Subnanometre-resolution structure of the intact Thermus thermophilus H+-driven ATP synthase

Ion-translocating rotary ATPases serve either as ATP synthases, using energy from a transmembrane ion motive force to create the cell's supply of ATP, or as transmembrane ion pumps that are powered by ATP hydrolysis. The members of this family of enzymes each contain two rotary motors: one that couples ion translocation to rotation and one that couples rotation to ATP synthesis or hydrolysis. During ATP synthesis, ion translocation through the membrane-bound region of the complex causes rotation of a central rotor that drives conformational changes and ATP synthesis in the catalytic region of the complex. There are no structural models available for the intact membrane region of any ion-translocating rotary ATPase. Here we present a 9.7?? resolution map of the H(+)-driven ATP synthase from Thermus thermophilus obtained by electron cryomicroscopy of single particles in ice. The 600-kilodalton complex has an overall subunit composition of A(3)B(3)CDE(2)FG(2)IL(12). The membrane-bound motor consists of a ring of L subunits and the carboxy-terminal region of subunit I, which are equivalent to the c and a subunits of most other rotary ATPases, respectively. The map shows that the ring contains 12 L subunits and that the I subunit has eight transmembrane helices. The L(12) ring and I subunit have a surprisingly small contact area in the middle of the membrane, with helices from the I subunit making contacts with two different L subunits. The transmembrane helices of subunit I form bundles that could serve as half-channels across the membrane, with the first half-channel conducting protons from the periplasm to the L(12) ring and the second half-channel conducting protons from the L(12) ring to the cytoplasm. This structure therefore suggests the mechanism by which a transmembrane proton motive force is converted to rotation in rotary ATPases.     

E. coli F1-ATPase interacts with a membrane protein component of a proton channel

The ATP synthases of bacteria, mitochondria and chloroplasts, which use the energy of a transmembrane proton gradient to power the synthesis of ATP, consist of an integral membrane component F0--thought to contain a proton channel--and a catalytic component, F1. To help investigate the way F0 and F1 are coupled, we have sequenced the b-subunit of the Escherichia coli F0, which seems to be the counterpart of a thermophilic bacteria F0 subunit thought to be essential for F1 binding. We report here that its sequence is remarkable, being hydrophobic around the N-terminus and highly charged in the remainder. We propose that the N-terminal segment lies in the membrane and the rest outside. The extramembranous section contains two adjacent stretches of 31 amino acids where the sequence is very similar: in the second of these stretches there is further internal homology. These duplicated stretches of the polypeptide probably fold into two alpha-helices which have many common features able to make contact with F1 subunits. Thus protein b occupies a central position in the enzyme, where it may be involved in proton translocation. It is possibly also important in biosynthetic assembly.     

ATP合成酶及其功能机制综述

在细胞能量转变过程中，F1F0-型ATP合成酶是一个关键酶。在ATP合成过程中，这个大的蛋白复合体利用质子梯度和相关的膜电势来合成ATP。这个酶结构的不同作用形式正在逐步阐明。一致的看法是这个酶由两个旋转发动机构成，一个在F1上，它将催化过程与内部的转子运动联系在一起，另一个在F0上，它将质子迁移与F0转子的运动联系在一起。虽然两个马达可以独立工作，但是它们必须结合在一起才能转换能量。从结构、基因和生化物理方面的研究中得出的关于这个旋转马达的功能的证据，在这里将作一个回顾，一些不确定的，关于酶机制尚留迷团的内容也将讨论如下。     

Mechanically driven ATP synthesis by F1-ATPase

ATP, the main biological energy currency, is synthesized from ADP and inorganic phosphate by ATP synthase in an energy-requiring reaction. The F1 portion of ATP synthase, also known as F1-ATPase, functions as a rotary molecular motor: in vitro its gamma-subunit rotates against the surrounding alpha3beta3 subunits, hydrolysing ATP in three separate catalytic sites on the beta-subunits. It is widely believed that reverse rotation of the gamma-subunit, driven by proton flow through the associated F(o) portion of ATP synthase, leads to ATP synthesis in biological systems. Here we present direct evidence for the chemical synthesis of ATP driven by mechanical energy. We attached a magnetic bead to the gamma-subunit of isolated F1 on a glass surface, and rotated the bead using electrical magnets. Rotation in the appropriate direction resulted in the appearance of ATP in the medium as detected by the luciferase-luciferin reaction. This shows that a vectorial force (torque) working at one particular point on a protein machine can influence a chemical reaction occurring in physically remote catalytic sites, driving the reaction far from equilibrium.     

Structure of the membrane domain of respiratory complex I

Complex I is the first and largest enzyme of the respiratory chain, coupling electron transfer between NADH and ubiquinone to the translocation of four protons across the membrane. It has a central role in cellular energy production and has been implicated in many human neurodegenerative diseases. The L-shaped enzyme consists of hydrophilic and membrane domains. Previously, we determined the structure of the hydrophilic domain. Here we report the crystal structure of the Esherichia coli complex I membrane domain at 3.0?? resolution. It includes six subunits, NuoL, NuoM, NuoN, NuoA, NuoJ and NuoK, with 55 transmembrane helices. The fold of the homologous antiporter-like subunits L, M and N is novel, with two inverted structural repeats of five transmembrane helices arranged, unusually, face-to-back. Each repeat includes a discontinuous transmembrane helix and forms half of a channel across the membrane. A network of conserved polar residues connects the two half-channels, completing the proton translocation pathway. Unexpectedly, lysines rather than carboxylate residues act as the main elements of the proton pump in these subunits. The fourth probable proton-translocation channel is at the interface of subunits N, K, J and A. The structure indicates that proton translocation in complex I, uniquely, involves coordinated conformational changes in six symmetrical structural elements.     

Proton-coupled electron transfer drives the proton pump of cytochrome c oxidase

Electron transfer in cell respiration is coupled to proton translocation across mitochondrial and bacterial membranes, which is a primary event of biological energy transduction. The resulting electrochemical proton gradient is used to power energy-requiring reactions, such as ATP synthesis. Cytochrome c oxidase is a key component of the respiratory chain, which harnesses dioxygen as a sink for electrons and links O2 reduction to proton pumping. Electrons from cytochrome c are transferred sequentially to the O2 reduction site of cytochrome c oxidase via two other metal centres, Cu(A) and haem a, and this is coupled to vectorial proton transfer across the membrane by a hitherto unknown mechanism. On the basis of the kinetics of proton uptake and release on the two aqueous sides of the membrane, it was recently suggested that proton pumping by cytochrome c oxidase is not mechanistically coupled to internal electron transfer. Here we have monitored translocation of electrical charge equivalents as well as electron transfer within cytochrome c oxidase in real time. The results show that electron transfer from haem a to the O2 reduction site initiates the proton pump mechanism by being kinetically linked to an internal vectorial proton transfer. This reaction drives the proton pump and occurs before relaxation steps in which protons are taken up from the aqueous space on one side of the membrane and released on the other.     

A mechanistic principle for proton pumping by cytochrome c oxidase

In aerobic organisms, cellular respiration involves electron transfer to oxygen through a series of membrane-bound protein complexes. The process maintains a transmembrane electrochemical proton gradient that is used, for example, in the synthesis of ATP. In mitochondria and many bacteria, the last enzyme complex in the electron transfer chain is cytochrome c oxidase (CytcO), which catalyses the four-electron reduction of O2 to H2O using electrons delivered by a water-soluble donor, cytochrome c. The electron transfer through CytcO, accompanied by proton uptake to form H2O drives the physical movement (pumping) of four protons across the membrane per reduced O2. So far, the molecular mechanism of such proton pumping driven by electron transfer has not been determined in any biological system. Here we show that proton pumping in CytcO is mechanistically coupled to proton transfer to O2 at the catalytic site, rather than to internal electron transfer. This scenario suggests a principle by which redox-driven proton pumps might operate and puts considerable constraints on possible molecular mechanisms by which CytcO translocates protons.     

Crystal structure of a catalytic intermediate of the maltose transporter

The maltose uptake system of Escherichia coli is a well-characterized member of the ATP-binding cassette transporter superfamily. Here we present the 2.8-A crystal structure of the intact maltose transporter in complex with the maltose-binding protein, maltose and ATP. This structure, stabilized by a mutation that prevents ATP hydrolysis, captures the ATP-binding cassette dimer in a closed, ATP-bound conformation. Maltose is occluded within a solvent-filled cavity at the interface of the two transmembrane subunits, about halfway into the lipid bilayer. The binding protein docks onto the entrance of the cavity in an open conformation and serves as a cap to ensure unidirectional translocation of the sugar molecule. These results provide direct evidence for a concerted mechanism of transport in which solute is transferred from the binding protein to the transmembrane subunits when the cassette dimer closes to hydrolyse ATP.     

Rebuilt AAA + motors reveal operating principles for ATP-fuelled machines

Hexameric ring-shaped ATPases of the AAA + (for ATPases associated with various cellular activities) superfamily power cellular processes in which macromolecular structures and complexes are dismantled or denatured, but the mechanisms used by these machine-like enzymes are poorly understood. By covalently linking active and inactive subunits of the ATPase ClpX to form hexamers, here we show that diverse geometric arrangements can support the enzymatic unfolding of protein substrates and translocation of the denatured polypeptide into the ClpP peptidase for degradation. These studies indicate that the ClpX power stroke is generated by ATP hydrolysis in a single subunit, rule out concerted and strict sequential ATP hydrolysis models, and provide evidence for a probabilistic sequence of nucleotide hydrolysis. This mechanism would allow any ClpX subunit in contact with a translocating polypeptide to hydrolyse ATP to drive substrate spooling into ClpP, and would prevent stalling if one subunit failed to bind or hydrolyse ATP. Energy-dependent machines with highly diverse quaternary architectures and molecular functions could operate by similar asymmetric mechanisms.     

Crystal structure of a membrane-embedded H+-translocating pyrophosphatase

H(+)-translocating pyrophosphatases (H(+)-PPases) are active proton transporters that establish a proton gradient across the endomembrane by means of pyrophosphate (PP(i)) hydrolysis. H(+)-PPases are found primarily as homodimers in the vacuolar membrane of plants and the plasma membrane of several protozoa and prokaryotes. The three-dimensional structure and detailed mechanisms underlying the enzymatic and proton translocation reactions of H(+)-PPases are unclear. Here we report the crystal structure of a Vigna radiata H(+)-PPase (VrH(+)-PPase) in complex with a non-hydrolysable substrate analogue, imidodiphosphate (IDP), at 2.35?? resolution. Each VrH(+)-PPase subunit consists of an integral membrane domain formed by 16 transmembrane helices. IDP is bound in the cytosolic region of each subunit and trapped by numerous charged residues and five Mg(2+) ions. A previously undescribed proton translocation pathway is formed by six core transmembrane helices. Proton pumping can be initialized by PP(i) hydrolysis, and H(+) is then transported into the vacuolar lumen through a pathway consisting of Arg?242, Asp?294, Lys?742 and Glu?301. We propose a working model of the mechanism for the coupling between proton pumping and PP(i) hydrolysis by H(+)-PPases.     

Highly coupled ATP synthesis by F1-ATPase single molecules

F1-ATPase is the smallest known rotary motor, and it rotates in an anticlockwise direction as it hydrolyses ATP. Single-molecule experiments point towards three catalytic events per turn, in agreement with the molecular structure of the complex. The physiological function of F1 is ATP synthesis. In the ubiquitous F0F1 complex, this energetically uphill reaction is driven by F0, the partner motor of F1, which forces the backward (clockwise) rotation of F1, leading to ATP synthesis. Here, we have devised an experiment combining single-molecule manipulation and microfabrication techniques to measure the yield of this mechanochemical transformation. Single F1 molecules were enclosed in femtolitre-sized hermetic chambers and rotated in a clockwise direction using magnetic tweezers. When the magnetic field was switched off, the F1 molecule underwent anticlockwise rotation at a speed proportional to the amount of synthesized ATP. At 10 Hz, the mechanochemical coupling efficiency was low for the alpha3beta3gamma subcomplex (F1-epsilon)), but reached up to 77% after reconstitution with the epsilon-subunit (F1+epsilon)). We provide here direct evidence that F1 is designed to tightly couple its catalytic reactions with the mechanical rotation. Our results suggest that the epsilon-subunit has an essential function during ATP synthesis.     

Cell-specific ATP7A transport sustains copper-dependent tyrosinase activity in melanosomes

Copper is a cofactor for many cellular enzymes and transporters. It can be loaded onto secreted and endomembrane cuproproteins by translocation from the cytosol into membrane-bound organelles by ATP7A or ATP7B transporters, the genes for which are mutated in the copper imbalance syndromes Menkes disease and Wilson disease, respectively. Endomembrane cuproproteins are thought to incorporate copper stably on transit through the trans-Golgi network, in which ATP7A accumulates by dynamic cycling through early endocytic compartments. Here we show that the pigment-cell-specific cuproenzyme tyrosinase acquires copper only transiently and inefficiently within the trans-Golgi network of mouse melanocytes. To catalyse melanin synthesis, tyrosinase is subsequently reloaded with copper within specialized organelles called melanosomes. Copper is supplied to melanosomes by ATP7A, a cohort of which localizes to melanosomes in a biogenesis of lysosome-related organelles complex-1 (BLOC-1)-dependent manner. These results indicate that cell-type-specific localization of a metal transporter is required to sustain metallation of an endomembrane cuproenzyme, providing a mechanism for exquisite spatial control of metalloenzyme activity. Moreover, because BLOC-1 subunits are mutated in subtypes of the genetic disease Hermansky-Pudlak syndrome, these results also show that defects in copper transporter localization contribute to hypopigmentation, and hence perhaps other systemic defects, in Hermansky-Pudlak syndrome.     

Mechanism of DNA translocation in a replicative hexameric helicase

The E1 protein of papillomavirus is a hexameric ring helicase belonging to the AAA + family. The mechanism that couples the ATP cycle to DNA translocation has been unclear. Here we present the crystal structure of the E1 hexamer with single-stranded DNA discretely bound within the hexamer channel and nucleotides at the subunit interfaces. This structure demonstrates that only one strand of DNA passes through the hexamer channel and that the DNA-binding hairpins of each subunit form a spiral 'staircase' that sequentially tracks the oligonucleotide backbone. Consecutively grouped ATP, ADP and apo configurations correlate with the height of the hairpin, suggesting a straightforward DNA translocation mechanism. Each subunit sequentially progresses through ATP, ADP and apo states while the associated DNA-binding hairpin travels from the top staircase position to the bottom, escorting one nucleotide of single-stranded DNA through the channel. These events permute sequentially around the ring from one subunit to the next.     

(Na/ + K+)ATPase has one functioning phosphorylation site per alpha subunit

(Na+ + K+)ATPase contains two different subunits, a catalytic subunit (alpha) and a subunit with uncertain function (beta). The enzyme binds ATP, ouabain and vanadate, and can be phosphorylated by ATP as well as by inorganic phosphate. From the previously reported maximal binding and phosphorylation capacities of 3.5--4.3 nmol P per mg protein (based on Lowry protein determination) and the earlier molecular weight value of approximately 250,000, a molar binding and phosphorylation capacity of 0.87--1.07 mol per mol enzyme was derived. As it is generally agreed that the enzyme molecule contains two alpha subunits or even a multiple of two, it has been suggested that the enzyme operates by means of a so-called "half-of-the-sites mechanism" whereby only of the two alpha subunits can be phosphorylated at any one time. We now present evidence that every alpha subunit can be phosphorylated simultaneously, which rules out the operation of such a mechanism.     

Structure of acid-sensing ion channel 1 at 1.9 A resolution and low pH

Acid-sensing ion channels (ASICs) are voltage-independent, proton-activated receptors that belong to the epithelial sodium channel/degenerin family of ion channels and are implicated in perception of pain, ischaemic stroke, mechanosensation, learning and memory. Here we report the low-pH crystal structure of a chicken ASIC1 deletion mutant at 1.9 A resolution. Each subunit of the chalice-shaped homotrimer is composed of short amino and carboxy termini, two transmembrane helices, a bound chloride ion and a disulphide-rich, multidomain extracellular region enriched in acidic residues and carboxyl-carboxylate pairs within 3 A, suggesting that at least one carboxyl group bears a proton. Electrophysiological studies on aspartate-to-asparagine mutants confirm that these carboxyl-carboxylate pairs participate in proton sensing. Between the acidic residues and the transmembrane pore lies a disulphide-rich 'thumb' domain poised to couple the binding of protons to the opening of the ion channel, thus demonstrating that proton activation involves long-range conformational changes.     

Insights into the subunit interactions of the chloroplast ATP synthase

Subunit interactions of the chloroplast F0F1- ATP synthase were studied using the yeast two-hybrid system. The coding sequences of all the nine subunits of spinach chloroplast ATP synthase were cloned in two-hybrid vectors. The vectors were transformed into the yeast strains HF7c and SFY526 by various pairwise combinations, and the protein interactions were analyzed by measuring the yeast growth on minimal SD medium without serine, lucine and histidine. Interactions of γ Subunit with wild type or two truncated mutants of γ sununit, △εN21 and △εC45, which lose their abilities to inhibit the ATP hydrolysis, were also detected by in vitro and in vivo binding assay. The present results are largely accordant to the common structure model of F0F1-ATP synthase. Different from that in the E. Coli F0F1-ATP synthase, the δ subunit of chloroplast ATP syn- thase could interact with β,γ,ε and all the CF0 subunits in the two-hybrid system. These results suggested that though the chloroplast ATP synthase shares the similar structure and composition of subunits with the enzyme from E. Coli, it may be different in the subunit interactions and con- formational change during catalysis between these two sources of ATP synthase. Based on the present results and our knowledge of structure model of E. Coli ATP synthase, a deduced structure model of chloroplast ATP synthase was proposed.     

Primary structure of Torpedo californica acetylcholinesterase deduced from its cDNA sequence

Acetylcholinesterase, an essential enzyme of the nervous system, rapidly terminates the action of acetylcholine released into the synapse. Acetylcholinesterase is also found (in lower abundance) in extrajunctional areas of muscle and nerve and on erythrocyte membranes. Hydrodynamic analyses of the native enzyme and characterization of its dissociated subunits have revealed multiple enzyme forms which can be divided into two classes: dimensionally asymmetric forms which are usually found within the synapse and contain a collagen-like structural subunit disulphide-linked to the catalytic subunits; and globular forms which appear to be widely distributed on the outer surface of cell membranes. Both forms have been characterized in the ray Torpedo californica and, although their catalytic behaviours seem to be identical, they differ slightly in amino-acid composition, peptide maps and reactivity with certain monoclonal antibodies. Here, we report the complete amino-acid sequence of an acetylcholinesterase inferred from the sequence of a complementary DNA clone. The 575-residue protein shows significant homology with the C-terminal portion of thyroglobulin.     

Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase

The enzyme F1-ATPase has been shown to be a rotary motor in which the central gamma-subunit rotates inside the cylinder made of alpha3beta3 subunits. At low ATP concentrations, the motor rotates in discrete 120 degrees steps, consistent with sequential ATP hydrolysis on the three beta-subunits. The mechanism of stepping is unknown. Here we show by high-speed imaging that the 120 degrees step consists of roughly 90 degrees and 30 degrees substeps, each taking only a fraction of a millisecond. ATP binding drives the 90 degrees substep, and the 30 degrees substep is probably driven by release of a hydrolysis product. The two substeps are separated by two reactions of about 1 ms, which together occupy most of the ATP hydrolysis cycle. This scheme probably applies to rotation at full speed ( approximately 130 revolutions per second at saturating ATP) down to occasional stepping at nanomolar ATP concentrations, and supports the binding-change model for ATP synthesis by reverse rotation of F1-ATPase.     

Ribosomal peptidyl transferase can withstand mutations at the putative catalytic nucleotide

Peptide bond formation is the principal reaction of protein synthesis. It takes place in the peptidyl transferase centre of the large (50S) ribosomal subunit. In the course of the reaction, the polypeptide is transferred from peptidyl transfer RNA to the alpha-amino group of amino acyl-tRNA. The crystallographic structure of the 50S subunit showed no proteins within 18 A from the active site, revealing peptidyl transferase as an RNA enzyme. Reported unique structural and biochemical features of the universally conserved adenine residue A2451 in 23S ribosomal RNA (Escherichia coli numbering) led to the proposal of a mechanism of rRNA catalysis that implicates this nucleotide as the principal catalytic residue. In vitro genetics allowed us to test the importance of A2451 for the overall rate of peptide bond formation. Here we report that large ribosomal subunits with mutated A2451 showed significant peptidyl transferase activity in several independent assays. Mutations at another nucleotide, G2447, which is essential to render catalytic properties to A2451 (refs 2, 3), also did not dramatically change the transpeptidation activity. As alterations of the putative catalytic residues do not severely affect the rate of peptidyl transfer the ribosome apparently promotes transpeptidation not through chemical catalysis, but by properly positioning the substrates of protein synthesis.     

ATP-induced helicase slippage reveals highly coordinated subunits

Helicases are vital enzymes that carry out strand separation of duplex nucleic acids during replication, repair and recombination. Bacteriophage T7 gene product 4 is a model hexameric helicase that has been observed to use dTTP, but not ATP, to unwind double-stranded (ds)DNA as it translocates from 5' to 3' along single-stranded (ss)DNA. Whether and how different subunits of the helicase coordinate their chemo-mechanical activities and DNA binding during translocation is still under debate. Here we address this question using a single-molecule approach to monitor helicase unwinding. We found that T7 helicase does in fact unwind dsDNA in the presence of ATP and that the unwinding rate is even faster than that with dTTP. However, unwinding traces showed a remarkable sawtooth pattern where processive unwinding was repeatedly interrupted by sudden slippage events, ultimately preventing unwinding over a substantial distance. This behaviour was not observed with dTTP alone and was greatly reduced when ATP solution was supplemented with a small amount of dTTP. These findings presented an opportunity to use nucleotide mixtures to investigate helicase subunit coordination. We found that T7 helicase binds and hydrolyses ATP and dTTP by competitive kinetics such that the unwinding rate is dictated simply by their respective maximum rates V(max), Michaelis constants K(M) and concentrations. In contrast, processivity does not follow a simple competitive behaviour and shows a cooperative dependence on nucleotide concentrations. This does not agree with an uncoordinated mechanism where each subunit functions independently, but supports a model where nearly all subunits coordinate their chemo-mechanical activities and DNA binding. Our data indicate that only one subunit at a time can accept a nucleotide while other subunits are nucleotide-ligated and thus they interact with the DNA to ensure processivity. Such subunit coordination may be general to many ring-shaped helicases and reveals a potential mechanism for regulation of DNA unwinding during replication.