This is a distinct difference from human mitochondrial F1, which also shows intervening dwell that probably corresponds to the short dwell of bMF1, at +65° from the binding pause. Furthermore, we conducted “stall-and-release” experiments with magnetic tweezers to reveal how the binding affinity and hydrolysis equilibrium are modulated by the γ rotation. Similar to thermophilic F1, bMF1 showed a strong exponential increase in ATP affinity, while the hydrolysis equilibrium did not change significantly. This indicates that the ATP binding process generates larger torque than the hydrolysis process.

scholarly journals The peptide sensor motif stabilizes the outward-facing conformation of TmrAB

2020 ◽  
Author(s):  
Cinthia R. Millan ◽  
Martina Francis ◽  
Valery F. Thompson ◽  
Tarjani M. Thaker ◽  
Thomas M. Tomasiak
Keyword(s):  
Conformational Changes ◽  
Biochemical Characterization ◽  
Atp Hydrolysis ◽  
Thermus Thermophilus ◽  
Atp Binding ◽  
Conformational Switching ◽  
Disulfide Crosslinking ◽  
Binding Domains ◽  
Functional Consequences ◽  
Atp Binding And Hydrolysis

ABSTRACTThe ATP binding cassette (ABC) family of transporters move diverse small molecules across membranes in nearly all organisms. Transport activity requires conformational switching between inward-facing and outward-facing states driven by ATP-dependent dimerization of two nucleotide binding domains (NBDs). The allosteric mechanism that connects ATP binding and hydrolysis in the NBDs to conformational changes in a substrate binding site in the transmembrane domains (TMDs) presents an unresolved question. Here we use sequence coevolution analyses together with biochemical characterization to investigate the role of a highly conserved motif called the peptide sensor in coordinating domain rearrangements in the heterodimeric peptide exporter from Thermus thermophilus, TmrAB. Mutations in the peptide sensor motif alter ATP hydrolysis rates as well as substrate release. Disulfide crosslinking, evolutionary trace, and evolutionary coupling analysis reveal that these effects likely destabilize a network between the peptide sensor motif and the Q-loop and X-loop, two known allosteric elements in the NBDs. We further find that disruption of this network in TmrA versus TmrB has different functional consequences, hinting at an intrinsic asymmetry in heterodimeric ABC transporters extending beyond that of the NBDs. These results support a mechanism in which the peptide sensor motifs help coordinate the transition of TmrAB to an outward open conformation, and each half of the transporter likely plays a different role in the conformational cycle of TmrAB.

scholarly journals Single-molecule Imaging of ATP-driven Stepping Rotation of FoF1-ATP Synthase Reconstituted into Supported Membrane

2009 ◽  
Vol 96 (3) ◽  
pp. 29a
Author(s):  
Ryota Iino ◽  
Khek-Chian Tham ◽  
Kazuhito V. Tabata ◽  
Hiroshi Ueno ◽  
Hiroyuki Noji
Keyword(s):  
Atp Synthase ◽  
Single Molecule ◽  
Single Molecule Imaging ◽  
Supported Membrane ◽  
Fof1 Atp Synthase

scholarly journals GyrB mutations in Staphylococcus aureus strains resistant to cyclothialidine, coumermycin, and novobiocin.

1996 ◽  
Vol 40 (4) ◽  
pp. 1060-1062 ◽  
Author(s):  
M Stieger ◽  
P Angehrn ◽  
B Wohlgensinger ◽  
H Gmünder
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Mode ◽  
Atp Binding ◽  
Subunit Gene ◽  
Binding Region ◽  
B Subunit ◽  
B Subunits ◽  
Atp Binding And Hydrolysis

The sequence of the gyrase B subunit gene from Staphylococcus aureus strains resistant to the gyrase B subunit inhibitors cyclothialidine, coumermycin, and novobiocin has been determined. The residues altered in the resistant gyrase B subunits map to the ATP-binding region, suggesting that the drugs inhibit ATP binding and hydrolysis. The pattern of cross-resistances indicates that the detailed binding mode of the compounds differs.

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.

Sign in / Sign up

Export Citation Format

Share Document