Structures of the thermophilic F1-ATPase epsilon subunit suggesting ATP-regulated arm motion of its C-terminal domain in F1

The epsilon subunit of bacterial and chloroplast F(o)F(1)-ATP synthases modulates their ATP hydrolysis activity. Here, we report the crystal structure of the ATP-bound epsilon subunit from a thermophilic Bacillus PS3 at 1.9-A resolution. The C-terminal two alpha-helices were folded into a hairpin, sitting on the beta sandwich structure, as reported for Escherichia coli. A previously undescribed ATP binding motif, I(L)DXXRA, recognizes ATP together with three arginine and one glutamate residues. The E. coli epsilon subunit binds ATP in a similar manner, as judged on NMR. We also determined solution structures of the C-terminal domain of the PS3 epsilon subunit and relaxation parameters of the whole molecule by NMR. The two helices fold into a hairpin in the presence of ATP but extend in the absence of ATP. The latter structure has more helical regions and is much more flexible than the former. These results suggest that the epsilon C-terminal domain can undergo an arm-like motion in response to an ATP concentration change and thereby contribute to regulation of F(o)F(1)-ATP synthase.     

F1-ATPase conformational cycle from simultaneous single-molecule FRET and rotation measurements

Despite extensive studies, the structural basis for the mechanochemical coupling in the rotary molecular motor F₁-ATPase (F₁) is still incomplete. We performed single-molecule FRET measurements to monitor conformational changes in the stator ring-α₃β₃, while simultaneously monitoring rotations of the central shaft-γ. In the ATP waiting dwell, two of three β-subunits simultaneously adopt low FRET nonclosed forms. By contrast, in the catalytic intermediate dwell, two β-subunits are simultaneously in a high FRET closed form. These differences allow us to assign crystal structures directly to both major dwell states, thus resolving a long-standing issue and establishing a firm connection between F₁ structure and the rotation angle of the motor. Remarkably, a structure of F₁ in an ε-inhibited state is consistent with the unique FRET signature of the ATP waiting dwell, while most crystal structures capture the structure in the catalytic dwell. Principal component analysis of the available crystal structures further clarifies the five-step conformational transitions of the αβ-dimer in the ATPase cycle, highlighting the two dominant modes: the opening/closing motions of β and the loosening/tightening motions at the αβ-interface. These results provide a new view of tripartite coupling among chemical reactions, stator conformations, and rotary angles in F₁-ATPase.ATP synthase (F₁F_o-ATPase) catalyzes ATP synthesis from ADP and P_i in cells. The isolated F₁ portion is called F₁-ATPase, because it also catalyzes the reverse reaction, ATP hydrolysis (1–3). The α₃β₃γ-catalytic core complex of F₁-ATPase (denoted F₁) is a rotary molecular motor in which three αβ-dimers are arranged around the central γ-shaft (4). Unidirectional rotation of γ is driven by the free energy derived from sequential ATP hydrolysis at catalytic sites in the three αβ-dimers (5–7). Under an external torque, F₁ synthesizes ATP coupled to the rotation of γ in the opposite direction (8). This reversible operation of F₁ is achieved by the tripartite mechanochemical coupling between chemical reactions at the catalytic sites of αβ, conformational changes in the stator ring-α₃β₃, and orientation of γ.A combination of the rotation assay (5, 6) and single-molecule fluorescence imaging techniques (9) has led to a detailed picture of the coupling between chemical reactions in α₃β₃ and the rotary angles (10, 11). One ATP hydrolysis reaction in α₃β₃ drives discrete 80° + 40° substeps of γ in bacterial F₁ (7). The 80° substep is mainly driven by the binding energy of ATP (7, 9). The dwell before the 80° substep is, therefore, named the ATP waiting dwell. Release of the product, ADP, occurs before completion of the 80° substep (9, 10). The angle-dependent affinity of ADP suggests that the ADP release event also contributes part of the energy for the 80° substep (10). The dwell before the 40° substep is called the catalytic dwell; it consists of two rate-limiting events: ATP cleavage and release of the product, P_i (10, 12). The 40° substep is accompanied by a decrease of P_i affinity, with release that, in turn, generates torque (10). The coupling scheme between chemical reactions in α₃β₃ and the rotary angles has, therefore, been almost completely established (10).In the coupling of chemical reactions and α₃β₃-conformations, the key concept is thought to be the binding change mechanism, in which three catalytic sites in F₁ undergo sequential transitions between conformational states with different affinities for nucleotides corresponding to different rotary angles (1). The binding change mechanism is supported by the first crystal structure of F₁, in which two βs adopt the closed form with nucleotides and the other β adopts the open form without a nucleotide (4). The 120° step of γ observed in the rotation assay further supports this mechanism (6).However, we still face significant gaps in the structural ATPase cycle. Previous studies have suggested that F₁ should adopt at least two distinct conformational states for the ATP waiting dwell (ATP waiting form) and the catalytic dwell (catalytic form) based on rotation and tilting angles of γ (13). Furthermore, based on indirect evidence, it has been pointed out that the first crystal structure should represent the catalytic form or forms similar to the catalytic intermediate states (14–18). Although the crystal structures of the α₃β₃γ-complex differ from each other in terms of their nucleotide binding states and detailed configurations of the residues, their global structures are similar to the first crystal structure (19), which leaves the structure of the ATP waiting form unresolved. Closing this gap in the conformational cycle will deepen our understanding of the coupling between chemical reactions, α₃β₃-conformations, conformations, and rotary angles, not least by providing critical input into the theoretical modeling of F₁ (20–26).Here, we use the FRET technique to elucidate the conformational transitions of α₃β₃-conformations in F₁. FRET involves excited-state energy transfer from one fluorescent dye (donor) to another (acceptor) through dipole–dipole interactions (27). Single-pair FRET measurements combined with single-molecule techniques have been used to investigate the dynamics of intramolecular conformational changes or intermolecular interactions at the single-molecule level (28–34), including for F_oF₁-ATP synthase (35–37). We perform single-molecule FRET measurement to monitor distance changes between two fluorescently labeled βs and simultaneously monitor the rotational steps of γ. The FRET data allow us to distinguish the ATP waiting form from the catalytic form and thus, relate these dwelling states to the respective crystal structures. A systematic comparison of the crystal structures reveals the structural basis of the ATPase cycle. This study provides a structural basis for tripartite coupling among chemical reactions, conformations in the stator, and rotary angles in bacterial F₁.     

Realistic simulations of the coupling between the protomotive force and the mechanical rotation of the F0-ATPase

The molecular origin of the action of the F(0) proton gradient-driven rotor presents a major puzzle despite significant structural advances. Although important conceptual models have provided guidelines of how such systems should work, it has been challenging to generate a structure-based molecular model using physical principles that will consistently lead to the unidirectional proton-driven rotational motion during ATP synthesis. This work uses a coarse-grained (CG) model to simulate the energetics of the F(0)-ATPase system in the combined space defined by the rotational coordinate and the proton transport (PTR) from the periplasmic side (P) to the cytoplasmic side (N). The model establishes the molecular origin of the rotation, showing that this effect is due to asymmetry in the energetics of the proton path rather than only the asymmetry of the interaction of the Asp on the c-ring helices and Arg on the subunit-a. The simulation provides a clear conceptual background for further exploration of the electrostatic basis of proton-driven mechanochemical systems.     

Engineering rotor ring stoichiometries in the ATP synthase

ATP synthase membrane rotors consist of a ring of c-subunits whose stoichiometry is constant for a given species but variable across different ones. We investigated the importance of c/c-subunit contacts by site-directed mutagenesis of a conserved stretch of glycines (GxGxGxGxG) in a bacterial c(11) ring. Structural and biochemical studies show a direct, specific influence on the c-subunit stoichiometry, revealing c(<11), c(12), c(13), c(14), and c(>14) rings. Molecular dynamics simulations rationalize this effect in terms of the energetics and geometry of the c-subunit interfaces. Quantitative data from a spectroscopic interaction study demonstrate that the complex assembly is independent of the c-ring size. Real-time ATP synthesis experiments in proteoliposomes show the mutant enzyme, harboring the larger c(12) instead of c(11), is functional at lower ion motive force. The high degree of compliance in the architecture of the ATP synthase rotor offers a rationale for the natural diversity of c-ring stoichiometries, which likely reflect adaptations to specific bioenergetic demands. These results provide the basis for bioengineering ATP synthases with customized ion-to-ATP ratios, by sequence modifications.     

Trapping the ATP binding state leads to a detailed understanding of the F1-ATPase mechanism

The rotary motor enzyme F_oF₁-ATP synthase uses the proton-motive force across a membrane to synthesize ATP from ADP and P_i (H₂PO₄⁻) under cellular conditions that favor the hydrolysis reaction by a factor of 2 × 10⁵. This remarkable ability to drive a reaction away from equilibrium by harnessing an external force differentiates it from an ordinary enzyme, which increases the rate of reaction without shifting the equilibrium. Hydrolysis takes place in the neighborhood of one conformation of the catalytic moiety F₁-ATPase, whose structure is known from crystallography. By use of molecular dynamics simulations we trap a second structure, which is rotated by 40° from the catalytic dwell conformation and represents the state associated with ATP binding, in accord with single-molecule experiments. Using the two structures, we show why P_i is not released immediately after ATP hydrolysis, but only after a subsequent 120° rotation, in agreement with experiment. A concerted conformational change of the α₃β₃ crown is shown to induce the 40° rotation of the γ-subunit only when the β_E subunit is empty, whereas with P_i bound, β_E serves as a latch to prevent the rotation of γ. The present results provide a rationalization of how F₁-ATPase achieves the coupling between the small changes in the active site of β_DP and the 40° rotation of γ.The molecular motor F_oF₁-ATP synthase is composed of two domains: a transmembrane portion (F_o), the rotation of which is induced by a proton gradient, and a globular catalytic moiety (F₁) that synthesizes and hydrolyzes ATP. The primary function of the proton-motive force acting on F_oF₁-ATP synthase is to provide the torque required to rotate the γ-subunit in the direction for ATP synthesis (1, 2). The catalytic moiety, F₁-ATPase, has an α₃β₃ “crown” composed of three α- and three β-subunits arranged in alternation around the γ-subunit, which has a globular base and an extended coiled-coil portion (3) (Fig. 1A). F₁-ATPase by itself binds ATP and hydrolyzes it to induce rotation of the γ-subunit (in the opposite direction from that for synthesis) on the millisecond time scale under optimum conditions (4, 5). All of the α- and β-subunits bind nucleotides, but only the three β-subunits are catalytically active. The original crystal structure (3) of F₁-ATPase from bovine heart mitochondria (MF₁) led to the identification of three conformations of the β-subunit: β_E (empty), β_TP (ATP analog bound), and β_DP (ADP bound); Fig. 1A. In the known structures of F₁-ATPase, which apparently are near the “catalytic dwell” state, the state in which catalysis occurs (6, 7), the β_E subunit conformation is partly to fully open and is very different from those of the β_TP and β_DP subunits, which are closed and very similar to each other (SI Appendix, SI1).Open in a separate windowFig. 1.(A) F₁-ATPase. The three β-subunits and the γ-subunit are shown (α-subunits are not shown for clarity): β_E (yellow), β_DP (orange), β_TP (gold), and γ (purple). To define the β_DP subunit conformation we use the angle between helix B (βT163-A176) and helix C (βT190-G204). The two helices are highlighted: helix B (blue) and helix C (gray); the B^C angle is depicted as a red angle. The β_DPH6 helix, whose orientation was reported to undergo a 20° change during the 40° substep γ-rotation, is highlighted as red. During the forced rotation simulations with an external torque, the force acts on the C_α atom of MF₁:γM25 (shown as a red sphere). The direction of the force is determined as the cross-product of the radial vector of γM25:C_α and the rotational axis (green). (B) Proposed 360° rotation cycle of F₁-ATPase showing the subunit conformations, as well as the binding–release of ligands and the hydrolysis of ATP. Starting from the binding of an ATP* to the β_E subunit in the ATP waiting state (0°), rotation of the γ-stalk by 200° (80°, 40°, 80°) leads to the transition of β_E (γ = 0°) via β_TP (γ = 80°) to β_DP (γ = 200°), the catalytic dwell state where hydrolysis of ATP* takes place. The hydrolysis product P_i* in the β_DP subunit is not released at this catalytic dwell (200°). Instead, the other hydrolysis product ADP* is released first after a 40° rotation [β_DP (200°) → β_HO (240°)]. Then, β_HO is transformed to β_E and P_i* is released after an additional 80° rotation to another catalytic dwell state (320°); the latter is shown in brackets outside the main cycle (see below). Finally, the release of P_i* from β_E leads to a 40° rotation that completes the 360° cycle (21, 41). The other subunits are going through corresponding cycles offset by 120° (β_DP) and 240° (β_TP), respectively. Here, the prime symbol when it appears on the β_DP and β_E conformations indicates that the conformation of corresponding subunits change slightly in or near the specified reaction steps. The γ-subunit is shown as a yellow oval, and its rotation during the hydrolysis cycle is indicated by a red arrow. The reaction steps occurring in or near the catalytic dwell and corresponding changes of ligands in each β-subunit are also shown in the 320° catalytic dwell: The first state (Left in the 320° catalytic dwell) has a bound ATP in β_DP′, and is thus referred to as a prehydrolysis state (the state before the hydrolysis of ATP during the catalytic dwell). The second state (Middle) represents the state after ATP hydrolysis (posthydrolysis state), and the third state (Right) presents the state after the release of P_i bound in β_E′ (postrelease state).     

Interacting cytoplasmic loops of subunits a and c of Escherichia coli F1F0 ATP synthase gate H+ transport to the cytoplasm

H⁺-transporting F₁F₀ ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F₀ and F₁. H⁺ transport at the subunit a–c interface in transmembranous F₀ drives rotation of a cylindrical c₁₀ oligomer within the membrane, which is coupled to rotation of subunit γ within the α₃β₃ sector of F₁ to mechanically drive ATP synthesis. F₁F₀ functions in a reversible manner, with ATP hydrolysis driving H⁺ transport. ATP-driven H⁺ transport in a select group of cysteine mutants in subunits a and c is inhibited after chelation of Ag⁺ and/or Cd⁺² with the substituted sulfhydryl groups. The H⁺ transport pathway mapped via these Ag⁺(Cd⁺²)-sensitive Cys extends from the transmembrane helices (TMHs) of subunits a and c into cytoplasmic loops connecting the TMHs, suggesting these loop regions could be involved in gating H⁺ release to the cytoplasm. Here, using select loop-region Cys from the single cytoplasmic loop of subunit c and multiple cytoplasmic loops of subunit a, we show that Cd⁺² directly inhibits passive H⁺ transport mediated by F₀ reconstituted in liposomes. Further, in extensions of previous studies, we show that the regions mediating passive H⁺ transport can be cross-linked to each other. We conclude that the loop-regions in subunits a and c that are implicated in H⁺ transport likely interact in a single structural domain, which then functions in gating H⁺ release to the cytoplasm.The F₁F₀-ATP synthase of oxidative phosphorylation uses the energy of a transmembrane electrochemical gradient of H⁺ or Na⁺ to mechanically drive the synthesis of ATP via two coupled rotary motors in the F₁ and F₀ sectors of the enzyme (1). H⁺ transport through the transmembrane F₀ sector is coupled to ATP synthesis or hydrolysis in the F₁ sector at the surface of the membrane. Homologous ATP synthases are found in mitochondria, chloroplasts, and many bacteria. In Escherichia coli and other eubacteria, F₁ consists of five subunits in an α₃β₃γδε stoichiometry. F₀ is composed of three subunits in a likely ratio of a₁b₂c₁₀ in E. coli and Bacillus PS3 (2, 3) or a₁b₂c₁₁ in the Na⁺ translocating Ilyobacter tartaricus ATP synthase (1, 4) and may contain as many as 15 c subunits in other bacterial species (5). Subunit c spans the membrane as a hairpin of two α-helices, with the first transmembrane helix (TMH) on the inside and the second TMH on the outside of the c ring (1, 4). The binding of Na⁺ or H⁺ occurs at an essential, membrane-embedded Glu or Asp on cTMH2. High-resolution X-ray structures of both Na⁺- and H⁺-binding c-rings have revealed the details and variations in the cation binding sites (4–8). In the H⁺-translocating E. coli enzyme, Asp-61 at the center of cTMH2 is thought to undergo protonation and deprotonation, as each subunit of the c ring moves past the stationary subunit a. In the functioning enzyme, the rotation of the c ring is thought to be driven by H⁺ transport at the subunit a/c interface. Subunit γ physically binds to the cytoplasmic surface of the c-ring, which results in the coupling of c-ring rotation with rotation of subunit γ within the α₃β₃ hexamer of F₁ to mechanically drive ATP synthesis (1).E. coli subunit a folds in the membrane with five TMHs and is thought to provide aqueous access channels to the H⁺-binding cAsp-61 residue (9, 10). Interaction of the conserved Arg-210 residue in aTMH4 with cTMH2 is thought to be critical during the deprotonation–protonation cycle of cAsp-61 (1, 11, 12). At this time, very limited biophysical or crystallographic information is available on the 3D arrangement of the TMHs in subunit a. TMHs 2–5 of subunit a pack in a four-helix bundle, which was initially defined by cross-linking (13), but now, such a bundle, packing at the periphery of the c-ring, has been viewed directly by high-resolution cryoelectron microscopy in the I. tartaricus enzyme (14). Previously published cross-linking experiments support the identification of aTMH4 and aTMH5 packing at the periphery of the c-ring and the identification of aTHM2 and aTMH3 as the other components of the four-helix bundle seen in these images (13, 15, 16). More recently, published cross-linking experiments identify the N-terminal α-helices of two b subunits, one of which packs at one surface of aTMH2 with close enough proximity to the c-ring to permit cross-linking (17). The other subunit b N-terminal helix packs on the opposite peripheral surface of aTMH2 in a position where it can also be cross-linked to aTMH3 (17). The last helix density shown in Hakulinen and colleagues (14) packs at the periphery of the c-ring next to aTMH5 and is very likely to be aTMH1.The aqueous accessibility of Cys residues introduced into the five TMHs of subunit a has been probed on the basis of their reactivity with and inhibitory effects of Ag⁺ and other thiolate-reactive agents (18–20). Two regions of aqueous access were found with distinctly different properties. One region in TMH4, extending from Asn-214 and Arg-210 at the center of the membrane to the cytoplasmic surface, contains Cys substitutions that are sensitive to inhibition by both N-ethylmaleimide (NEM) and Ag⁺ (18–20; Fig. 1). These NEM- and Ag⁺-sensitive residues in TMH4 pack at or near the peripheral face and cytoplasmic side of the modeled four-helix bundle (11, 13). A second set of Ag⁺-sensitive substitutions in subunit a mapped to the opposite face and periplasmic side of aTMH4 (18, 19), and Ag⁺-sensitive substitutions were also found in TMHs 2, 3, and 5, where they extend from the center of the membrane to the periplasmic surface (19, 20). The Ag⁺-sensitive substitutions on the periplasmic side of TMHs 2–5 cluster at the interior of the four-helix bundle predicted by cross-linking and could interact to form a continuous aqueous pathway extending from the periplasmic surface to the central region of the lipid bilayer (11, 13, 19, 20). We have proposed that the movement of H⁺ from the periplasmic half-channel and binding to the single ionized Asp61 in the c-ring is mediated by a swiveling of TMHs at the a–c subunit interface (16, 21–24). This gating is thought to be coupled with ionization of a protonated cAsp61 in the adjacent subunit of the c-ring and with release of the H⁺ into the cytoplasmic half-channel at the subunit a–c interface. The route of aqueous access to the cytoplasmic side of the c subunit packing at the a–c interface has also been mapped by the chemical probing of Cys substitutions and, more recently, by molecular dynamics simulations (22, 25, 26).Open in a separate windowFig. 1.The predicted topology of subunit a in the E. coli inner membrane. The location of the most Ag⁺-sensitive Cys substitutions are highlighted in red (>85% inhibition) or orange (66–85% inhibition). The five proposed TMHs are shown in boxes, each with a span of 21 amino acids, which is the minimum length required to span the hydrophobic core of a lipid bilayer. The α-helical segments shown in loops 1–2 and 3–4 are consistent with the predictions of TALOS, based on backbone chemical shifts seen by NMR (29). Others have also predicted extensive α-helical regions in these loops (12, 30), but the possible positions remain largely speculative. aArg210 is highlighted in green. Figure is modified from those shown previously (21, 23, 24, 27).We have also reported Ag⁺-sensitive Cys substitutions in two cytoplasmic loops of subunit a (27) and, more recently, in the cytoplasmic loop of subunit c (28). The mechanism by which Ag⁺ inhibited F₁F₀-mediated H⁺ transport was uncertain. Several of these substitutions were also sensitive to inhibition by Cd⁺², and these substitutions provided a means of testing whether Cd⁺² directly inhibits passive H⁺ transport through F₀ (28). In the case of two subunit c loop substitutions, Cd⁺² was shown to directly inhibit passive F₀-mediated transport activity. In this study, we have extended the survey to Cd⁺²-sensitive Cys substitutions in cytoplasmic loops of subunit a. We report four loop substitutions in which Cd⁺² inhibits passive F₀-mediated H⁺ transport. Further, in two cases, we show cross-linking between pairs of Cys substitutions, which lie in subunits a and c, respectively, and which individually mediate passive H⁺ transport activity. These results suggest that the a and c loops, which gate H⁺ release to the cytoplasm, fold into a single domain at the surface of F₀.     

Structure of the rotor ring modified with N,N'-dicyclohexylcarbodiimide of the Na+-transporting vacuolar ATPase

The prokaryotic V-ATPase of Enterococcus hirae, closely related to the eukaryotic enzymes, provides a unique opportunity to study the ion-translocation mechanism because it transports Na⁺, which can be detected by radioisotope () experiments and X-ray crystallography. In this study, we demonstrated that the binding affinity of the rotor ring (K ring) for decreased approximately 30-fold by reaction with N,N^′-dicyclohexylcarbodiimide (DCCD), and determined the crystal structures of Na⁺-bound and Na⁺-unbound K rings modified with DCCD at 2.4- and 3.1-Å resolutions, respectively. Overall these structures were similar, indicating that there is no global conformational change associated with release of Na⁺ from the DCCD-K ring. A conserved glutamate residue (E139) within all 10 ion-binding pockets of the K ring was neutralized by modification with DCCD, and formed an “open” conformation by losing hydrogen bonds with the Y68 and T64 side chains, resulting in low affinity for Na⁺. This open conformation is likely to be comparable to that of neutralized E139 forming a salt bridge with the conserved arginine of the stator during the ion-translocation process. Based on these findings, we proposed the ion-translocation model that the binding affinity for Na⁺ decreases due to the neutralization of E139, thus releasing bound Na⁺, and that the structures of Na⁺-bound and Na⁺-unbound DCCD-K rings are corresponding to intermediate states before and after release of Na⁺ during rotational catalysis of V-ATPase, respectively.     

How subunit coupling produces the gamma-subunit rotary motion in F1-ATPase

F(o)F(1)-ATP synthase manufactures the energy "currency," ATP, of living cells. The soluble F(1) portion, called F(1)-ATPase, can act as a rotary motor, with ATP binding, hydrolysis, and product release, inducing a torque on the gamma-subunit. A coarse-grained plastic network model is used to show at a residue level of detail how the conformational changes of the catalytic beta-subunits act on the gamma-subunit through repulsive van der Waals interactions to generate a torque that drives unidirectional rotation, as observed experimentally. The simulations suggest that the calculated 85 degrees substep rotation is driven primarily by ATP binding and that the subsequent 35 degrees substep rotation is produced by product release from one beta-subunit and a concomitant binding pocket expansion of another beta-subunit. The results of the simulation agree with single-molecule experiments [see, for example, Adachi K, et al. (2007) Cell 130:309-321] and support a tri-site rotary mechanism for F(1)-ATPase under physiological condition.     

Ecto-F₁-ATPase: a moonlighting protein complex and an unexpected apoA-I receptor

Mitochondrial ATP synthase has been recently detected at the surface of different cell types, where it is a high affinity receptor for apoA-I, the major protein component in high density lipoproteins (HDL). Cell surface ATP synthase (namely ecto-F₁-ATPase) expression is related to different biological effects, such as regulation of HDL uptake by hepatocytes, endothelial cell proliferation or antitumor activity of Vγ9/Vδ2 T lymphocytes. This paper reviews the recently discovered functions and regulations of ecto-F₁-ATPase. Particularly, the role of the F₁-ATPase pathway(s) in HDL-cholesterol uptake and apoA-I-mediated endothelial protection suggests its potential importance in reverse cholesterol transport and its regulation might represent a potential therapeutic target for HDL-related therapy for cardiovascular diseases. Therefore, it is timely for us to better understand how this ecto-enzyme and downstream pathways are regulated and to develop pharmacologic interventions.     

Diverse genetic basis of field-evolved resistance to Bt cotton in cotton bollworm from China

Evolution of pest resistance reduces the efficacy of insecticidal proteins from Bacillus thuringiensis (Bt) used in sprays or in transgenic crops. Although several pests have evolved resistance to Bt crops in the field, information about the genetic basis of field-evolved resistance to Bt crops has been limited. In particular, laboratory-selected resistance to Bt toxin Cry1Ac based on recessive mutations in a gene encoding a toxin-binding cadherin protein has been identified in three major cotton pests, but previous work has not determined if such mutations are associated with field-selected resistance to Bt cotton. Here we show that the most common resistance alleles in field populations of cotton bollworm, Helicoverpa armigera, selected with Bt cotton in northern China, had recessive cadherin mutations, including the deletion mutation identified via laboratory selection. However, unlike all previously studied cadherin resistance alleles, one field-selected cadherin resistance allele conferred nonrecessive resistance. We also detected nonrecessive resistance that was not genetically linked with the cadherin locus. In field-selected populations, recessive cadherin alleles accounted for 75-84% of resistance alleles detected. However, most resistance alleles occurred in heterozygotes and 59-94% of resistant individuals carried at least one nonrecessive resistance allele. The results suggest that resistance management strategies must account for diverse resistance alleles in field-selected populations, including nonrecessive alleles.     

A genetic basis for atrophy: dominant non-responsiveness and helicobacter induced gastritis in F1 hybrid mice

BACKGROUND: The importance of host factors in helicobacter induced gastritis has been shown in animal models. Infection of most mouse strains with Helicobacter felis results in a functional atrophic gastritis, while other strains remain gastritis free. AIMS: To investigate these host factors further by using genetic crosses of responder and non-responder mice. METHODS: F(1) hybrids of the non-responder CBA/Ca strain and three strains of mice known to develop H felis induced gastritis were infected for three months with H felis. Gastritis was assessed by histopathology and serum antibody responses by ELISA. RESULTS: Infection of CBA/Ca mice and F(1) hybrids induced little or no gastritis. Analyses of the antibody responses in these mice revealed virtually undetectable anti-helicobacter antibody levels despite colonisation with high numbers of H felis. In contrast, infection of H felis responsive strains induced gastritis and a significant humoral immune response. CONCLUSIONS: The non-responsiveness of CBA/Ca mice to H felis infection is dominantly inherited. The lack of gastritis in CBA mice and their offspring is probably due to active suppression of the immune response normally mounted against H felis. Investigation of these mechanisms will provide important insights relevant to induction of gastric atrophy and cancer in humans.     

Correlation between the conformational states of F1-ATPase as determined from its crystal structure and single-molecule rotation

F₁-ATPase is a rotary molecular motor driven by ATP hydrolysis that rotates the γ-subunit against the α₃β₃ ring. The crystal structures of F₁, which provide the structural basis for the catalysis mechanism, have shown essentially 1 stable conformational state. In contrast, single-molecule studies have revealed that F₁ has 2 stable conformational states: ATP-binding dwell state and catalytic dwell state. Although structural and single-molecule studies are crucial for the understanding of the molecular mechanism of F₁, it remains unclear as to which catalytic state the crystal structure represents. To address this issue, we introduced cysteine residues at βE391 and γR84 of F₁ from thermophilic Bacillus PS3. In the crystal structures of the mitochondrial F₁, the corresponding residues in the ADP-bound β (β_DP) and γ were in direct contact. The βE190D mutation was additionally introduced into the β to slow ATP hydrolysis. By incorporating a single copy of the mutant β-subunit, the chimera F₁, α₃β₂β(E190D/E391C)γ(R84C), was prepared. In single-molecule rotation assay, chimera F₁ showed a catalytic dwell pause in every turn because of the slowed ATP hydrolysis of β(E190D/E391C). When the mutant β and γ were cross-linked through a disulfide bond between βE391C and γR84C, F₁ paused the rotation at the catalytic dwell angle of β(E190D/E391C), indicating that the crystal structure represents the catalytic dwell state and that β_DP is the catalytically active form. The former point was again confirmed in experiments where F₁ rotation was inhibited by adenosine-5′-(β,γ-imino)-triphosphate and/or azide, the most commonly used inhibitors for the crystallization of F₁.     

Increased ATPase activity produced by mutations at arginine-1380 in nucleotide-binding domain 2 of ABCC8 causes neonatal diabetes

Gain-of-function mutations in the genes encoding the ATP-sensitive potassium (K(ATP)) channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) are a common cause of neonatal diabetes mellitus. Here we investigate the molecular mechanism by which two heterozygous mutations in the second nucleotide-binding domain (NBD2) of SUR1 (R1380L and R1380C) separately cause neonatal diabetes. SUR1 is a channel regulator that modulates the gating of the pore formed by Kir6.2. K(ATP) channel activity is inhibited by ATP binding to Kir6.2 but is stimulated by MgADP binding, or by MgATP binding and hydrolysis, at the NBDs of SUR1. Functional analysis of purified NBD2 showed that each mutation enhances MgATP hydrolysis by purified isolated fusion proteins of maltose-binding protein and NBD2. Inhibition of ATP hydrolysis by MgADP was unaffected by mutation of R1380, but inhibition by beryllium fluoride (which traps the ATPase cycle in the prehydrolytic state) was reduced. MgADP-dependent activation of K(ATP) channel activity was unaffected. These data suggest that the R1380L and R1380C mutations enhance the off-rate of P(i), thereby enhancing the hydrolytic rate. Molecular modeling studies supported this idea. Because mutant channels were inhibited less strongly by MgATP, this would increase K(ATP) currents in pancreatic beta cells, thus reducing insulin secretion and producing diabetes.     

ATP-driven stepwise rotation of FoF1-ATP synthase

FoF1-ATP synthase (FoF1) is a motor enzyme that couples ATP synthesis/hydrolysis with a transmembrane proton translocation. F1, a water-soluble ATPase portion of FoF1, rotates by repeating ATP-waiting dwell, 80 degrees substep rotation, catalytic dwell, and 40 degrees -substep rotation. Compared with F1, rotation of FoF1 has yet been poorly understood, and, here, we analyzed ATP-driven rotations of FoF1. Rotation was probed with an 80-nm bead attached to the ring of c subunits in the immobilized FoF1 and recorded with a submillisecond fast camera. The rotation rates at various ATP concentrations obeyed the curve defined by a Km of approximately 30 microM and a Vmax of approximately 350 revolutions per second (at 37 degrees C). At low ATP, ATP-waiting dwell was seen and the kon-ATP was estimated to be 3.6 x 10(7) M(-1) x s(-1). At high ATP, fast, poorly defined stepwise motions were observed that probably reflect the catalytic dwells. When a slowly hydrolyzable substrate, adenosine 5'-[gamma-thio]triphosphate, was used, the catalytic dwells consisting of two events were seen more clearly at the angular position of approximately 80 degrees . The rotational behavior of FoF1 resembles that of F1. This finding indicates that "friction" in Fo motor is negligible during the ATP-driven rotation. Tributyltin chloride, a specific inhibitor of proton translocation, slowed the rotation rate by 96%. However, dwells at clearly defined angular positions were not observed under these conditions, indicating that inhibition by tributyltin chloride is complex.     

Structural study on the architecture of the bacterial ATP synthase Fo motor

We purified the F_o complex from the Ilyobacter tartaricus Na⁺-translocating F₁F_o-ATP synthase and performed a biochemical and structural study. Laser-induced liquid bead ion desorption MS analysis demonstrates that all three subunits of the isolated F_o complex were present and in native stoichiometry (ab₂c₁₁). Cryoelectron microscopy of 2D crystals yielded a projection map at a resolution of 7.0 Å showing electron densities from the c₁₁ rotor ring and up to seven adjacent helices. A bundle of four helices belongs to the stator a-subunit and is in contact with c₁₁. A fifth helix adjacent to the four-helix bundle interacts very closely with a c-subunit helix, which slightly shifts its position toward the ring center. Atomic force microscopy confirms the presence of the F_o stator, and a height profile reveals that it protrudes less from the membrane than c₁₁. The data limit the dimensions of the subunit a/c-ring interface: Three helices from the stator region are in contact with three c₁₁ helices. The location and distances of the stator helices impose spatial restrictions on the bacterial F_o complex.     

Prostaglandin F2α elevates blood pressure and promotes atherosclerosis

Little is known about prostaglandin F_2α in cardiovascular homeostasis. Prostaglandin F_2α dose-dependently elevates blood pressure in WT mice via activation of the F prostanoid (FP) receptor. The FP is expressed in preglomerular arterioles, renal collecting ducts, and the hypothalamus. Deletion of the FP reduces blood pressure, coincident with a reduction in plasma renin concentration, angiotensin, and aldosterone, despite a compensatory up-regulation of AT1 receptors and an augmented hypertensive response to infused angiotensin II. Plasma and urinary osmolality are decreased in FP KOs that exhibit mild polyuria and polydipsia. Atherogenesis is retarded by deletion of the FP, despite the absence of detectable receptor expression in aorta or in atherosclerotic lesions in Ldlr KOs. Although vascular TNF_α, inducible nitric oxide enzyme and TGF_β are reduced and lesional macrophages are depleted in the FP/Ldlr double KOs, this result reflects the reduction in lesion burden, as the FP is not expressed on macrophages and its deletion does not alter macrophage cytokine generation. Blockade of the FP offers an approach to the treatment of hypertension and its attendant systemic vascular disease.     

Thermodynamic efficiency and mechanochemical coupling of F1-ATPase

F(1)-ATPase is a nanosized biological energy transducer working as part of F(o)F(1)-ATP synthase. Its rotary machinery transduces energy between chemical free energy and mechanical work and plays a central role in the cellular energy transduction by synthesizing most ATP in virtually all organisms. However, information about its energetics is limited compared to that of the reaction scheme. Actually, fundamental questions such as how efficiently F(1)-ATPase transduces free energy remain unanswered. Here, we demonstrated reversible rotations of isolated F(1)-ATPase in discrete 120° steps by precisely controlling both the external torque and the chemical potential of ATP hydrolysis as a model system of F(o)F(1)-ATP synthase. We found that the maximum work performed by F(1)-ATPase per 120° step is nearly equal to the thermodynamical maximum work that can be extracted from a single ATP hydrolysis under a broad range of conditions. Our results suggested a 100% free-energy transduction efficiency and a tight mechanochemical coupling of F(1)-ATPase.