The membrane rotor ring from the vacuolar-type (V-type) sodium ion-pumping adenosine triphosphatase (Na⁺-ATPase) from Enterococcus hirae consists of 10 NtpK subunits, which are homologs of the ...16-kilodalton and 8-kilodalton proteolipids found in other V-ATPases and in F₁Fsubscript o- or F-ATPases, respectively. Each NtpK subunit has four transmembrane alpha helices, with a sodium ion bound between helices 2 and 4 at a site buried deeply in the membrane that includes the essential residue glutamate-139. This site is probably connected to the membrane surface by two half-channels in subunit NtpI, against which the ring rotates. Symmetry mismatch between the rotor and catalytic domains appears to be an intrinsic feature of both V- and F-ATPases.
The structure of the complex between bovine mitochondrial F₁-ATPase and a stator subcomplex has been determined at a resolution of 3.2 Å. The resolved region of the stator contains residues 122-207 ...of subunit b; residues 5-25 and 35-57 of F₆; 3 segments of subunit d from residues 30-40, 65-74, and 85-91; and residues 1-146 and 169-189 of the oligomycin sensitivity conferral protein (OSCP). The stator subcomplex represents its membrane distal part, and its structure has been augmented with an earlier structure of a subcomplex containing residues 79-183, 3-123, and 5-70 of subunits b, d, and F₆, respectively, which extends to the surface of the inner membrane of the mitochondrion. The N-terminal domain of the OSCP links the stator with F₁-ATPase via α-helical interactions with the N-terminal region of subunit αE. Its C-terminal domain makes extensive helix-helix interactions with the C-terminal α-helix of subunit b from residues 190-207. Subunit b extends as a continuous 160-Å long α-helix from residue 188 back to residue 79 near to the surface of the inner mitochondrial membrane. This helix appears to be stiffened by other α-helices in subunits d and F₆, but the structure can bend inward toward the F₁ domain around residue 146 of subunit b. The linker region between the 2 domains of the OSCP also appears to be flexible, enabling the stator to adjust its shape as it passes over the changing profile of the F₁ domain during a catalytic cycle. The structure of the membrane extrinsic part of bovine ATP synthase is now complete.
How Azide Inhibits ATP Hydrolysis by the F-ATPases Bowler, Matthew W.; Montgomery, Martin G.; Leslie, Andrew G. W. ...
Proceedings of the National Academy of Sciences - PNAS,
06/2006, Letnik:
103, Številka:
23
Journal Article
Recenzirano
Odprti dostop
In the structure of bovine F1-ATPase determined at 1.95-Å resolution with crystals grown in the presence of ADP, 5'-adenylylimidodiphosphate, and azide, the azide anion interacts with the β-phosphate ...of ADP and with residues in the ADP-binding catalytic subunit,$\beta_{DP}$. It occupies a position between the catalytically essential amino acids, β-Lys-162 in the P loop and the "arginine finger" residue, α-Arg-373, similar to the site occupied by the γ-phosphate in the ATP-binding subunit,$\beta_{TP}$. Its presence in the$\beta_{DP}$-Subunit tightens the binding of the side chains to the nucleotide, enhancing its affinity and thereby stabilizing the state with bound ADP. This mechanism of inhibition appears to be common to many other ATPases, including ABC transporters, SecA, and DNA topoisomerase IIα. It also explains the stimulatory effect of azide on ATP-sensitive potassium channels by enhancing the binding of ADP.
The hydrolysis of ATP by the ATP synthase in mitochondria is inhibited by a protein called IF ₁. Bovine IF ₁ has 84 amino acids, and its N-terminal inhibitory region is intrinsically disordered. In a ...known structure of bovine F ₁-ATPase inhibited with residues 1–60 of IF ₁, the inhibitory region from residues 1–50 is mainly α-helical and buried deeply at the α DPβ DP-catalytic interface, where it forms extensive interactions with five of the nine subunits of F ₁-ATPase but mainly with the β DP-subunit. As described here, on the basis of two structures of inhibited complexes formed in the presence of large molar excesses of residues 1–60 of IF ₁ and of a version of IF ₁ with the mutation K39A, it appears that the intrinsically disordered inhibitory region interacts first with the α Eβ E-catalytic interface, the most open of the three catalytic interfaces, where the available interactions with the enzyme allow it to form an α-helix from residues 31–49. Then, in response to the hydrolysis of an ATP molecule and the associated partial closure of the interface to the α TPβ TP state, the extent of the folded α-helical region of IF ₁ increases to residues 23–50 as more interactions with the enzyme become possible. Finally, in response to the hydrolysis of a second ATP molecule and a concomitant 120° rotation of the γ-subunit, the interface closes further to the α DPβ DP-state, allowing more interactions to form between the enzyme and IF ₁. The structure of IF ₁ now extends to its maximally folded state found in the previously observed inhibited complex.
The crystal structure of the F₁-catalytic domain of the adenosine triphosphate (ATP) synthase has been determined from Mycobacterium smegmatis which hydrolyzes ATP very poorly. The structure of the ...α₃β₃-component of the catalytic domain is similar to those in active F₁-ATPases in Escherichia coli and Geobacillus stearothermophilus. However, its ε-subunit differs from those in these two active bacterial F₁-ATPases as an ATP molecule is not bound to the two α-helices forming its C-terminal domain, probably because they are shorter than those in active enzymes and they lack an amino acid that contributes to the ATP binding site in active enzymes. In E. coli and G. stearothermophilus, the α-helices adopt an “up” state where the α-helices enter the α₃β₃-domain and prevent the rotor from turning. The mycobacterial F₁-ATPase is most similar to the F₁-ATPase from Caldalkalibacillus thermarum, which also hydrolyzes ATP poorly. The βE-subunits in both enzymes are in the usual “open” conformation but appear to be occupied uniquely by the combination of an adenosine 5′-diphosphate molecule with no magnesium ion plus phosphate. This occupation is consistent with the finding that their rotors have been arrested at the same point in their rotary catalytic cycles. These bound hydrolytic products are probably the basis of the inhibition of ATP hydrolysis. It can be envisaged that specific as yet unidentified small molecules might bind to the F₁ domain in Mycobacterium tuberculosis, prevent ATP synthesis, and inhibit the growth of the pathogen.
iMOSFLM is a graphical user interface to the diffraction data‐integration program MOSFLM. It is designed to simplify data processing by dividing the process into a series of steps, which are normally ...carried out sequentially. Each step has its own display pane, allowing control over parameters that influence that step and providing graphical feedback to the user. Suitable values for integration parameters are set automatically, but additional menus provide a detailed level of control for experienced users. The image display and the interfaces to the different tasks (indexing, strategy calculation, cell refinement, integration and history) are described. The most important parameters for each step and the best way of assessing success or failure are discussed.
β-adrenergic receptors (βARs) are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins upon binding catecholamine agonist ligands such as adrenaline and noradrenaline. Synthetic ...ligands have been developed that either activate or inhibit βARs for the treatment of asthma, hypertension or cardiac dysfunction. These ligands are classified as either full agonists, partial agonists or antagonists, depending on whether the cellular response is similar to that of the native ligand, reduced or inhibited, respectively. However, the structural basis for these different ligand efficacies is unknown. Here we present four crystal structures of the thermostabilized turkey (Meleagris gallopavo) β(1)-adrenergic receptor (β(1)AR-m23) bound to the full agonists carmoterol and isoprenaline and the partial agonists salbutamol and dobutamine. In each case, agonist binding induces a 1 Å contraction of the catecholamine-binding pocket relative to the antagonist bound receptor. Full agonists can form hydrogen bonds with two conserved serine residues in transmembrane helix 5 (Ser(5.42) and Ser(5.46)), but partial agonists only interact with Ser(5.42) (superscripts refer to Ballesteros-Weinstein numbering). The structures provide an understanding of the pharmacological differences between different ligand classes, illuminating how GPCRs function and providing a solid foundation for the structure-based design of novel ligands with predictable efficacies.
The rotation of the central stalk of F₁-ATPase is driven by energy derived from the sequential binding of an ATP molecule to its three catalytic sites and the release of the products of hydrolysis. ...In human F₁-ATPase, each 360° rotation consists of three 120° steps composed of substeps of about 65°, 25°, and 30°, with intervening ATP binding, phosphate release, and catalytic dwells, respectively. The F₁-ATPase inhibitor protein, IF₁, halts the rotary cycle at the catalytic dwell. The human and bovine enzymes are essentially identical, and the structure of bovine F₁-ATPase inhibited by IF₁ represents the catalytic dwell state. Another structure, described here, of bovine F₁-ATPase inhibited by an ATP analog and the phosphate analog, thiophosphate, represents the phosphate binding dwell. Thiophosphate is bound to a site in the αEβE-catalytic interface, whereas in F₁-ATPase inhibited with IF₁, the equivalent site is changed subtly and the enzyme is incapable of binding thiophosphate. These two structures provide a molecular mechanism of how phosphate release generates a rotary substep as follows. In the active enzyme, phosphate release from the βE-subunit is accompanied by a rearrangement of the structure of its binding site that prevents released phosphate from rebinding. The associated extrusion of a loop in the βE-subunit disrupts interactions in the αEβE-catalytic interface and opens it to its fullest extent. Other rearrangements disrupt interactions between the γ-subunit and the C-terminal domain of the αE-subunit. To restore most of these interactions, and to make compensatory new ones, the γ-subunit rotates through 25°–30°.
Mitochondrial complex I (proton-pumping NADH:ubiquinone oxidoreductase) is an essential respiratory enzyme. Mammalian complex I contains 45 subunits: 14 conserved “core” subunits and 31 ...“supernumerary” subunits. The structure ofBos tauruscomplex I, determined to 5-Å resolution by electron cryomicroscopy, described the structure of the mammalian core enzyme and allowed the assignment of 14 supernumerary subunits. Here, we describe the 6.8-Å resolution X-ray crystallography structure of subcomplex Iβ, a large portion of the membrane domain ofB. tauruscomplex I that contains two core subunits and a cohort of supernumerary subunits. By comparing the structures and composition of subcomplex Iβ and complex I, supported by comparisons withYarrowia lipolyticacomplex I, we propose assignments for eight further supernumerary subunits in the structure. Our new assignments include two CHCH-domain containing subunits that contain disulfide bridges between CX₉C motifs; they are processed by the Mia40 oxidative-folding pathway in the intermembrane space and probably stabilize the membrane domain. We also assign subunit B22, an LYR protein, to the matrix face of the membrane domain. We reveal that subunit B22 anchors an acyl carrier protein (ACP) to the complex, replicating the LYR protein–ACP structural module that was identified previously in the hydrophilic domain. Thus, we significantly extend knowledge of how the mammalian supernumerary subunits are arranged around the core enzyme, and provide insights into their roles in biogenesis and regulation.
Thermophilic DNA polymerases of the polB family are of great importance in biotechnological applications including high-fidelity PCR. Of particular interest is the relative promiscuity of engineered ...versions of the exo- form of polymerases from the Thermo- and Pyrococcales families towards non-canonical substrates, which enables key advances in Next-generation sequencing. Despite this there is a paucity of structural information to guide further engineering of this group of polymerases. Here we report two structures, of the apo form and of a binary complex of a previously described variant (E10) of Pyrococcus furiosus (Pfu) polymerase with an ability to fully replace dCTP with Cyanine dye-labeled dCTP (Cy3-dCTP or Cy5-dCTP) in PCR and synthesise highly fluorescent "CyDNA" densely decorated with cyanine dye heterocycles. The apo form of Pfu-E10 closely matches reported apo form structures of wild-type Pfu. In contrast, the binary complex (in the replicative state with a duplex DNA oligonucleotide) reveals a closing movement of the thumb domain, increasing the contact surface with the nascent DNA duplex strand. Modelling based on the binary complex suggests how bulky fluorophores may be accommodated during processive synthesis and has aided the identification of residues important for the synthesis of unnatural nucleic acid polymers.
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