17 research outputs found
The structure of subunit E of the pyrococcus horikoshii OT3 A-ATP synthase gives insight into the elasticity of the peripheral stalk
No full textA1AO ATP synthases are the major energy converters of archaea. They are composed of an A1 region that synthesizes ATP and an integral part AO that conducts ions. Subunit E is a component of the peripheral stalk that links the A1 with the AO part of the A-ATP synthase. We have determined the crystal structure of the entire subunit E (PhE) of the Pyrococcus horikoshii OT3 A-ATP synthase at 3.6 Å resolution. The structure reveals an extended S-shaped N-terminal α-helix with 112.29 Å in length, followed by a globular head group. The S-shaped feature, common in elastic connectors and spacers, would facilitate the storage of transient elastic energy during rotary motion in the enzyme. The structure has been superimposed into the asymmetric peripheral stalks of the three-dimensional reconstruction of the Pyrococcus furiosus enzyme, revealing that the S-shaped subunit PhE fits well into the bent peripheral stalk, whereas the previously solved E subunit structure (3.1 Å resolution) of Thermus thermophilus A-ATP synthase is well accommodated in the density of the straight stator domain. The different features of the two stalk subunits are discussed in light of a novel coupling mechanism in A-ATP synthases proposed to differ from the Wankel engine of F-ATP synthases
Spectroscopic and crystallographic studies of the mutant R416W give insight into the nucleotide binding traits of subunit B of the A(1)A(O) ATP synthase
No full textKumar A, Manimekalai MSS, Balakrishna AM, et al. Spectroscopic and crystallographic studies of the mutant R416W give insight into the nucleotide binding traits of subunit B of the A(1)A(O) ATP synthase. PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS. 2009;75(4):807-819.A strategically placed tryptophan in position of Arg416 was used as an optical probe to monitor adenosine triphosphate and adenosine-diphosphate binding to subunit B of the A(1)A(O) adenosine triphosphate (ATP) synthase from Methanosarcina mazei Go1. Tryptophan fluorescence and fluorescence correlation spectroscopy gave binding constants indicating a preferred binding of ATP over ADP to the protein. The X-ray crystal structure of the R416W mutant protein in the presence of ATP was solved to 2.1 angstrom resolution, showing the substituted Trp-residue inside the predicted adenine-binding pocket. The cocrystallized ATP molecule could be trapped in a so-called transition nucleotide-binding state. The high resolution structure shows the phosphate residues of the ATP near the P-loop region (S150-E158) and its adenine ring forms pi-pi interaction with Phe149. This transition binding position of ATP could be confirmed by tryptophan emission spectra using the subunit B mutant F149W. The trapped ATP position, similar to the one of the binding region of the antibiotic efrapeptin in F1FO ATP synthases, is discussed in light of a transition nucleotide-binding state of ATP while on its way to the final binding pocket. Finally, the inhibitory effect of efrapeptin C in ATPase activity of a reconstituted A(3)B(3)- and A(3)B(R416W)(3)-subcomplex, composed of subunit A and the B subunit mutant R416W, of the A(1)A(O) ATP synthase is shown
Crystallographic insight into the catalytic mechanism of subunit A of the A-ATP synthase and the P-loop switch in evolution
No full textCryo-EM structures of prokaryotic ligand-gated ion channel GLIC provide insights into gating in a lipid environment
No full textAbstract GLIC, a proton-activated prokaryotic ligand-gated ion channel, served as a model system for understanding the eukaryotic counterparts due to their structural and functional similarities. Despite extensive studies conducted on GLIC, the molecular mechanism of channel gating in the lipid environment requires further investigation. Here, we present the cryo-EM structures of nanodisc-reconstituted GLIC at neutral and acidic pH in the resolution range of 2.6 – 3.4 Å. In our apo state at pH 7.5, the extracellular domain (ECD) displays conformational variations compared to the existing apo structures. At pH 4.0, three distinct conformational states (C1, C2 and O states) are identified. The protonated structures exhibit a compacted and counter-clockwise rotated ECD compared with our apo state. A gradual widening of the pore in the TMD is observed upon reducing the pH, with the widest pore in O state, accompanied by several layers of water pentagons. The pore radius and molecular dynamics (MD) simulations suggest that the O state represents an open conductive state. We also observe state-dependent interactions between several lipids and proteins that may be involved in the regulation of channel gating. Our results provide comprehensive insights into the importance of lipids impact on gating
Crystal structure of subunits D and F in complex gives insight into energy transmission of the eukaryotic V-ATPase from Saccharomyces cerevisiae
No full textEukaryotic V1VO-ATPases hydrolyze ATP in the V1 domain coupled to ion pumping in VO. A unique mode of regulation of V-ATPases is the reversible disassembly of V1 and VO, which reduces ATPase activity and causes silencing of ion conduction. The subunits D and F are proposed to be key in these enzymatic processes. Here, we describe the structures of two conformations of the subunit DF assembly of Saccharomyces cerevisiae (ScDF) V-ATPase at 3.1 Å resolution. Subunit D (ScD) consists of a long pair of α-helices connected by a short helix (79IGYQVQE85) as well as a β-hairpin region, which is flanked by two flexible loops. The long pair of helices is composed of the N-terminal α-helix and the C-terminal helix, showing structural alterations in the two ScDF structures. The entire subunit F (ScF) consists of an N-terminal domain of four β-strands (β1–β4) connected by four α-helices (α1–α4). α1 and β2 are connected via the loop 26GQITPETQEK35, which is unique in eukaryotic V-ATPases. Adjacent to the N-terminal domain is a flexible loop, followed by a C-terminal α-helix (α5). A perpendicular and extended conformation of helix α5 was observed in the two crystal structures and in solution x-ray scattering experiments, respectively. Fitted into the nucleotide-bound A3B3 structure of the related A-ATP synthase from Enterococcus hirae, the arrangements of the ScDF molecules reflect their central function in ATPase-coupled ion conduction. Furthermore, the flexibility of the terminal helices of both subunits as well as the loop 26GQITPETQEK35 provides information about the regulatory step of reversible V1VO disassembly.MOE (Min. of Education, S’pore)Published versio
Redox chemistry of Mycobacterium tuberculosis alkylhydroperoxide reductase E (AhpE): Structural and mechanistic insight into a mycoredoxin-1 independent reductive pathway of AhpE via mycothiol
No full textMycobacterium tuberculosis (Mtb) has the ability to persist within the human host for a long time in a dormant stage and re-merges when the immune system is compromised. The pathogenic bacterium employs an elaborate antioxidant defence machinery composed of the mycothiol- and thioredoxin system in addition to a superoxide dismutase, a catalase, and peroxiredoxins (Prxs). Among the family of Peroxiredoxins, Mtb expresses a 1-cysteine peroxiredoxin, known as alkylhydroperoxide reductase E (MtAhpE), and defined as a potential tuberculosis drug target. The reduced MtAhpE (MtAhpE-SH) scavenges peroxides to become converted to MtAhpE-SOH. To provide continuous availability of MtAhpE-SH, MtAhpE-SOH has to become reduced. Here, we used NMR spectroscopy to delineate the reduced (MtAhpE-SH), sulphenic (MtAhpE-SOH) and sulphinic (MtAhpE-SO2H) states of MtAhpE through cysteinyl-labelling, and provide for the first time evidence of a mycothiol-dependent mechanism of MtAhpE reduction. This is confirmed by crystallographic studies, wherein MtAhpE was crystallized in the presence of mycothiol and the structure was solved at 2.43Å resolution. Combined with NMR-studies, the crystallographic structures reveal conformational changes of important residues during the catalytic cycle of MtAhpE. In addition, alterations of the overall protein in solution due to redox modulation are observed by small angle X-ray scattering (SAXS) studies. Finally, by employing SAXS and dynamic light scattering, insight is provided into the most probable physiological oligomeric state of MtAhpE necessary for activity, being also discussed in the context of concerted substrate binding inside the dimeric MtAhpE.MOE (Min. of Education, S’pore)Accepted versio
Crystallographic and solution structure of the N‐terminal domain of the Rel protein from <i>Mycobacterium tuberculosis</i>
No full textCrystallization and preliminary X-ray crystallographic analysis of subunit F (F1-94), an essential coupling subunit of the eukaryotic V1VO-ATPase from Saccharomyces cerevisiae
No full textV-ATPases are very complex multi-subunit enzymes which function as proton-pumping rotary nanomotors. The rotary and coupling subunit F (F1-94) was crystallized by the hanging-drop vapour-diffusion method. The native crystals diffracted to a resolution of 2.64 Å and belonged to space group C2221, with unit-cell parameters a = 47.21, b = 160.26, c = 102.49 Å. The selenomethionyl form of the F1-94 I69M mutant diffracted to a resolution of 2.3 Å and belonged to space group C2221, with unit-cell parameters a = 47.22, b = 160.83, c = 102.74 Å. Initial phasing and model building suggested the presence of four molecules in the asymmetric unit.Published versio
Crystallographic structure of the turbine C-ring from spinach chloroplast F-ATP synthase
No full textIn eukaryotic and prokaryotic cells, F-ATP synthases provide energy through the synthesis of ATP. The chloroplast F-ATP synthase (CF1FO-ATP synthase) of plants is integrated into the thylakoid membrane via its FO-domain subunits a, b, b’ and c. Subunit c with a stoichiometry of 14 and subunit a form the gate for H+-pumping, enabling the coupling of electrochemical energy with ATP synthesis in the F1 sector.
Here we report the crystallization and structure determination of the c14-ring of subunit c of the CF1FO-ATP synthase from spinach chloroplasts. The crystals belonged to space group C2, with unit-cell parameters a=144.420, b=99.295, c=123.51 Å, and β=104.34° and diffracted to 4.5 Å resolution. Each c-ring contains 14 monomers in the asymmetric unit. The length of the c-ring is 60.32 Å, with an outer ring diameter 52.30 Å and an inner ring width of 40 Å.Published versio
Atomic structure and enzymatic insights into the vancomycin-resistant Enterococcus faecalis (V583) alkylhydroperoxide reductase subunit C
No full textThe Enterococcus faecalis alkyl hydroperoxide reductase complex (AhpR) with its subunits AhpC (EfAhpC) and AhpF (EfAhpF) are of paramount importance to restore redox homeostasis. Recently, the novel phenomenon of swapping of the catalytic domains of EfAhpF was uncovered. Here, we visualized its counterpart EfAhpC (187 residues) from the vancomycin-resistant E. faecalis (V583) bacterium by electron microscopy and demonstrate, that in contrast to other bacterial AhpCs, EfAhpC forms a stable decamer-ring irrespective of the redox state. The first crystallographic structure (2.8Å resolution) of the C-terminal truncated form (EfAhpC1-172) confirms the decamer ring and provides new insight into a transition state in-between a fully folded to a locally unfolded conformation in the catalytic center due to redox modulation. Amino acid substitutions of residues in the N- and C-termini as well as the oligomeric interphase of EfAhpC provide information into their structural and enzymatic roles. Mutagenesis, enzymatic and biophysical studies reveal the effect of the unusual existence of four cysteines in EfAhpC, which might optimize the functional adaptation of the E. faecalis enzyme under various physiological conditions.MOE (Min. of Education, S’pore
