The βG156C Substitution in the F1-ATPase from the Thermophilic Bacillus PS3 Affects Catalytic Site Cooperativity by Destabilizing the Closed Conformation of the Catalytic Site

Bandyopadhyay, Sanjay; Valder, Carolina R.; Huynh, Hue G.; Ren, Huimiao; Allison, William S.

doi:10.1021/bi026243g

Cited by 33 publications

(26 citation statements)

References 31 publications

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“…Bandyopadhyay et al (26) have suggested that endogenous nucleotides bound at the noncatalytic sites of ␤Y331W-EcF 1 are responsible for the apparent lack of asymmetry in the nucleotide binding in the absence of Mg 2ϩ reported earlier (24). Our results make it more likely that the main reason that the catalytic-site asymmetry had not been detected in the absence of Mg 2ϩ was the presence of Ϸ17 mM sulfate that competed with nucleotide binding to the high-affinity site.…”

Section: Discussionmentioning

confidence: 43%

“…2, circles) show that the catalytic sites of ␤Y331W-dEcF 1 do exhibit asymmetry in nucleotide binding in the absence of Mg 2ϩ . Different affinities of the catalytic sites for a nucleotide in the absence of Mg 2ϩ have also been observed with the homologous ␤Y341W-mutant ␣ 3 ␤ 3 ␥-subcomplex of TF 1 (26). We did not attempt to measure K d values for MgATP as has been done previously (18,27) because the rapid formation of catalytic site ADP makes it uncertain what nucleotide affinity is being measured.…”

Section: Resultsmentioning

confidence: 96%

“…This is likely due to the presence of Ϸ20 mM sulfate in these experiments, which would have already reduced nucleotide affinity significantly. (25,27,29,34) and MgAMPPNP (27,32) and titration of the homologous ␤Y341W-mutant ␣ 3 ␤ 3 ␥-subcomplex of TF 1 with MgADP and MgATP (26,35,36). Each component could logically represent binding at only one catalytic site.…”

Section: Discussionmentioning

confidence: 99%

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Studies of nucleotide binding to the catalytic sites of Escherichia coli βY331W-F ₁ -ATPase using fluorescence quenching

Bulygin

Milgrom

2007

Proc. Natl. Acad. Sci. U.S.A.

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ATP synthase ͉ bi-site catalysis ͉ phosphate ͉ sulfate ͉ tri-site catalysis D uring oxidative and photophosphorylation, F o F 1 -ATP synthase is responsible for reversible synthesis of ATP coupled to transmembrane movement of H ϩ (or, in some bacteria, Na ϩ ). The enzyme can be separated into two multisubunit components, the membranous factor F o , which in bacteria consists of a ring of c subunits and an a and b subunit in a stoichiometry of ab 2 c n , and the soluble factor F 1 with a subunit composition of ␣ 3 ␤ 3 ␥␦. Energy transformation by F o F 1 is best described by the binding change mechanism (1) and involves a rotation of a complex of subunits (␥c n , rotor) within the rest of the enzyme (stator) (2, 3). F 1 is the catalytic component of ATP synthase and, when isolated, is capable only of net ATP hydrolysis. In the crystal structure of F 1 from beef heart mitochondria (MF 1 ), six nucleotide-binding sites are located at the interfaces between ␣ and ␤ subunits that are arranged alternately around an asymmetrical ␣-helical-coiled coil formed by ␥-subunit (4). Three of the nucleotide-binding sites formed mostly by side chains of ␤ subunits are catalytic, and another three primarily on ␣ subunits are noncatalytic (5).Three different conformations of ␤ subunits in the crystal structure of MF 1 are associated with asymmetric interactions with the ␥ subunit and are thought to represent distinct states that each of the catalytic sites sequentially assumes during catalytic cycle according to the binding change mechanism (4). That the ␥ subunit plays a crucial role in determining properties of the catalytic sites is supported by the crystal structure of ␣ 3 ␤ 3 -subcomplex of F 1 from the thermophilic Bacillus PS3 (TF 1 ). In this structure, all of the ␤ subunits are in very similar conformations, and the three catalytic sites are in a state similar to one of the three states (open) found in the crystal structure of MF 1 (6). In addition, the strong positive catalytic cooperativity in catalysis by F 1 (7,8) that is mediated by a rotation of ␥ subunit (9, 10) is lacking in the ␣ 3 ␤ 3 -subcomplex. Interactions between ␣ and ␤ subunits are also considered as contributing to the modulation of the state of catalytic sites in F 1 (11,12).Whether asymmetric interactions of the three ␤ subunits with ␥, together with ␣/␤ interactions, are sufficient to induce asymmetry in the properties of the catalytic sites is not clear. According to Senior and colleagues (13-15), intersubunit interactions in F 1 alone cannot induce nucleotide-binding heterogeneity at catalytic sites. Based on observations obtained with ␤Y331W-mutant F 1 from Escherichia coli (EcF 1 ), it has been argued that in the absence of Mg 2ϩ , all three catalytic sites bind nucleotides with identical and low affinity and are in a similar state. Mg 2ϩ is considered to play a crucial role in inducing asymmetry between catalytic sites, with high-affinity nucleotide binding at a single catalytic site. This role, according to (15), extends beyond Mg 2ϩ being simpl...

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Section: Discussionmentioning

confidence: 43%

Section: Resultsmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Studies of nucleotide binding to the catalytic sites of Escherichia coli βY331W-F ₁ -ATPase using fluorescence quenching

Bulygin

Milgrom

2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Consequently, the total binding energy was compared with the binding free energy of the complexes using the fluorescence spectroscopy quenching method[20], [21]. As shown in Table 2 , the Molecular Mechanics Generalized Born Surface Area (MM-GBSA) calculation predicted that mutants wound bind more weakly to the inhibitors than to the wild type (WT)-HL systems with estimated ΔG bind values of −8.4, −4.2, −1.4, −8.9, −4.0, −2.3 kcal/mol for R104A-OLG, R104A-ORA, R104A-ORB, G126A-OLG, G126A-ORA, and G126A-ORB, respectively.…”

Section: Resultsmentioning

confidence: 99%

Molecular Modeling Reveals the Novel Inhibition Mechanism and Binding Mode of Three Natural Compounds to Staphylococcal α-Hemolysin

et al. 2013

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α-Hemolysin (α-HL) is a self-assembling, channel-forming toxin that is produced as a soluble monomer by Staphylococcus aureus strains. Until now, α-HL has been a significant virulence target for the treatment of S. aureus infection. In our previous report, we demonstrated that some natural compounds could bind to α-HL. Due to the binding of those compounds, the conformational transition of α-HL from the monomer to the oligomer was blocked, which resulted in inhibition of the hemolytic activity of α-HL. However, these results have not indicated how the binding of the α-HL inhibitors influence the conformational transition of the whole protein during the oligomerization process. In this study, we found that three natural compounds, Oroxylin A 7-O-glucuronide (OLG), Oroxin A (ORA), and Oroxin B (ORB), when inhibiting the hemolytic activity of α-HL, could bind to the “stem” region of α-HL. This was completed using conventional Molecular Dynamics (MD) simulations. By interacting with the novel binding sites of α-HL, the ligands could form strong interactions with both sides of the binding cavity. The results of the principal component analysis (PCA) indicated that because of the inhibitors that bind to the “stem” region of α-HL, the conformational transition of α-HL from the monomer to the oligomer was restricted. This caused the inhibition of the hemolytic activity of α-HL. This novel inhibition mechanism has been confirmed by both the steered MD simulations and the experimental data obtained from a deoxycholate-induced oligomerization assay. This study can facilitate the design of new antibacterial drugs against S. aureus.

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“…A 280-nm excitation wavelength with a 5-nm band-pass and a 345-nm emission wavelength with a 10-nm band-pass were used for the measurements. The details of the protocol used to perform the measurements were as previously described (Bandyopadhyay et al, 2002; Jurasekova et al, 2009). …”

Section: Methodsmentioning

confidence: 99%

Phloretin Attenuates Listeria monocytogenes Virulence Both In vitro and In vivo by Simultaneously Targeting Listeriolysin O and Sortase A

Wang

Liu

Teng

et al. 2017

Front. Cell. Infect. Microbiol.

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The critical roles of sortase A (SrtA) and listeriolysin O (LLO) in Listeria monocytogenes pathogenicity render these two virulence factors as ideal targets for the development of anti-virulence agents against L. monocytogenes infection. Additionally, the structures of SrtA and LLO are highly conserved among the members of sortase enzyme family and cholesterol dependent toxin family. Here, phloretin, a natural polyphenolic compound derived from apples and pears that has little anti-L. monocytogenes activity, was identified to simultaneously inhibit LLO expression and neutralize SrtA catalytic activity. Phloretin neutralized SrtA activity by causing a conformational change in the protein's active pocket, which prevented engagement with its substrate. Treatment with phloretin simultaneously reduced L. monocytogenes invasion into host cells and blocked the escape of vacuole-entrapped L. monocytogenes into cytoplasm. Further, L. monocytogenes-infected mice that received phloretin showed lower mortality, decreased bacterial burden and reduced pathological injury. Our results demonstrate that phloretin is a promising anti-infective therapeutic for infections caused by L. monocytogenes due to its simultaneous targeting of SrtA and LLO, which may result in fewer side effects than those caused by other antibiotics.

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The βG¹⁵⁶C Substitution in the F₁-ATPase from the Thermophilic Bacillus PS3 Affects Catalytic Site Cooperativity by Destabilizing the Closed Conformation of the Catalytic Site

Cited by 33 publications

References 31 publications

Studies of nucleotide binding to the catalytic sites of Escherichia coli βY331W-F ₁ -ATPase using fluorescence quenching

Studies of nucleotide binding to the catalytic sites of Escherichia coli βY331W-F ₁ -ATPase using fluorescence quenching

Molecular Modeling Reveals the Novel Inhibition Mechanism and Binding Mode of Three Natural Compounds to Staphylococcal α-Hemolysin

Phloretin Attenuates Listeria monocytogenes Virulence Both In vitro and In vivo by Simultaneously Targeting Listeriolysin O and Sortase A

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The βG156C Substitution in the F1-ATPase from the Thermophilic Bacillus PS3 Affects Catalytic Site Cooperativity by Destabilizing the Closed Conformation of the Catalytic Site

Cited by 33 publications

References 31 publications

Studies of nucleotide binding to the catalytic sites of Escherichia coli βY331W-F 1 -ATPase using fluorescence quenching

Studies of nucleotide binding to the catalytic sites of Escherichia coli βY331W-F 1 -ATPase using fluorescence quenching

Molecular Modeling Reveals the Novel Inhibition Mechanism and Binding Mode of Three Natural Compounds to Staphylococcal α-Hemolysin

Phloretin Attenuates Listeria monocytogenes Virulence Both In vitro and In vivo by Simultaneously Targeting Listeriolysin O and Sortase A

Contact Info

Product

Resources

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The βG¹⁵⁶C Substitution in the F₁-ATPase from the Thermophilic Bacillus PS3 Affects Catalytic Site Cooperativity by Destabilizing the Closed Conformation of the Catalytic Site

Studies of nucleotide binding to the catalytic sites of Escherichia coli βY331W-F ₁ -ATPase using fluorescence quenching

Studies of nucleotide binding to the catalytic sites of Escherichia coli βY331W-F ₁ -ATPase using fluorescence quenching