Severe MgADP inhibition of Bacillus subtilis F1-ATPase is not due to the absence of nucleotide binding to the noncatalytic nucleotide binding sites.
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ABSTRACT:
SUBMITTER: Ishikawa T
PROVIDER: S-EPMC4171097 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:MgADP inhibition, which is considered as a part of the regulatory system of ATP synthase, is a well-known process common to all F1-ATPases, a soluble component of ATP synthase. The entrapment of inhibitory MgADP at catalytic sites terminates catalysis. Regulation by the ? subunit is a common mechanism among F1-ATPases from bacteria and plants. The relationship between these two forms of regulatory mechanisms is obscure because it is difficult to distinguish which is active at a particular moment. Here, using F1-ATPase from Bacillus subtilis (BF1), which is strongly affected by MgADP inhibition, we can distinguish MgADP inhibition from regulation by the ? subunit. The ? subunit did not inhibit but activated BF1. We conclude that the ? subunit relieves BF1 from MgADP inhibition.
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Project description:Molecular machines fueled by NTP play pivotal roles in a wide range of cellular activities. One common feature among NTP-driven molecular machines is that NTP binding is a major force-generating step among the elementary reaction steps comprising NTP hydrolysis. To understand the mechanism in detail,in this study, we conducted a single-molecule rotation assay of the ATP-driven rotary motor protein F1-ATPase using uridine triphosphate (UTP) and a base-free nucleotide (ribose triphosphate) to investigate the impact of a pyrimidine base or base depletion on kinetics and force generation. Although the binding rates of UTP and ribose triphosphate were 10(3) and 10(6) times, respectively, slower than that of ATP, they supported rotation, generating torque comparable to that generated by ATP. Affinity change of F1 to UTP coupled with rotation was determined, and the results again were comparable to those for ATP, suggesting that F1 exerts torque upon the affinity change to UTP via rotation similar to ATP-driven rotation. Thus, the adenine-ring significantly enhances the binding rate, although it is not directly involved in force generation. Taking into account the findings from another study on F1 with mutated phosphate-binding residues, it was proposed that progressive bond formation between the phosphate region and catalytic residues is responsible for the rotation-coupled change in affinity.
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Project description:Mitochondrial F(1)-ATPase contains a hexamer of alternating alpha and beta subunits. The assembly of this structure requires two specialized chaperones, Atp11p and Atp12p, that bind transiently to beta and alpha. In the absence of Atp11p and Atp12p, the hexamer is not formed, and alpha and beta precipitate as large insoluble aggregates. An early model for the mechanism of chaperone-mediated F(1) assembly (Wang, Z. G., Sheluho, D., Gatti, D. L., and Ackerman, S. H. (2000) EMBO J. 19, 1486-1493) hypothesized that the chaperones themselves look very much like the alpha and beta subunits, and proposed an exchange of Atp11p for alpha and of Atp12p for beta; the driving force for the exchange was expected to be a higher affinity of alpha and beta for each other than for the respective chaperone partners. One important feature of this model was the prediction that as long as Atp11p is bound to beta and Atp12p is bound to alpha, the two F(1) subunits cannot interact at either the catalytic site or the noncatalytic site interface. Here we present the structures of Atp11p from Candida glabrata and Atp12p from Paracoccus denitrificans, and we show that some features of the Wang model are correct, namely that binding of the chaperones to alpha and beta prevents further interactions between these F(1) subunits. However, Atp11p and Atp12p do not resemble alpha or beta, and it is instead the F(1) gamma subunit that initiates the release of the chaperones from alpha and beta and their further assembly into the mature complex.
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Project description:Identification of the specific WalR (YycF) binding regions on the B. subtilis chromosome during exponential and phosphate starvation growth phases. The data serves to extend the WalRK regulon in Bacillus subtilis and its role in cell wall metabolism, as well as implying a role in several other cellular processes.
2015-01-26 | GSE65272 | GEO
Project description:EngA proteins form a unique family of bacterial GTPases with two GTP-binding domains in tandem, namely GD1 and GD2, followed by a KH (K-homology) domain. They have been shown to interact with the bacterial ribosome and to be involved in its biogenesis. Most prokaryotic EngA possess a high GTPase activity in contrast to eukaryotic GTPases that act mainly as molecular switches. Here, we have purified and characterized the GTPase activity of the Bacillus subtilis EngA and two shortened EngA variants that only contain GD1 or GD2-KH. Interestingly, the GTPase activity of GD1 alone is similar to that of the whole EngA, whereas GD2-KH has a 150-fold lower GTPase activity. At physiological concentration, potassium strongly stimulates the GTPase activity of each protein construct. Interestingly, it affects neither the affinities for nucleotides nor the monomeric status of EngA or the GD1 domain. Thus, potassium likely acts as a chemical GTPase-activating element as proposed for another bacterial GTPase like MnmE. However, unlike MnmE, potassium does not promote dimerization of EngA. In addition, we solved two crystal structures of full-length EngA. One of them contained for the first time a GTP-like analogue bound to GD2 while GD1 was free. Surprisingly, its overall fold was similar to a previously solved structure with GDP bound to both sites. Our data indicate that a significant structural change must occur upon K(+) binding to GD2, and a comparison with T. maritima EngA and MnmE structures allowed us to propose a model explaining the chemical basis for the different GTPase activities of GD1 and GD2.
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Project description:The hydrolysis of ATP by the ATP synthase in mitochondria is inhibited by a protein called IF1. Bovine IF1 has 84 amino acids, and its N-terminal inhibitory region is intrinsically disordered. In a known structure of bovine F1-ATPase inhibited with residues 1-60 of IF1, the inhibitory region from residues 1-50 is mainly α-helical and buried deeply at the α(DP)β(DP)-catalytic interface, where it forms extensive interactions with five of the nine subunits of F1-ATPase but mainly with the β(DP)-subunit. As described here, on the basis of two structures of inhibited complexes formed in the presence of large molar excesses of residues 1-60 of IF1 and of a version of IF1 with the mutation K39A, it appears that the intrinsically disordered inhibitory region interacts first with the αEβE-catalytic interface, the most open of the three catalytic interfaces, where the available interactions with the enzyme allow it to form an α-helix from residues 31-49. Then, in response to the hydrolysis of an ATP molecule and the associated partial closure of the interface to the αTPβTP state, the extent of the folded α-helical region of IF1 increases to residues 23-50 as more interactions with the enzyme become possible. Finally, in response to the hydrolysis of a second ATP molecule and a concomitant 120° rotation of the γ-subunit, the interface closes further to the α(DP)β(DP)-state, allowing more interactions to form between the enzyme and IF1. The structure of IF1 now extends to its maximally folded state found in the previously observed inhibited complex.
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Project description:Carbohydrates from plant cell walls are often found as heteropolysaccharides intertwined with each other. For competitive advantage against other microorganisms, and ability to fully exploit available carbon and energy sources, Bacillus subtilis possesses a high number of proteins dedicated to the uptake of mono- and oligosaccharides. Here, we characterize transporter complexes, belonging to the ATP-binding cassette (ABC) superfamily, involved in the uptake of oligosaccharides commonly found in pectin. The uptake of these carbohydrates is shown to be MsmX-dependent, assigning a key role in pectin mobilization for MsmX, a multipurpose ATPase serving several distinct ABC-type I sugar importers. Mutagenesis analysis of the transmembrane domains of the AraNPQ MsmX-dependent importer revealed putative residues for MsmX interaction. Interestingly however, although MsmX is shown to be essential for energizing various ABC transporters we found that a second B. subtilis ATPase, YurJ, is able to complement its function when placed in trans at a different locus of the chromosome.
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Project description:F1-ATPase, the catalytic complex of the ATP synthase, is a molecular motor that can consume ATP to drive rotation of the γ-subunit inside the ring of three αβ-subunit heterodimers in 120° power strokes. To elucidate the mechanism of ATPase-powered rotation, we determined the angular velocity as a function of rotational position from single-molecule data collected at 200,000 frames per second with unprecedented signal-to-noise. Power stroke rotation is more complex than previously understood. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur during the power stroke. The decreases in angular velocity that occurred with the lower-affinity substrate ITP, which could not be explained by an increase in substrate-binding dwells, provides direct evidence that rotation depends on substrate binding affinity. The presence of elevated ADP concentrations not only increased dwells at 35° from the catalytic dwell consistent with competitive product inhibition but also decreased the angular velocity from 85° to 120°, indicating that ADP can remain bound to the catalytic site where product release occurs for the duration of the power stroke. The angular velocity profile also supports a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F1 structures 2HLD1 and 1H8E (Protein Data Bank).
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