Subunit Rotation Research Articles

Inverse Regulation of Rotation of F1-ATPase by the Mutation at the Regulatory Region on the γ Subunit of Chloroplast ATP Synthase

In F1-ATPase, the rotation of the central axis subunit gamma relative to the surrounding alpha3beta3 subunits is coupled to ATP hydrolysis. We previously reported that the introduced regulatory region of the gamma subunit of chloroplast F1-ATPase can modulate rotation of the gamma subunit of the thermophilic bacterial F1-ATPase (Bald, D., Noji, H., Yoshida, M., Hirono-Hara, Y., and Hisabori, T. (2001) J. Biol. Chem. 276, 39505-39507). The attenuated enzyme activity of this chimeric enzyme under oxidizing conditions was characterized by frequent and long pauses of rotation of gamma. In this study, we report an inverse regulation of the gamma subunit rotation in the newly engineered F1-chimeric complex whose three negatively charged residues Glu210-Asp211-Glu212 adjacent to two cysteine residues of the regulatory region derived from chloroplast F1-ATPase gamma were deleted. ATP hydrolysis activity of the mutant complex was stimulated up to 2-fold by the formation of the disulfide bond at the regulatory region by oxidation. We successfully observed inverse redox switching of rotation of gamma using this mutant complex. The complex exhibited long and frequent pauses in its gamma rotation when reduced, but the rotation rates between pauses remained unaltered. Hence, the suppression or activation of the redox-sensitive F1-ATPase can be explained in terms of the change in the rotation behavior at a single molecule level. These results obtained by the single molecule analysis of the redox regulation provide further insights into the regulation mechanism of the rotary enzyme.

Dynamics of EF-G interaction with the ribosome explored by classification of a heterogeneous cryo-EM dataset

A method of supervised classification using two available structure templates was applied to investigate the possible heterogeneity existing in a large cryo-EM dataset of an Escherichia coli 70S ribosome–EF-G complex. Two subpopulations showing the ribosome in distinct conformational states, related by a ratchet-like rotation of the 30S subunit with respect to the 50S subunit, were extracted from the original dataset. The possible presence of additional intermediate states is discussed.

Insights into the Molecular Mechanism of Rotation in the F o Sector of ATP Synthase

F 1F o-ATP synthase is a ubiquitous membrane protein complex that efficiently converts a cell's transmembrane proton gradient into chemical energy stored as ATP. The protein is made of two molecular motors, F o and F 1, which are coupled by a central stalk. The membrane unit, F o, converts the transmembrane electrochemical potential into mechanical rotation of a rotor in F o and the physically connected central stalk. Based on available data of individual components, we have built an all-atom model of F o and investigated through molecular dynamics simulations and mathematical modeling the mechanism of torque generation in F o. The mechanism that emerged generates the torque at the interface of the a- and c-subunits of F o through side groups aSer-206, aArg-210, and aAsn-214 of the a-subunit and side groups cAsp-61 of the c-subunits. The mechanism couples protonation/deprotonation of two cAsp-61 side groups, juxtaposed to the a-subunit at any moment in time, to rotations of individual c-subunit helices as well as rotation of the entire c-subunit. The aArg-210 side group orients the cAsp-61 side groups and, thereby, establishes proton transfer via aSer-206 and aAsn-214 to proton half-channels, while preventing direct proton transfer between the half-channels. A mathematical model proves the feasibility of torque generation by the stated mechanism against loads typical during ATP synthesis; the essential model characteristics, e.g., helix and subunit rotation and associated friction constants, have been tested and furnished by steered molecular dynamics simulations.

An Exceptional Variability in the Motor of Archaeal A1A0ATPases: From Multimeric to Monomeric Rotors Comprising 6–13 Ion Binding Sites

The motor domain of A1A0 ATPases is composed of only two subunits, the stator subunit I and the rotor subunit c. Recent studies on the molecular biology of the A0 domains revealed the surprising finding that the gene encoding subunit c underwent several multiplication events leading to rotor subunits comprising 2, 3, or even 13 hairpin domains suggesting multimeric in different stoichiometry as well as monomeric rotors. The number of ion translocating groups per rotor ranges from 13 to 6. Furthermore, as deduced from the gene sequences H(+)-as well as Na(+)-driven rotors are found in archaea. Features previously thought to be distinctive for A0, F0 or V0 are all found in A0 suggesting that the differences encountered in the three classes of ATPases today emerged already very early in evolution. The extraordinary features and exceptional structural and functional variability in the rotor of A1A0 ATPases may have arisen as an adaptation to different cellular needs and the extreme physicochemical conditions in the early history of life.

Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase

Synthesis of ATP from ADP and phosphate, catalyzed by F(0)F(1)-ATP synthases, is the most abundant physiological reaction in almost any cell. F(0)F(1)-ATP synthases are membrane-bound enzymes that use the energy derived from an electrochemical proton gradient for ATP formation. We incorporated double-labeled F(0)F(1)-ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis. The gamma subunit rotates stepwise during proton transport-powered ATP synthesis, showing three distinct distances to the b subunits in repeating sequences. The average durations of these steps correspond to catalytic turnover times upon ATP synthesis as well as ATP hydrolysis. The direction of rotation during ATP synthesis is opposite to that of ATP hydrolysis.

Chemomechanical coupling in F1-ATPase revealed by simultaneous observation of nucleotide kinetics and rotation.

F(1)-ATPase is a rotary molecular motor in which unidirectional rotation of the central gamma subunit is powered by ATP hydrolysis in three catalytic sites arranged 120 degrees apart around gamma. To study how hydrolysis reactions produce mechanical rotation, we observed rotation under an optical microscope to see which of the three sites bound and released a fluorescent ATP analog. Assuming that the analog mimics authentic ATP, the following scheme emerges: (i) in the ATP-waiting state, one site, dictated by the orientation of gamma, is empty, whereas the other two bind a nucleotide; (ii) ATP binding to the empty site drives an approximately 80 degrees rotation of gamma; (iii) this triggers a reaction(s), hydrolysis and/or phosphate release, but not ADP release in the site that bound ATP one step earlier; (iv) completion of this reaction induces further approximately 40 degrees rotation.

Crystal Structures of the Group II Chaperonin from Thermococcus strain KS-1: Steric Hindrance by the Substituted Amino Acid, and Inter-subunit Rearrangement between Two Crystal Forms

The crystal structures of the group II chaperonins consisting of the α subunit with amino acid substitutions of G65C and/or I125T from the hyperthermophilic archaeum Thermococcus strain KS-1 were determined. These mutants have been shown to be active in ATP hydrolysis but inactive in protein folding. The structures were shown to be double-ring hexadecamers in an extremely closed form, which was consistent with the crystal structure of native α 8β 8-chaperonin from Thermoplasma acidophilum. Comparisons of the present structures with the atomic structures of the GroEL 14–GroES 7–(ADP) 7 complex revealed that the deficiency in protein-folding activity with the G65C amino acid substitution is caused by the steric hindrance of the local conformational change in an equatorial domain. We concluded that this mutant chaperonin with G65C substitution is deprived of the smooth conformational change in the refolding-reaction cycle. We obtained a new form of crystal with a distinct space group at a lower concentration of sulfate ion in the presence of nucleotide. The crystal structure obtained at the lower concentration of sulfate ion tilts outward, and has much looser inter-subunit contacts compared with those in the presence of a higher concentration of sulfate ion. Such subunit rotation has never been characterized in group II chaperonins. The crystal structure obtained at the lower concentration of sulfate ion tilts outward, and has much looser inter-subunit contacts compared with those in the presence of a higher concentration of sulfate ion.

Interactions among γR268, γQ269, and the β Subunit Catch Loop of Escherichia coli F1-ATPase Are Important for Catalytic Activity

Removal of the ability to form a salt bridge or hydrogen bonds between the beta subunit catch loop (beta Y297-D305) and the gamma subunit of Escherichia coli F1Fo-ATP synthase significantly altered the ability of the enzyme to hydrolyze ATP and the bacteria to grow via oxidative phosphorylation. Residues beta T304, beta D305, beta D302, gamma Q269, and gamma R268 were found to be very important for ATP hydrolysis catalyzed by soluble F1-ATPase, and the latter four residues were also very important for oxidative phosphorylation. The greatest effects on catalytic activity were observed by the substitution of side chains that contribute to the shortest and/or multiple H-bonds as well as the salt bridge. Residue beta D305 would not tolerate substitution with Val or Ser and had extremely low activity as beta D305E, suggesting that this residue is particularly important for synthesis and hydrolysis activity. These results provide evidence that tight winding of the gamma subunit coiled-coil is important to the rate-limiting step in ATP hydrolysis and are consistent with an escapement mechanism for ATP synthesis in which alpha beta gamma intersubunit interactions provide a means to make substrate binding a prerequisite of proton gradient-driven gamma subunit rotation.

Crystal structure of UDP-N-acetylglucosamine acyltransferase from Helicobacter pylori.

Crystal structure of UDP-N-acetylglucosamine acyltransferase from Helicobacter pylori.

Microwave spectrum, structure, and internal motion of the open-shell van der Waals complex Ar–ClO2

The rotational spectrum of the open-shell complex argon–chlorine dioxide has been measured between 5–24 GHz with a pulsed nozzle Fourier transform microwave spectrometer. This complex displays a- and c-type spectra, the latter of which is shifted by internal motion of the ClO2 unit. Structure information has been gained from the analysis of the rotational constants and of the fine and hyperfine structure parameters of the Ar–35ClO2 and Ar–37ClO2 complexes. Changes of the electronic structure of ClO2 upon complexation are shown to be very small. The observed internal motion effect was analyzed with a model of an internal rotation of the ClO2 subunit around its a axis. In addition, the structure of the complex and the barrier hindering internal motion has also been explored by ab initio calculations. The results of the analysis of the structure and internal motion of Ar–ClO2 were compared with those of the similar argon complexes of SO2, O3, and NO2.

Agonist-induced conformational changes in the extracellular domain of alpha 7 nicotinic acetylcholine receptors.

The molecular mechanisms that couple agonist binding to the gating of Cys-loop ionotropic receptors are not well understood. The crystal structure of the acetylcholine (ACh) binding protein has provided insights into the structure of the extracellular domain of nicotinic receptors and a framework for testing mechanisms of activation. Key ligand binding residues are located at the C-terminal end of the beta9 strand. At the N-terminal end of this strand (loop 9) is a conserved glutamate [E172 in chick alpha7 nicotinic acetylcholine receptors (nAChRs)] that is important for modulating activation. We hypothesize that agonist binding induces the movement of loop 9. To test this, we used the substituted-cysteine accessibility method to examine agonist-dependent changes in the modification of cysteines introduced in loop 9 of L247T alpha7 nAChRs. In the absence of agonist, ACh-evoked responses of E172C/L247T alpha7 nAChRs were inhibited by 2-trimethylammonioethylmethane thiosulfonate (MTSET). Agonist coapplication with MTSET reduced the extent and rate of modification. The dose-dependence of ACh activation was nearly identical with that of ACh-dependent protection from modification. ACh increased the inhibition by methanethiosulfonate reagents of N170C and did not change inhibition of G171C receptors. The antagonist dihydro-beta-erythroidine did not mimic the effects of ACh. Combined with a structural model, the data suggest that receptor activation includes subunit rotation and/or intrasubunit conformational changes that move N170 to a more accessible position and E172 to a more protected position away from the vestibule. Thus, loop 9, located near the junction between the extracellular and transmembrane domains, participates in conformational changes triggered by ligand binding.

Rotation of the Proteolipid Ring in the V-ATPase

V0V1-ATPase is a proton-translocating ATPase responsible for acidification of eukaryotic intracellular compartments and for ATP synthesis in archaea and some eubacteria. We demonstrated recently the rotation of the central stalk subunits in V1, a catalytic sector of V0V1-ATPase (Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2312-2315), but the rotation of the proteolipid ring, a predicted counterpart rotor in the membrane V0 sector, has remained to be proven. V0V1-ATPase that retained sensitivity to N',N'-dicyclohexylcarbodiimide was isolated from Thermus thermophilus, immobilized onto a glass surface through the N termini of the A subunits of V1, and decorated with a bead attached to a proteolipid subunit of V0. Rotation of beads was observed in the presence of ATP, and direction of rotation was always counterclockwise viewed from the membrane side. The rotation proceeded at approximately 3.0 rev/s in average at 4 mm ATP and was abolished by N',N'-dicyclohexylcarbodiimide treatment. Thus, the rotation of the central stalk in V1 accompanies rotation of a proteolipid ring of V0 in the functioning V0V1-ATPase.

ATP synthesis driven by proton transport in F 1F 0-ATP synthase

Topical questions in ATP synthase research are: (1) how do protons cause subunit rotation and how does rotation generate ATP synthesis from ADP+Pi? (2) How does hydrolysis of ATP generate subunit rotation and how does rotation bring about uphill transport of protons? The finding that ATP synthase is not just an enzyme but rather a unique nanomotor is attracting a diverse group of researchers keen to find answers. Here we review the most recent work on rapidly developing areas within the field and present proposals for enzymatic and mechanoenzymatic mechanisms.

Ligand-linked structural transitions in crystals of a cooperative dimeric hemoglobin.

Cooperative ligand binding in the dimeric hemoglobin (HbI) from the blood clam Scapharca inaequivalvis is mediated primarily by tertiary structural changes, but with a small quaternary rearrangement (approximately 3 degrees), based on analysis of distinct crystal forms for ligated and unligated molecules. We report here ligand transition structures in both crystal forms. Binding CO to unligated HbI crystals results in a structure that approaches, but does not attain, the full allosteric transition. In contrast, removing CO from the HbI-CO crystals results in a structure that possesses all the key low affinity attributes previously identified from analysis of HbI crystals grown in the unligated state. Subsequent binding of CO shows the reversibility of this process. The observed structural changes include the quaternary rearrangement even under the constraints of lattice interactions, demonstrating that subunit rotation is an integral component of the ligand-linked structural transition in HbI. Analysis of both crystal forms, along with data from HbI mutants, suggests that the quaternary structural change is linked to the movement of the heme group, supporting a hypothesis that the heme movement is the central event that triggers cooperative ligand binding in this hemoglobin dimer. These results show both the effects of a crystal lattice in limiting quaternary structural transitions and provide the first example of complete allosteric transitions within another crystal lattice.

Molecular origin of auxetic behavior in tetrahedral framework silicates.

Recent analytical models for the Poisson's ratios (nu(ij)) of tetrahedral frameworks are applied to alpha-cristobalite and alpha-quartz for the first time. Rotation and dilation of the SiO4 tetrahedral subunits are considered. Each mechanism leads to negative nu(31) values, whereas negative and positive values are possible when they act concurrently. The concurrent model is in excellent agreement with experiment and explains the dichotomy between negative and positive nu(31) values in alpha-cristobalite and alpha-quartz, respectively. The predicted strain-dependent trends confirm those from molecular modeling.

Structures and interactions of proteins involved in the coupling function of the protonmotive F(o)F(1)-ATP synthase.

The mitochondrial F(1)F(o) ATP synthase complex has a key role in cellular energy metabolism. The general architecture of the enzyme is conserved among species and consists of a globular catalytic moiety F(1), protruding out of the inner side of the membrane, a membrane integral proton translocating moiety F(o), and a stalk connecting F(1) to F(o). The X-ray crystallographic analysis of the structure of the bovine mitochondrial F(1) ATPase has provided a structural basis for the binding-change rotary mechanism of the catalytic process in F(1), in which the gamma subunit rotates in the central cavity of the F(1) alpha3/beta3 hexamer. Rotation of gamma and eta subunits in the E. coli enzyme and of, gamma and delta subunits in the mitochondrial enzyme, is driven, during ATP synthesis, by proton motive rotation of an oligomer of c subunits (10-12 copies) within the F(o) base piece. Average analysis of electron microscopy images and cross-linking results have revealed that, in addition to a central stalk, contributed by gamma and delta/eta subunits, there is a second lateral one connecting the peripheries of F(o) and F(1). To gain deeper insight into the mechanism of coupling between proton translocation and catalytic activity (ATP synthesis and hydrolysis), studies have been undertaken on the role of F(1) and F(o) subunits which contribute to the structural and functional connection between the catalytic sector F(1) and the proton translocating moiety F(o). These studies, which employed limited proteolysis, chemical cross-linking and functional analysis of the native and reconstituted F(1)F(o) complex, as well as isolated F(1), have shown that the N-terminus of alpha subunits, located at the top of the F(1) hexamer is essential for energy coupling in the F(1)F(o) complex. The alpha N-terminus domain appears to be connected to F(o) by OSCP (F(o) subunit conferring sensitivity of the complex to oligomycin). In turn, OSCP contacts F(o)I-PVP(b) and d subunits, with which it constitutes a structure surrounding the central gamma and delta rotary shaft. Cross-linking of F(o)I-PVP(b) and gamma subunits causes a dramatic enhancement of downhill proton translocation decoupled from ATP synthesis but is without effect on ATP driven uphill proton transport. This would indicate the existence of different rate-limiting steps in the two directions of proton translocation through F(o). In mitochondria, futile ATP hydrolysis by the F(1)F(o) complex is inhibited by the ATPase inhibitor protein (IF(1)), which reversibly binds at one side of the F(1)F(o) connection. The trans-membrane deltapH component of the respiratory deltap displaces IF(1) from the complex; in particular the matrix pH is the critical factor for IF(1)association and its related inhibitory activity. The 42L-58K segment of the IF(1) has been shown to be the most active segment of the protein; it interacts with the surface of one alpha/beta pairs of F(1), thus inhibiting, with the same pH dependence as the natural IF(1), the conformational interconversions of the catalytic sites involved in ATP hydrolysis. IF(1) has a relevant physiopathological role for the conservation of the cellular ATP pool in ischemic tissues. Under these conditions IF(1), which appears to be over expressed, prevents dissipation of the glycolytic ATP.

Synthesis of Novel Mixed Metal Cluster Complexes of Osmium and Mercury: Formation of the Wheel-Shaped Cluster Complexes Os6(μ6-Hg)(μ-PR2)2(CO)20 (R = Ph, i-Bu)

The reaction of [PPN][Os3(μ-PR2)(CO)10] (R = Ph 1a, i-Bu 1b) with HgCl2 in THF at −90 °C under exclusion of light gave the spirocyclic cluster μ4-Hg[Os3(μ-PR2)(μ-CO)(CO)9]2 (R = Ph 2a, i-Bu 2b). In these compounds the μ4-Hg bridges one Os−Os edge of each Os3 subunit. The remaining four edges are bridged by the μ-CO and μ-PPh2 ligands, respectively. 13C NMR spectra of 13CO-enriched 2a recorded at room temperature indicate an intramolecular rotation of the Os3 subunits around the axis running through the mercury atom and the midpoints of the Os−Os vectors it bridges. When 2a is heated in toluene or 1,4-dioxane, a rearrangement of the metal skeleton takes place. The μ4-Hg moiety is shifted to another edge of one of the Os3 rings, giving the isomer (CO)10(μ-PPh2)Os3(μ4-Hg)Os3(μ-PPh2)(μ-CO)(CO)9, 3a. The kinetics of this process were measured by UV/vis spectroscopy at four different temperatures in toluene and 1,4-dioxane, respectively. The reaction is of first order with a rate constant of 6.66(2) × 10-5 s-1 in toluene at 331.6(1) K. The thermodynamic parameters ΔH⧧ = 97(5) kJ mol-1, ΔS⧧ = −33(13) J K-1 mol-1, and ΔG⧧ = 107(6) kJ mol-1 were derived from an Eyring plot. The analogous cluster complex 3b with μ-P(i-Bu)2 bridges has been obtained, too. Its structure has been confirmed by single-crystal X-ray analysis. 2a and 2b are highly photosensitive in solution. Upon exposure to daylight, they rearrange to give the novel wheel-shaped cluster complexes Os6(μ6-Hg)(μ-PR2)2(CO)20 (R = Ph 4a, i-Bu 4b). The structure of 4a was confirmed by single-crystal X-ray analysis. In 4a the Hg atom is located at the center of a Os6 ring. The ring is corrugated with the mercury atom in bonding distance to all osmium atoms. The course of the reaction was monitored for the conversion of 2a by UV/vis spectra exhibiting two isosbestic points indicating an intramolecular reaction.

Modeling the transmembrane domain of bacterial chemoreceptors

Bacterial chemoreceptors signal across the membrane by conformational changes that traverse a four-helix transmembrane domain. High-resolution structures are available for the chemoreceptor periplasmic domain and part of the cytoplasmic domain but not for the transmembrane domain. Thus, we constructed molecular models of the transmembrane domains of chemoreceptors Trg and Tar, using coordinates of an unrelated four-helix coiled coil as a template and the X-ray structure of a chemoreceptor periplasmic domain to establish register and positioning. We tested the models using the extensive data for cross-linking propensities between cysteines introduced into adjacent transmembrane helices, and we found that many aspects of the models corresponded with experimental observations. The one striking disparity, the register of transmembrane helix 2 (TM2) relative to its partner transmembrane helix 1, could be corrected by sliding TM2 along its long axis toward the periplasm. The correction implied that axial sliding of TM2, the signaling movement indicated by a large body of data, was of greater magnitude than previously thought. The refined models were used to assess effects of inter-helical disulfides on the two ligand-induced conformational changes observed in alternative crystal structures of periplasmic domains: axial sliding within a subunit and subunit rotation. Analyses using a measure of disulfide potential energy provided strong support for the helical sliding model of transmembrane signaling but indicated that subunit rotation could be involved in other ligand-induced effects. Those analyses plus modeled distances between diagnostic cysteine pairs indicated a magnitude for TM2 sliding in transmembrane signaling of several angstroms.

Cross-linking of the endogenous inhibitor protein (IF1) with rotor (gamma, epsilon) and stator (alpha) subunits of the mitochondrial ATP synthase.

The location of the endogenous inhibitor protein (IF1) in the rotor/stator architecture of the bovine mitochondrial ATP synthase was studied by reversible cross-linking with dithiobis(succinimidylpropionate) in soluble F1I and intact F1F0I complexes of submitochondrial particles. Reducing two-dimensional electrophoresis, Western blotting, and fluorescent cysteine labeling showed formation of alpha-IF1, IF1-IF1, gamma-IF1, and epsilon-IF1 cross-linkages in soluble F1I and in native F1F0I complexes. Cross-linking blocked the release of IF1 from its inhibitory site and therefore the activation of F1I and F1F0I complexes in a dithiothreitol-sensitive process. These results show that the endogenous IF1 is at a distance < or = 12 angstroms to gamma and epsilon subunits of the central rotor of the native mitochondrial ATP synthase. This finding strongly suggests that, without excluding the classical assumption that IF1 inhibits conformational changes of the catalytic beta subunits, the inhibitory mechanism of IF1 may involve the interference with rotation of the central stalk.

Rotational and vibrational dynamics of methane in helium nanodroplets

High resolution infrared laser spectroscopy has been used to study the rotational and vibrational dynamics of methane in helium nanodroplets. Because of the nuclear spin symmetry, four transitions ( P(1), Q(1), R(0) and R(1)) are observed. While the first three have widths of approximately 0.2 cm −1 , which we attribute to moderately fast vibrational relaxation, the significantly larger width of the R(1) transition suggests even faster rotational relaxation for the latter. At higher methane pressures in the pick-up cell, three bands are observed that are attributed to clusters of methane. The spectra suggest that nearly free rotation of the monomer sub-units is still occurring in these larger clusters.