Molecular mechanism of a potassium channel gating through activation gate-selectivity filter coupling.

Potassium channels are presumed to have two allosterically coupled gates, the activation gate and the selectivity filter gate, that control channel opening, closing, and inactivation. However, the molecular mechanism of how these gates regulate K+ ion flow through the channel remains poorly understood. An activation process, occurring at the selectivity filter, has been recently proposed for several potassium channels. Here, we use X-ray crystallography and extensive molecular dynamics simulations, to study ion permeation through a potassium channel MthK, for various opening levels of both gates. We find that the channel conductance is controlled at the selectivity filter, whose conformation depends on the activation gate. The crosstalk between the gates is mediated through a collective motion of channel helices, involving hydrophobic contacts between an isoleucine and a conserved threonine in the selectivity filter. We propose a gating model of selectivity filter-activated potassium channels, including pharmacologically relevant two-pore domain (K2P) and big potassium (BK) channels.

How [Fe]-Hydrogenase from Methanothermobacter is Protected Against Light and Oxidative Stress.

[Fe]-hydrogenase (Hmd) catalyzes the reversible hydrogenation of methenyltetrahydromethanopterin (methenyl-H4 MPT+ ) with H2 . Hmd contains the iron-guanylylpyridinol (FeGP) cofactor, which is sensitive to light and oxidative stress. A natural protection mechanism is reported for Hmd based on structural and biophysical data. Hmd from Methanothermobacter marburgensis (mHmd) was found in a hexameric state, where an expanded oligomerization loop is detached from the dimer core and intrudes into the active site of a neighboring dimer. An aspartic acid residue from the loop ligates to FeII of the FeGP cofactor and thus blocks the postulated H2 -binding site. In solution, this enzyme is in a hexamer-to-dimer equilibrium. Lower enzyme concentrations, and the presence of methenyl-H4 MPT+ , shift the equilibrium toward the active dimer side. At higher enzyme concentrations-as present in the cell-the enzyme is predominantly in the inactive hexameric state and is thereby protected against light and oxidative stress.

Didehydroaspartate Modification in Methyl-Coenzyme†M Reductase Catalyzing Methane Formation.

All methanogenic and methanotrophic archaea known to date contain methyl-coenzyme M reductase (MCR) that catalyzes the reversible reduction of methyl-coenzyme M to methane. This enzyme contains the nickel porphinoid F430 as a prosthetic group and, highly conserved, a thioglycine and four methylated amino acid residues near the active site. We describe herein the presence of a novel post-translationally modified amino acid, didehydroaspartate, adjacent to the thioglycine as revealed by mass spectrometry and high-resolution X-ray crystallography. Upon chemical reduction, the didehydroaspartate residue was converted into aspartate. Didehydroaspartate was found in MCR I and II from Methanothermobacter marburgensis and in MCR of phylogenetically distantly related Methanosarcina barkeri but not in MCR I and II of Methanothermobacter wolfeii, which indicates that didehydroaspartate is dispensable but might have a role in fine-tuning the active site to increase the catalytic efficiency.

Molecular characterization of methanogenic N(5)-methyl-tetrahydromethanopterin: Coenzyme M methyltransferase.

Methanogenic archaea share one ion gradient forming reaction in their energy metabolism catalyzed by the membrane-spanning multisubunit complex N(5)-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH or simply Mtr). In this reaction the methyl group transfer from methyl-tetrahydromethanopterin to coenzyme M mediated by cobalamin is coupled with the vectorial translocation of Na(+) across the cytoplasmic membrane. No detailed structural and mechanistic data are reported about this process. In the present work we describe a procedure to provide a highly pure and homogenous Mtr complex on the basis of a selective removal of the only soluble subunit MtrH with the membrane perturbing agent dimethyl maleic anhydride and a subsequent two-step chromatographic purification. A molecular mass determination of the Mtr complex by laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) and size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) resulted in a (MtrABCDEFG)3 heterotrimeric complex of ca. 430kDa with both techniques. Taking into account that the membrane protein complex contains various firmly bound small molecules, predominantly detergent molecules, the stoichiometry of the subunits is most likely 1:1. A schematic model for the subunit arrangement within the MtrABCDEFG protomer was deduced from the mass of Mtr subcomplexes obtained by harsh IR-laser LILBID-MS.

Advanced electron paramagnetic resonance on the catalytic iron-sulfur cluster bound to the CCG domain of heterodisulfide reductase and succinate: quinone reductase.

Heterodisulfide reductase (Hdr) is a key enzyme in the energy metabolism of methanogenic archaea. The enzyme catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) to the thiol coenzymes M (CoM-SH) and B (CoB-SH). Cleavage of CoM-S-S-CoB at an unusual FeS cluster reveals unique substrate chemistry. The cluster is fixed by cysteines of two cysteine-rich CCG domain sequence motifs (CX31-39CCX35-36CXXC) of subunit HdrB of the Methanothermobacter marburgensis HdrABC complex. We report on Q-band (34 GHz) (57)Fe electron-nuclear double resonance (ENDOR) spectroscopic measurements on the oxidized form of the cluster found in HdrABC and in two other CCG-domain-containing proteins, recombinant HdrB of Hdr from M. marburgensis and recombinant SdhE of succinate: quinone reductase from Sulfolobus solfataricus P2. The spectra at 34 GHz show clearly improved resolution arising from the absence of proton resonances and polarization effects. Systematic spectral simulations of 34 GHz data combined with previous 9 GHz data allowed the unambiguous assignment of four (57)Fe hyperfine couplings to the cluster in all three proteins. (13)C Mims ENDOR spectra of labelled CoM-SH were consistent with the attachment of the substrate to the cluster in HdrABC, whereas in the other two proteins no substrate is present. (57)Fe resonances in all three systems revealed unusually large (57)Fe ENDOR hyperfine splitting as compared to known systems. The results infer that the cluster's unique magnetic properties arise from the CCG binding motif.

Conformational changes in orotidine 5'-monophosphate decarboxylase: a structure-based explanation for how the 5'-phosphate group activates the enzyme.

The binding of a ligand to orotidine 5'-monophosphate decarboxylase (OMPDC) is accompanied by a conformational change from an open, inactive conformation (E(o)) to a closed, active conformation (E(c)). As the substrate traverses the reaction coordinate to form the stabilized vinyl carbanion/carbene intermediate, interactions that destabilize the carboxylate group of the substrate and stabilize the intermediate (in the E(c)·S(‡) complex) are enforced. Focusing on the OMPDC from Methanothermobacter thermautotrophicus, we find the "remote" 5'-phosphate group of the substrate activates the enzyme 2.4 × 10(8)-fold; the activation is equivalently described by an intrinsic binding energy (IBE) of 11.4 kcal/mol. We studied residues in the activation that (1) directly contact the 5'-phosphate group, (2) participate in a hydrophobic cluster near the base of the active site loop that sequesters the bound substrate from the solvent, and (3) form hydrogen bonding interactions across the interface between the "mobile" and "fixed" half-barrel domains of the (ß/α)(8)-barrel structure. Our data support a model in which the IBE provided by the 5'-phosphate group is used to allow interactions both near the N-terminus of the active site loop and across the domain interface that stabilize both the E(c)·S and E(c)·S(‡) complexes relative to the E(o)·S complex. The conclusion that the IBE of the 5'-phosphate group provides stabilization to both the E(c)·S and E(c)·S(‡) complexes, not just the E(c)·S(‡) complex, is central to understanding the structural origins of enzymatic catalysis as well as the requirements for the de novo design of enzymes that catalyze novel reactions.

Structure and activity of a novel archaeal ß-CASP protein with N-terminal KH domains.

MTH1203, a ß-CASP metallo-ß-lactamase family nuclease from the archaeon Methanothermobacter thermautotrophicus, was identified as a putative nuclease that might contribute to RNA processing. The crystal structure of MTH1203 reveals that, in addition to the metallo-ß-lactamase nuclease and the ß-CASP domains, it contains two contiguous KH domains that are unique to MTH1203 and its orthologs. RNA-binding experiments indicate that MTH1203 preferentially binds U-rich sequences with a dissociation constant in the micromolar range. In vitro nuclease activity assays demonstrated that MTH1203 is a zinc-dependent nuclease. MTH1203 is also shown to be a dimer and, significantly, this dimerization enhances the nuclease activity. Transcription termination in archaea produces mRNA transcripts with U-rich 3' ends that could be degraded by MTH1203 considering its RNA-binding specificity. We hypothesize that this nuclease degrades mRNAs of proteins targeted for degradation and so regulates archaeal RNA turnover, possibly in concert with the exosome.

Structure-function relationship in an archaebacterial methionine sulphoxide reductase B.

Oxidation of methionine to methionine sulphoxide (MetSO) may lead to loss of molecular integrity and function. This oxidation can be 'repaired' by methionine sulphoxide reductases (MSRs), which reduce MetSO back to methionine. Two structurally unrelated classes of MSRs, MSRA and MSRB, show stereoselectivity towards the S and the R enantiomer of the sulphoxide respectively. Interestingly, these enzymes were even maintained throughout evolution in anaerobic organisms. Here, the activity and the nuclear magnetic resonance (NMR) structure of MTH711, a zinc containing MSRB from the thermophilic, methanogenic archaebacterium Methanothermobacter thermoautotrophicus, are described. The structure appears more rigid as compared with similar MSRBs from aerobic and mesophilic organisms. No significant structural differences between the oxidized and the reduced MTH711 state can be deduced from our NMR data. A stable sulphenic acid is formed at the catalytic Cys residue upon oxidation of the enzyme with MetSO. The two non-zinc-binding cysteines outside the catalytic centre are not necessary for activity of MTH711 and are not situated close enough to the active-site cysteine to serve in regenerating the active centre via the formation of an intramolecular disulphide bond. These findings imply a reaction cycle that differs from that observed for other MSRBs.

Preliminary crystallography confirms that the archaeal DNA-binding and tryptophan-sensing regulator TrpY is a dimer.

TrpY regulates the transcription of the metabolically expensive tryptophan-biosynthetic operon in the thermophilic archaeon Methanothermobacter thermautotrophicus. TrpY was crystallized using the hanging-drop method with ammonium sulfate as the precipitant. The crystals belonged to the tetragonal space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 87, c = 147 Å, and diffracted to 2.9 Šresolution. The possible packing of molecules within the cell based on the values of the Matthews coefficient (V(M)) and analysis of the self-rotation function are consistent with the asymmetric unit being a dimer. Determining the structure of TrpY in detail will provide insight into the mechanisms of DNA binding, tryptophan sensing and transcription regulation at high temperature by this novel archaeal protein.

Effects of 3-hydroxy-3-methylglutaryl-coenzyme a reductase inhibitor pravastatin on membrane lipids and membrane associated functions of Methanothermobacter thermautotrophicus.

The role of archaeal membrane and its lipid constituents was investigated in bioenergetic functions of Methanothermobacter thermautotrophicus. The effects were determined of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, pravastatin, on lipid composition, and its impact on some bioenergetic functions of treated cells. Pravastatin remarkably inhibited the growth of M. thermautotrophicus. On membrane level, pravastatin treatment modulated the composition of the mixture of squalene and hydrosqualene derivatives as well as the activities of ATPase, A1Ao-ATP synthase and Na+/H+ antiporter. SDS-PAGE of chloroform-methanol extracts of membranes from control and pravastatin-treated cells revealed changes in the amount of AtpK proteolipids, which suggests that pravastatin modifies cell-membrane composition, hereby modulating the properties of some membrane-bound enzymes participating in energy transformation in methanoarchaea.

Observation of organometallic and radical intermediates formed during the reaction of methyl-coenzyme M reductase with bromoethanesulfonate.

Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the final step of methane formation, in which methyl-coenzyme M (2-methylthioethanesulfonate, methyl-SCoM) is reduced with coenzyme B (N-(7-mercaptoheptanoyl)threonine phosphate, CoBSH) to form methane and the heterodisulfide CoBS-SCoM. The active dimeric form of MCR contains two Ni(I)-F(430) prosthetic groups, one in each monomer. This report describes studies of the reaction of the active Ni(I) state of MCR (MCR(red1)) with BES (2-bromoethanesulfonate) and CoBSH or its analogue, CoB(6)SH (N-(6-mercaptohexanoyl)threonine phosphate), by transient kinetic measurements using EPR and UV-visible spectroscopy and by global fits of the data. This reaction is shown to lead to the formation of three intermediates, the first of which is assigned as an alkyl-Ni(III) species that forms as the active Ni(I)-MCR(red1) state of the enzyme decays. Subsequently, a radical (MCR(BES) radical) is formed that was characterized by multifrequency electron paramagnetic resonance (EPR) studies at X- ( approximately 9 GHz), Q- ( approximately 35 GHz), and D- ( approximately 130 GHz) bands and by electron-nuclear double resonance (ENDOR) spectroscopy. The MCR(BES) radical is characterized by g-values at 2.00340 and 1.99832 and includes a strongly coupled nonexchangeable proton with a hyperfine coupling constant of 50 MHz. Based on transient kinetic measurements, the formation and decay of the radical coincide with a species that exhibits absorption peaks at 426 and 575 nm. Isotopic substitution, multifrequency EPR, and ENDOR spectroscopic experiments rule out the possibility that MCR(BES) is a tyrosyl radical and indicate that if a tyrosyl radical is formed during the reaction, it does not accumulate to detectable levels. The results provide support for a hybrid mechanism of methanogenesis by MCR that includes both alkyl-Ni and radical intermediates.

Comparing RNA secondary structures using a relaxed base-pair score.

The use of free energy-based algorithms to compute RNA secondary structures produces, in general, large numbers of foldings. Recent research has addressed the problem of grouping structures into a small number of clusters and computing a representative folding for each cluster. At the heart of this problem is the need to compute a quantity that measures the difference between pairs of foldings. We introduce a new concept, the relaxed base-pair (RBP) score, designed to give a more biologically realistic measure of the difference between structures than the base-pair (BP) metric, which simply counts the number of base pairs in one structure but not the other. The degree of relaxation is determined by a single relaxation parameter, t. When t = 0, (no relaxation) our method is the same as the BP metric. At the other extreme, a very large value of t will give a distance of 0 for identical structures and 1 for structures that differ. Scores can be recomputed with different values of t, at virtually no extra computation cost, to yield satisfactory results. Our results indicate that relaxed measures give more stable and more meaningful clusters than the BP metric. We also use the RBP score to compute representative foldings for each cluster.

Differential stability of TATA box binding proteins from archaea with different optimal growth temperatures.

The TATA box binding protein (TBP) is involved in promoter recognition, the first step of transcription initiation. TBP is universally conserved and essential in archaea and eukaryotes. In archaea, TBPs have to be stable and to function in species that cover an extremely wide range of optimal growth temperatures (OGTs), from below 0 degrees C to more than 100 degrees C. Thus, the archaeal TBP family is ideally suited to study the evolutionary adaptation of proteins to an extremely wide range of temperatures. We characterized the thermostability of one mesophilic and one thermophilic TBP by infrared spectroscopy. Transition temperatures (T(m)s) of thermal unfolding have been determined using TBPs from Methanosarcina mazei (OGT 37 degrees C) and from Methanothermobacter thermautotrophicus (OGT 65 degrees C). Furthermore, the influence of protein and salt concentration on thermostability has been characterized. Together with previous studies, our results reveal that the T(m)s of archaeal TBPs are closely correlated with the OGTs of the respective species. Noteworthy, this is also true for the TBP from M. mazei representing the first characterized TBP from a mesophilic archaeon. In contrast, the only characterized eukaryotic TBP of the mesophilic plant Arabidopsis thaliana has a T(m) more than 40 degrees C above the OGT.

ATP hydrolysis and DNA binding confer thermostability on the MCM helicase.

The minichromosome maintenance (MCM) helicase is the replicative helicase in archaea. The enzyme utilizes the energy derived from ATP hydrolysis to translocate along one strand of the DNA and unwind the complementary strand. Here, the effect of DNA and ATP on the thermostability of the Methanothermobacter thermautotrophicus MCM protein was determined by differential scanning calorimetry. The MCM protein shows a single thermal transition at 67 degrees C. The stability is dramatically altered with the appearance of a second thermal transition up to 10 degrees C higher in the presence of DNA and either ATP or ADP-AlF(4)(-), a transition-state analogue of ATP, bound to MCM. In the presence of DNA and ADP or the nonhydrolyzable ATP analogues ATPgammaS and AMP-PNP, however, only a single thermal transition is observed at temperatures slightly higher than the transition temperature of MCM alone. Thus, the results suggest that ATP hydrolysis proceeds through a transition state that decouples an interaction between the N-terminal DNA binding domain and the C-terminal catalytic domain in the presence of DNA.

Isolation and characterization of a N,N'-dicyclohexylcarbodiimide-resistant mutant of Methanothermobacter thermautotrophicus with alterations to the ATP synthesis machinery.

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant toward the ATP-synthase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) was isolated. DCCD normally inhibits methanogenic electron-transport-driven ATP synthesis, however, the DCCD-resistant strain exhibited methanogenesis in the presence of 300 micromol/L DCCD. Total ATP synthesis was shown to be higher in the mutant strain, both in the presence and absence of DCCD. These results suggested a modification in the ATP-synthesizing system of the mutant strain. Using Blue Native PAGE combined with MALDI TOF/TOF mass spectrometry, increased concentrations of both the A(1) and A(o) subcomplexes of the A(1)A(o)-type synthase were identified in the mutant strain. However, no alterations were found in the structural genes (atp) for the A(1)A(o) ATP synthase. The results imply that DCCD resistance is a consequence of increased A(1)A(o) ATP synthase expression, and suggest that genes involved in regulating synthase expression are responsible for DCCD resistance.

Expression, purification, crystallization and preliminary X-ray studies of the TAN1 orthologue from Methanothermobacter thermautotrophicus.

MTH909 is the Methanothermobacter thermautotrophicus orthologue of Saccharomyces cerevisiae TAN1, which is required for N(4)-acetylcytidine formation in tRNA. The protein consists of an N-terminal near-ferredoxin-like domain and a C-terminal THUMP domain. Unlike most other proteins containing the THUMP domain, TAN1 lacks any catalytic domains and has been proposed to form a complex with a catalytic protein that is capable of making base modifications. MTH909 has been cloned, overexpressed and purified. The molecule exists as a monomer in solution. X-ray data were collected to 2.85 A resolution from a native crystal belonging to space group P6(1)22 (or P6(5)22), with unit-cell parameters a = 69.9, c = 408.5 A.

Expression, purification, crystallization and preliminary X-ray analysis of an archaeal protein homologous to plant nicotianamine synthase.

In plants, nicotianamine synthase (NAS) plays a key role in metal homeostasis as it catalyzes the formation of nicotianamine, an important iron and nickel chelator and a precursor of plant phytosiderophores. Here, the crystallization of a protein from Methanothermobacter thermoautotrophicus (MTH675; referred to here as MtNAS) that appears to be homologous to plant NAS is reported. Purification of this protein showed a monomer-dimer equilibrium that could be displaced by using a reducing agent such as DTT. Crystals belonging to space group P2(1)2(1)2(1) and containing dimers of MtNAS were grown by the vapour-diffusion method using polyethylene glycol 3350 as precipitant. A complete native X-ray data set was collected to 1.7 A resolution at a synchrotron source.

Spectroscopic and computational studies of reduction of the metal versus the tetrapyrrole ring of coenzyme F430 from methyl-coenzyme M reductase.

Methyl-coenzyme M reductase (MCR) catalyzes the final step in methane biosynthesis by methanogenic archaea and contains a redox-active nickel tetrahydrocorphin, coenzyme F430, at its active site. Spectroscopic and computational methods have been used to study a novel form of the coenzyme, called F330, which is obtained by reducing F430 with sodium borohydride (NaBH4). F330 exhibits a prominent absorption peak at 330 nm, which is blue shifted by 100 nm relative to F430. Mass spectrometric studies demonstrate that the tetrapyrrole ring in F330 has undergone reduction, on the basis of the incorporation of protium (or deuterium), upon treatment of F430 with NaBH4 (or NaBD4). One- and two-dimensional NMR studies show that the site of reduction is the exocyclic ketone group of the tetrahydrocorphin. Resonance Raman studies indicate that elimination of this pi-bond increases the overall pi-bond order in the conjugative framework. X-ray absorption, magnetic circular dichroism, and computational results show that F330 contains low-spin Ni(II). Thus, conversion of F430 to F330 reduces the hydrocorphin ring but not the metal. Conversely, reduction of F430 with Ti(III) citrate to generate F380 (corresponding to the active MCR(red1) state) reduces the Ni(II) to Ni(I) but does not reduce the tetrapyrrole ring system, which is consistent with other studies [Piskorski, R., and Jaun, B. (2003) J. Am. Chem. Soc. 125, 13120-13125; Craft, J. L., et al. (2004) J. Biol. Inorg. Chem. 9, 77-89]. The distinct origins of the absorption band shifts associated with the formation of F330 and F380 are discussed within the framework of our computational results. These studies on the nature of the product(s) of reduction of F430 are of interest in the context of the mechanism of methane formation by MCR and in relation to the chemistry of hydroporphinoid systems in general. The spectroscopic and time-dependent DFT calculations add important insight into the electronic structure of the nickel hydrocorphinate in its Ni(II) and Ni(I) valence states.

Protein complexes in the archaeon Methanothermobacter thermautotrophicus analyzed by blue native/SDS-PAGE and mass spectrometry.

Methanothermobacter thermautotrophicus is a thermophilic archaeon that produces methane as the end product of its primary metabolism. The biochemistry of methane formation has been extensively studied and is catalyzed by individual enzymes and proteins that are organized in protein complexes. Although much is known of the protein complexes involved in methanogenesis, only limited information is available on the associations of proteins involved in other cell processes of M. thermautotrophicus. To visualize and identify interacting and individual proteins of M. thermautotrophicus on a proteome-wide scale, protein preparations were separated using blue native electrophoresis followed by SDS-PAGE. A total of 361 proteins, corresponding to almost 20% of the predicted proteome, was identified using peptide mass fingerprinting after MALDI-TOF MS. All previously characterized complexes involved in energy generation could be visualized. Furthermore the expression and association of the heterodisulfide reductase and methylviologen-reducing hydrogenase complexes depended on culture conditions. Also homomeric supercomplexes of the ATP synthase stalk subcomplex and the N5-methyl-5,6,7,8-tetrahydromethanopterin:coenzyme M methyltransferase complex were separated. Chemical cross-linking experiments confirmed that the multimerization of both complexes was not experimentally induced. A considerable number of previously uncharacterized protein complexes were reproducibly visualized. These included an exosome-like complex consisting of four exosome core subunits, which associated with a tRNA-intron endonuclease, thereby expanding the constituency of archaeal exosomes. The results presented show the presence of novel complexes and demonstrate the added value of including blue native gel electrophoresis followed by SDS-PAGE in discovering protein complexes that are involved in catabolic, anabolic, and general cell processes.

Crystal structure of Mil (Mth680): internal duplication and similarity between the Imp4/Brix domain and the anticodon-binding domain of class IIa aminoacyl-tRNA synthetases.

Proteins of the Imp4/Brix superfamily are involved in ribosomal RNA processing, an essential function in all cells. We report the first structure of an Imp4/Brix superfamily protein, the Mil (for Methanothermobacter thermautotrophicus Imp4-like) protein (gene product Mth680), from the archaeon M. thermautotrophicus. The amino- and carboxy-terminal halves of Mil show significant structural similarity to one another, suggesting an origin by means of an ancestral duplication. Both halves show the same fold as the anticodon-binding domain of class IIa aminoacyl-tRNA synthetases, with greater conservation seen in the N-terminal half. This structural similarity, together with the charge distribution in Mil, suggests that Imp4/Brix superfamily proteins could bind single-stranded segments of RNA along a concave surface formed by the N-terminal half of their beta-sheet and a central alpha-helix. The crystal structure of Mil is incompatible with the presence, in the Imp4/Brix domain, of a helix-turn-helix motif that was proposed to comprise the RNA-binding moiety of the Imp4/Brix proteins.