Neither helix in the coiled coil region of the axle of F1-ATPase plays a significant role in torque production

F₁-ATPase is an ATP-driven rotary molecular motor in which the central γ-subunit rotates inside the cylinder made of α₃β₃ subunits. The amino and carboxy termini of the γ-subunit form the axle, an α-helical coiled coil that deeply penetrates the stator cylinder. We previously truncated the axle step by step, starting with the longer carboxy terminus and then cutting both termini at the same levels, resulting in a slower yet considerably powerful rotation. Here we examine the role of each helix by truncating only the carboxy terminus by 25-40 amino-acid residues. Longer truncation impaired the stability of the motor complex severely: 40 deletions failed to yield rotating the complex. Up to 36 deletions, however, the mutants produced an apparent torque at nearly half of the wild-type torque, independent of truncation length. Time-averaged rotary speeds were low because of load-dependent stumbling at 120° intervals, even with saturating ATP. Comparison with our previous work indicates that half the normal torque is produced at the orifice of the stator. The very tip of the carboxy terminus adds the other half, whereas neither helix in the middle of the axle contributes much to torque generation and the rapid progress of catalysis. None of the residues of the entire axle played a specific decisive role in rotation.     

None of the Rotor Residues of F1-ATPase Are Essential for Torque Generation

F₁-ATPase is a powerful rotary molecular motor that can rotate an object several hundred times as large as the motor itself against the viscous friction of water. Forced reverse rotation has been shown to lead to ATP synthesis, implying that the mechanical work against the motor’s high torque can be converted into the chemical energy of ATP. The minimal composition of the motor protein is α₃β₃γ subunits, where the central rotor subunit γ turns inside a stator cylinder made of alternately arranged α₃β₃ subunits using the energy derived from ATP hydrolysis. The rotor consists of an axle, a coiled coil of the amino- and carboxyl-terminal α-helices of γ, which deeply penetrates the stator cylinder, and a globular protrusion that juts out from the stator. Previous work has shown that, for a thermophilic F₁, significant portions of the axle can be truncated and the motor still rotates a submicron sized bead duplex, indicating generation of up to half the wild-type (WT) torque. Here, we inquire if any specific interactions between the stator and the rest of the rotor are needed for the generation of a sizable torque. We truncated the protruding portion of the rotor and replaced part of the remaining axle residues such that every residue of the rotor has been deleted or replaced in this or previous truncation mutants. This protrusionless construct showed an unloaded rotary speed about a quarter of the WT, and generated one-third to one-half of the WT torque. No residue-specific interactions are needed for this much performance. F₁ is so designed that the basic rotor-stator interactions for torque generation and control of catalysis rely solely upon the shape and size of the rotor at very low resolution. Additional tailored interactions augment the torque to allow ATP synthesis under physiological conditions.     

Role of the DELSEED Loop in Torque Transmission of F1-ATPase

F₁-ATPase is an ATP-driven rotary motor that generates torque at the interface between the catalytic β-subunits and the rotor γ-subunit. The β-subunit inwardly rotates the C-terminal domain upon nucleotide binding/dissociation; hence, the region of the C-terminal domain that is in direct contact with γ—termed the DELSEED loop—is thought to play a critical role in torque transmission. We substituted all the DELSEED loop residues with alanine to diminish specific DELSEED loop-γ interactions and with glycine to disrupt the loop structure. All the mutants rotated unidirectionally with kinetic parameters comparable to those of the wild-type F₁, suggesting that the specific interactions between DELSEED loop and γ is not involved in cooperative interplays between the catalytic β-subunits. Glycine substitution mutants generated half the torque of the wild-type F₁, whereas the alanine mutant generated comparable torque. Fluctuation analyses of the glycine/alanine mutants revealed that the γ-subunit was less tightly held in the α₃β₃-stator ring of the glycine mutant than in the wild-type F₁ and the alanine mutant. Molecular dynamics simulation showed that the DELSEED loop was disordered by the glycine substitution, whereas it formed an α-helix in the alanine mutant. Our results emphasize the importance of loop rigidity for efficient torque transmissions.     

None of the Rotor Residues of F1-ATPase Are Essential for Torque Generation

F₁-ATPase is a powerful rotary molecular motor that can rotate an object several hundred times as large as the motor itself against the viscous friction of water. Forced reverse rotation has been shown to lead to ATP synthesis, implying that the mechanical work against the motor’s high torque can be converted into the chemical energy of ATP. The minimal composition of the motor protein is α₃β₃γ subunits, where the central rotor subunit γ turns inside a stator cylinder made of alternately arranged α₃β₃ subunits using the energy derived from ATP hydrolysis. The rotor consists of an axle, a coiled coil of the amino- and carboxyl-terminal α-helices of γ, which deeply penetrates the stator cylinder, and a globular protrusion that juts out from the stator. Previous work has shown that, for a thermophilic F₁, significant portions of the axle can be truncated and the motor still rotates a submicron sized bead duplex, indicating generation of up to half the wild-type (WT) torque. Here, we inquire if any specific interactions between the stator and the rest of the rotor are needed for the generation of a sizable torque. We truncated the protruding portion of the rotor and replaced part of the remaining axle residues such that every residue of the rotor has been deleted or replaced in this or previous truncation mutants. This protrusionless construct showed an unloaded rotary speed about a quarter of the WT, and generated one-third to one-half of the WT torque. No residue-specific interactions are needed for this much performance. F₁ is so designed that the basic rotor-stator interactions for torque generation and control of catalysis rely solely upon the shape and size of the rotor at very low resolution. Additional tailored interactions augment the torque to allow ATP synthesis under physiological conditions.     

Rotary catalysis of the stator ring of F1-ATPase

F₁-ATPase is a rotary motor protein in which 3 catalytic β-subunits in a stator α₃β₃ ring undergo unidirectional and cooperative conformational changes to rotate the rotor γ-subunit upon adenosine triphosphate hydrolysis. The prevailing view of the mechanism behind this rotary catalysis elevated the γ-subunit as a “dictator” completely controlling the chemical and conformational states of the 3 catalytic β-subunits. However, our recent observations using high-speed atomic force microscopy clearly revealed that the 3 β-subunits undergo cyclic conformational changes even in the absence of the rotor γ-subunit, thus dethroning it from its dictatorial position. Here, we introduce our results in detail and discuss the possible operating principle behind the F₁-ATPase, along with structurally related hexameric ATPases, also mentioning the possibility of generating hybrid nanomotors. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

N-terminal half of the cGMP phosphodiesterase gamma-subunit contributes to stabilization of the GTPase-accelerating protein complex

In the visual signal terminating transition state, the cyclic GMP phosphodiesterase (PDE6) inhibitory γ-subunit (PDEγ) stimulates GTPase activity of the α-subunit of transducin (αt) by enhancing the interaction between αt and its regulator of G protein signaling (RGS9), which is constitutively bound to the type 5 G protein β-subunit (β5). Although it is known from a crystal structure of partial molecules that the PDEγ C terminus contacts with both αt and RGS9, contributions from the intrinsically disordered PDEγ N-terminal half remain unclear. In this study, we were able to investigate this issue using a photolabel transfer strategy that allows for mapping the interface of full-length proteins. We observed label transfer from PDEγ N-terminal positions 50, 30, and 16 to RGS9·β5 in the GTPase-accelerating protein (GAP) complex composed of PDEγ·αt·RGS9·β5. In support of a direct PDEγ N-terminal interaction with RGS9·β5, the PDEγ N-terminal peptide PDEγ(1-61) abolished label transfer to RGS9·β5, and another N-terminal peptide, PDEγ(10-30), disassembled the GAP complex in label transfer and pulldown experiments. Furthermore, we determined that the PDEγ C-terminal interaction with αt was enhanced whereas the N-terminal interaction was weakened upon changing the αt conformation from the signaling state to the transition state. This "rearrangement" of PDEγ domain interactions with αt appears to facilitate the interaction of the PDEγ N-terminal half with RGS9·β5 and hence its contribution to optimal stabilization of the GAP complex.     

The rotor tip inside a bearing of a thermophilic F1-ATPase is dispensable for torque generation

F(1)-ATPase is an ATP-driven rotary molecular motor in which the central gamma-subunit rotates inside a stator cylinder made of alpha(3)beta(3) subunits. To elucidate the role of rotor-stator interactions in torque generation, we truncated the gamma-subunit at its carboxyl terminus, which forms an alpha helix that penetrates deeply into the stator cylinder. We used an alpha(3)beta(3)gamma subcomplex of F(1)-ATPase derived from thermophilic Bacillus PS3 and expressed it in Escherichia coli. We could obtain purified subcomplexes in which 14, 17, or 21 amino-acid residues were deleted. The rotary characteristics of the truncated mutants, monitored by attaching a duplex of 0.49-microm beads to the gamma-subunit, did not differ greatly from those of the wild-type over the ATP concentrations of 20 nM-2 mM, the most conspicuous effect being approximately 50% reduction in torque and approximately 70% reduction in the rate of ATP binding upon deletion of 21 residues. The ATP hydrolysis activity estimated in bulk samples was more seriously affected. The 21-deletion mutant, in particular, was >10-fold less active, but this is likely due to instability of this subcomplex. For torque generation, though not for rapid catalysis, most of the rotor-stator contacts on the deeper half of the penetrating portion of the gamma-subunit are dispensable.     

Potency of GABA at human recombinant GABAA receptors expressed in Xenopus oocytes: a mini review

GABA_A receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory neurotransmission in the central nervous system. They are thought to be composed of 2 alpha (α), 2 beta (β) subunits and one other such as a gamma (γ) or delta (δ) subunit. The potency of GABA is influenced by the subunit composition. However, there are no reported systematic studies that evaluate GABA potency on a comprehensive number of subunit combinations expressed in Xenopus oocytes, despite the wide use of this heterologous expression system in structure–function studies and drug discovery. Thus, the aim of this study was to conduct a systematic characterization of the potency of GABA at 43 human recombinant GABA_A receptor combinations expressed in Xenopus oocytes using the two-electrode voltage clamp technique. The results show that the α-subunits and to a lesser extent, the β-subunits influence GABA potency. Of the binary and ternary combinations with and without the γ2L subunit, the α6/γ2L-containing receptors were the most sensitive to GABA, while the β2- or β3-subunit conferred higher sensitivity to GABA than receptors containing the β1-subunit with the exception of the α2β1γ2L and α6β1γ2L subtypes. Of the δ-subunit containing GABA_A receptors, α4/δ-containing GABA_A receptors displayed highest GABA sensitivity, with mid-nanomolar concentrations activating α4β1δ and α4β3δ receptors. At α4β2δ, GABA had low micromolar activity.     

Unique assignment of inter-subunit association in GABAA α1β3γ2 receptors determined by molecular modeling

Recent publications defined requirements for inter-subunit contacts in a benzodiazepine-sensitive GABA_A receptor (GABA_ARα1β3γ2). There is strong evidence that the heteropentameric receptor contains two α1, two β3, and one γ2 subunit. However, the available data do not distinguish two possibilities: When viewed clockwise from an extracellular viewpoint the subunits could be arranged in either γ2β3α1β3α1 or γ2α1β3α1β3 configurations. Here we use molecular modeling to thread the relevant GABA_AR subunit sequences onto a template of homopentameric subunits in the crystal structure of the acetylcholine binding protein (AChBP). The GABA_A sequences are known to have 15-18% identity with the acetylcholine binding protein and nearly all residues that are conserved within the nAChR family are present in AChBP. The correctly aligned GABA_A sequences were threaded onto the AChBP template in the γ2β3α1β3α1 or γ2α1β3α1β3  arrangements. Only the γ2α1β3α1β3 arrangement satisfied three known criteria: (1) α1 His¹⁰² binds at the γ2 subunit interface in proximity to γ2 residues Thr¹⁴², Phe⁷⁷, and Met¹³⁰; (2) α1 residues 80-100 bind near γ2 residues 91-104; and (3) α1 residues 58-67 bind near the β3 subunit interface. In addition to predicting the most likely inter-subunit arrangement, the model predicts which residues form the GABA and benzodiazepine binding sites.     

GPCR-induced dissociation of G-protein subunits in early stage signal transduction

G-protein coupled receptors (GPCRs) form a ternary complex of agonist, receptor and G-proteins during primary signal transduction at the cell membrane. Downstream signalling is thought to be preceded by the process of dissociation of Gα and Gβγ subunits, thus exposing new surfaces to interact with downstream effectors. We demonstrate here for the first time, the dissociation of heterotrimeric G-protein subunits (i.e., Gα and Gβγ) following agonist-induced GPCR (α_2A-adrenergic receptor; α_2A-AR) activation in a cell-free assay system. α_2A-AR membranes were reconstituted with the G-proteins (±hexahistidine-tagged) Gα_i1 and Gβ₁γ₂ and functional signalling was determined following activation of the reconstituted receptor:G-protein complex with the potent agonist UK-14304, and [³⁵S]GTPγS. In the presence of Ni²⁺-coated agarose beads, the activated his-tagged Gα_i1his-[³⁵S]GTPγS complex was captured on the Ni²⁺-presenting surface. When his-tagged Gβ₁γ₂ (Gβ₁γ₂his) was used with Gα_i1, the [³⁵S]GTPγS-bound Gα_i1 was not present on the Ni²⁺-coated beads, but rather, it was separated from the β₁γ₂(his)-beads, demonstrating receptor-induced dissociation of Gα and Gβγ subunits. Treatment of the reconstituted α_2A-AR membranes containing Gβ₁γ₂his:Gα_i1 with imidazole confirmed the specificity for the Ni²⁺:G-protein surface dissociation of Gα_i1 from Gβ₁γ₂his. These data demonstrate for the first time, the complete dissociation of the G-protein subunits and extend observations on the role of G-proteins in the assembly and disassembly of the ternary complex in the primary events of GPCR signalling.     

Crystallographic characterization of the α,γ C12 helix in hybrid peptide sequences

The solid‐state conformations of two αγ hybrid peptides Boc‐[Aib‐γ⁴(R)Ile]₄‐OMe 1 and Boc‐[Aib‐γ⁴(R)Ile]₅‐OMe 2 are described. Peptides 1 and 2 adopt C₁₂‐helical conformations in crystals. The structure of octapeptide 1 is stabilized by six intramolecular 4 → 1 hydrogen bonds, forming 12 atom C₁₂ motifs. The structure of peptide 2 reveals the formation of eight successive C₁₂ hydrogen‐bonded turns. Average backbone dihedral angles for αγ C₁₂ helices are peptide 1 , Aib; φ (°) = ?57.2 ± 0.8, ψ (°) = ?44.5 ± 4.7; γ⁴(R)Ile; φ (°) = ?127.3 ± 7.3, θ₁ (°) = 58.5 ± 12.1, θ₂ (°) = 67.6 ± 10.1, ψ (°) = ?126.2 ± 16.1; peptide 2 , Aib; φ (°) = ?58.8 ± 5.1, ψ (°) = ?40.3 ± 5.5; ψ⁴(R)Ile; φ (°) = ?123.9 ± 2.7, θ₁ (°) = 53.3 θ 4.9, θ ₂ (°) = 61.2 ± 1.6, ψ (°) = ?121.8 ± 5.1. The tendency of γ⁴‐substituted residues to adopt gauche–gauche conformations about the C^α–C^β and C^β–C^γ bonds facilitates helical folding. The αγ C₁₂ helix is a backbone expanded analog of α peptide 3₁₀ helix. The hydrogen bond parameters for α peptide 3₁₀ and α‐helices are compared with those for αγ hybrid C₁₂ helix. Copyright © 2016 European Peptide Society and John Wiley & Sons.     

Flexibility within the Rotor and Stators of the Vacuolar H+-ATPase

The V-ATPase is a membrane-bound protein complex which pumps protons across the membrane to generate a large proton motive force through the coupling of an ATP-driven 3-stroke rotary motor (V₁) to a multistroke proton pump (V_o). This is done with near 100% efficiency, which is achieved in part by flexibility within the central rotor axle and stator connections, allowing the system to flex to minimise the free energy loss of conformational changes during catalysis. We have used electron microscopy to reveal distinctive bending along the V-ATPase complex, leading to angular displacement of the V₁ domain relative to the V_o domain to a maximum of ~30°. This has been complemented by elastic network normal mode analysis that shows both flexing and twisting with the compliance being located in the rotor axle, stator filaments, or both. This study provides direct evidence of flexibility within the V-ATPase and by implication in related rotary ATPases, a feature predicted to be important for regulation and their high energetic efficiencies.     

Solution structure analysis of the periplasmic region of bacterial flagellar motor stators by small angle X-ray scattering

The bacterial flagellar motor drives the rotation of helical flagellar filaments to propel bacteria through viscous media. It consists of a dynamic population of mechanosensitive stators that are embedded in the inner membrane and activate in response to external load. This entails assembly around the rotor, anchoring to the peptidoglycan layer to counteract torque from the rotor and opening of a cation channel to facilitate an influx of cations, which is converted into mechanical rotation. Stator complexes are comprised of four copies of an integral membrane A subunit and two copies of a B subunit. Each B subunit includes a C-terminal OmpA-like peptidoglycan-binding (PGB) domain. This is thought to be linked to a single N-terminal transmembrane helix by a long unstructured peptide, which allows the PGB domain to bind to the peptidoglycan layer during stator anchoring. The high-resolution crystal structures of flagellar motor PGB domains from Salmonella enterica (MotB_C2) and Vibrio alginolyticus (PomB_C5) have previously been elucidated. Here, we use small-angle X-ray scattering (SAXS). We show that unlike MotB_C2, the dimeric conformation of the PomB_C5 in solution differs to its crystal structure, and explore the functional relevance by characterising gain-of-function mutants as well as wild-type constructs of various lengths. These provide new insight into the conformational diversity of flagellar motor PGB domains and experimental verification of their overall topology.     

A novel penicillin acylase from the environmental gene pool with improved synthetic properties

A new penicillin acylase was isolated by cloning and functional screening of DNA isolated from a sand soil enrichment culture. Sequence analysis of this enzyme, PAS2, revealed homology to a group of prominent penicillin G acylases, including the intensively studied enzyme of Escherichia coli ATCC 11105. Accordingly, PAS2 was found to be an Ntn-hydrolase with an N-terminal serine as the catalytic nucleophile, located on its 61.9 kDa β-subunit. The α-subunit was shown to have a molecular mass of 25.5 kDa.To evaluate the biocatalytic performance of the new enzyme, the complex kinetic parameters α, β₀, and γ were determined for the kinetically controlled synthesis of a number of important semi-synthetic penicillins and cephalosporins. While α is a measure for the relative affinity of the enzyme for the activated acyl donor (AD), β₀ and γ quantify the efficiency of acyl-transfer to the β-lactam nucleophile. Compared to the penicillin acylase of E. coli, PAS2 showed superior potential for the synthesis of 6-aminopenicillanic acid (6-APA)-derived antibiotics, allowing the accumulation of up to 2.3-fold more target product at significantly higher conversion rates. In the synthesis of amoxicillin, for instance, 1.6-fold more antibiotic was formed using the new enzyme, making PAS2 an interesting candidate for biocatalytic application.     

Heterooligomers of the muscarinic receptor and G proteins purified from porcine atria

Muscarinic receptor extracted from porcine atria in digitonin-cholate copurified with Gα_o, Gα_i1-3, and caveolins. The presence of complexes was confirmed by coimmunoprecipitation of the receptor, α-subunits, and caveolins in various combinations. Homooligomers of α_i2 were detected on Western blots, and heterooligomers of α_i2 and α_o were identified by coimmunoprecipitation; thus, a complex may contain at least two α-subunits. Other combinations of α-subunit were not detected. The ratio of total α-subunit to receptor was near 1, as measured by [³⁵S]GTPγS and the antagonist [³H]quinuclidinylbenzilate, and the binding of [³⁵S]GTPγS was manifestly biphasic. The ratio of α_o to α_i1,2 also was near 1, as determined from the intensity of Western blots. Cardiac muscarinic receptors therefore can be purified as a mixture of complexes that contain caveolins and oligomers of α-subunit, some of which are heteromeric. Each complex would appear to contain equal numbers of α-subunit and the receptor.     

A stimulatory subunit in the polypeptide elongation factor-1 of the chick brain

Abstract: The higher-molecular-weight elongation factor-1 (EF-1_H) of the chick brain was observed to contain three subunits (denominated α, β, and γ), contrary to a previous report that the brain EF-1_H consisted of aggregates of low-molecular-weight elongation factor- 1 (EF-1_L). Crude EF-1_H, obtained from 20-day embryonic brain, was treated with 0.4 M ammonium chloride and 0.1 mM GTP, and EF-1_βγ, was obtained using a DEAE-Sephadex column equilibrated in 0.025 mM GTP. Both EF-1_β, and EF-1_γ, were isolated by means of a DE-52 column equilibrated in 6 M urea and were found to have molecular weights of 2.8 and 4.8 × 10⁴, respectively. EF-1_β and EF-1_γ were also obtained from young rat and calf brains by the same procedures. The molecular weight of the isolated EF-1_α was 5 × 10⁴. It was found that EF-1_β stimulated the two EF-1_α-dependent reactions, i.e., phenylalanyl-tRNA binding (reaction 1) and polyphenylalanine synthesis (reaction 2), and also stimulated the nucleotide exchange reaction in the EF- 1_α-guanine nucleotide binary complex (reaction 3). The degrees of stimulation of reactions 1, 2, and 3 by the addition of EF-1_β were 2 to 3 times, about 18 times, and 2 to 3 times as much as with EF-1_α alone, respectively. The amino acid compositions of EF-1_α -1_β, and -1_γ and EF-2 were very similar to those of other eukaryotic tissues. Thus the constituents and properties of EFs of the brain were found to be basically similar to those of other tissues of eukaryotes, although EF-1_β, and EF-1, had not been reported in the brain. A possible physiological significance of EF-1_β during brain development is also discussed.     

MALE ALTERNATIVE REPRODUCTIVE STRATEGIES IN A MARINE ISOPOD CRUSTACEAN (PARACERCEIS SCULPTA): THE USE OF GENETIC MARKERS TO MEASURE DIFFERENCES IN FERTILIZATION SUCCESS AMONG α-, β-, AND γ-MALES

Three discrete male morphs coexist in Paracerceis sculpta, a marine isopod crustacean inhabiting the northern Gulf of California. Ornamented α-males establish themselves in the spongocoels of intertidal sponges, where females congregate to breed. Smaller β-males, resembling sexually mature females, enter spongocoels by deception, while tiny γ-males invade spongocoels by stealth. Isopods breed year-round, and the operational sex ratio fluctuates widely over short durations. When females are abundant, receptive females accumulate in spongocoels, and these spongocoels are preferentially invaded by β- and γ-males. To test the hypothesis that the density of receptive females affects relative fertilization success among male morphs, individual β- and γ-males, heterozygous for a dominant cuticular pigmentation allele, were placed in artificial spongocoels with an unmarked α-male and densities of one, two, and three unmarked, receptive females. The fertilization success of each male was determined by counting the number of marked and unmarked progeny each female produced. Alpha-males guard females effectively and sire nearly all young when one female is in a spongocoel. The success of β- and γ-males increases, however, and may even exceed the success of α-males when two or three females are present. The regular occurrence of more than one receptive female in the harems of α-males may contribute to the persistence of β- and γ-males in this species.