Discovery, structure and mechanism of a tetraether lipid synthase.

Archaea synthesize isoprenoid-based ether-linked membrane lipids, which enable them to withstand extreme environmental conditions, such as high temperatures, high salinity, and low or high pH values1-5. In some archaea, such as Methanocaldococcus jannaschii, these lipids are further modified by forming carbon-carbon bonds between the termini of two lipid tails within one glycerophospholipid to generate the macrocyclic archaeol or forming two carbon-carbon bonds between the termini of two lipid tails from two glycerophospholipids to generate the macrocycle glycerol dibiphytanyl glycerol tetraether (GDGT)1,2. GDGT contains two 40-carbon lipid chains (biphytanyl chains) that span both leaflets of the membrane, providing enhanced stability to extreme conditions. How these specialized lipids are formed has puzzled scientists for decades. The reaction necessitates the coupling of two completely inert sp3-hybridized carbon centres, which, to our knowledge, has not been observed in nature. Here we show that the gene product of mj0619 from M. jannaschii, which encodes a radical S-adenosylmethionine enzyme, is responsible for biphytanyl chain formation during synthesis of both the macrocyclic archaeol and GDGT membrane lipids6. Structures of the enzyme show the presence of four metallocofactors: three [Fe4S4] clusters and one mononuclear rubredoxin-like iron ion. In vitro mechanistic studies show that Csp3-Csp3 bond formation takes place on fully saturated archaeal lipid substrates and involves an intermediate bond between the substrate carbon and a sulfur of one of the [Fe4S4] clusters. Our results not only establish the biosynthetic route for tetraether formation but also improve the use of GDGT in GDGT-based paleoclimatology indices7-10.

An archaellum filament composed of two alternating subunits.

Archaea use a molecular machine, called the archaellum, to swim. The archaellum consists of an ATP-powered intracellular motor that drives the rotation of an extracellular filament composed of multiple copies of proteins named archaellins. In many species, several archaellin homologs are encoded in the same operon; however, previous structural studies indicated that archaellum filaments mainly consist of only one protein species. Here, we use electron cryo-microscopy to elucidate the structure of the archaellum from Methanocaldococcus villosus at 3.08 Å resolution. The filament is composed of two alternating archaellins, suggesting that the architecture and assembly of archaella is more complex than previously thought. Moreover, we identify structural elements that may contribute to the filament's flexibility.

Filamentous chaperone protein-based hydrogel stabilizes enzymes against thermal inactivation.

We report a filamentous chaperone-based protein hydrogel capable of stabilizing enzymes against thermal inactivation. The hydrogel backbone consists of a thermostable chaperone protein, the gamma-prefoldin (γPFD) from Methanocaldococcus jannaschii, which self-assembles into a fibrous structure. Specific coiled-coil interactions engineered into the wildtype γPFD trigger the formation of a cross-linked network of protein filaments. The structure of the filamentous chaperone is preserved through the designed coiled-coil interactions. The resulting hydrogel enables entrapped enzymes to retain greater activity after exposure to high temperatures, presumably by virtue of the inherent chaperone activity of the γPFD.

A general strategy to red-shift green fluorescent protein-based biosensors.

Compared with green fluorescent protein-based biosensors, red fluorescent protein (RFP)-based biosensors are inherently advantageous because of reduced phototoxicity, decreased autofluorescence and enhanced tissue penetration. However, existing RFP-based biosensors often suffer from small dynamic ranges, mislocalization and undesired photoconversion. In addition, the choice of available RFP-based biosensors is limited, and development of each biosensor requires substantial effort. Herein, we describe a general and convenient method, which introduces a genetically encoded noncanonical amino acid, 3-aminotyrosine, to the chromophores of green fluorescent protein-like proteins and biosensors for spontaneous and efficient green-to-red conversion. We demonstrated that this method could be used to quickly expand the repertoire of RFP-based biosensors. With little optimization, the 3-aminotyrosine-modified biosensors preserved the molecular brightness, dynamic range and responsiveness of their green fluorescent predecessors. We further applied spectrally resolved biosensors for multiplexed imaging of metabolic dynamics in pancreatic ß-cells.

Structure of the ribosomal P stalk base in archaean Methanococcus jannaschii.

Complexes of archaeal ribosomal proteins uL11 and uL10/P0 (the two-domain N-terminal fragment of uL10, uL10NTF/P0NTF) with the adjacent 74 nucleotides of 23S rRNA fragment (23SrRNA(74)) from Methanococcus jannaschii (Mja) were obtained, crystallized and their structures were studied. The comparative structural analysis of the complexes of Mja uL10NTF•23SrRNA(74) and Mja uL10NTF•uL11•23SrRNA(74) shows that the insertion of uL11 in the binary complex does not change the conformation of the 23S rRNA fragment. On the other hand, the interaction with this specific RNA fragment leads to the restructuring of uL11 compared to the structure of this protein in the free state. Besides, although analysis confirmed the mobility of uL10/P0 domain II, disproved the assumption that it may be in contact with rRNA or uL11. In addition, the Mja uL10NTF•uL11•23SrRNA(74) complex was cocrystallized with the antibiotic thiostrepton, and the structure of this complex was solved. The thiostrepton binding site in this archaeal complex was found between the 23S rRNA and the N-terminal domain (NTD) of the Mja uL11 protein, similar to its binding site in the one of bacterial ribosome complex with thiostrepton. Upon binding of thiostrepton, the NTD of uL11 shifts toward rRNA by 7 Å. Such a shift may be the cause of the inhibitory effect of the antibiotic on the recruitment of translation factors to the GTPase-activating region in archaeal ribosomes, similar to its inhibitory effect on protein synthesis in bacterial ribosomes.

Mechanical unfolding of a knotted protein unveils the kinetic and thermodynamic consequences of threading a polypeptide chain.

Knots are remarkable topological features in nature. The presence of knots in crystallographic structures of proteins have stimulated considerable research to determine the kinetic and thermodynamic consequences of threading a polypeptide chain. By mechanically manipulating MJ0366, a small single domain protein harboring a shallow trefoil knot, we allow the protein to refold from either the knotted or the unknotted denatured state to characterize the free energy profile associated to both folding pathways. By comparing the stability of the native state with reference to the knotted and unknotted denatured state we find that knotting the polypeptide chain of MJ0366 increase the folding energy barrier in a magnitude close to the energy cost of forming a knot randomly in the denatured state. These results support that a protein knot can be formed during a single cooperative step of folding but occurs at the expenses of a large increment on the free energy barrier.

Role of disordered regions in transferring tyrosine to its cognate tRNA.

Aminoacyl tRNA synthetase (AARS) plays an important role in transferring each amino acid to its cognate tRNA. Specifically, tyrosyl tRNA synthetase (TyrRS) is involved in various functions including protection from DNA damage due to oxidative stress, protein synthesis and cell signaling and can be an attractive target for controlling the pathogens by early inhibition of translation. TyrRS has two disordered regions, which lack a stable 3D structure in solution, and are involved in tRNA synthetase catalysis and stability. One of the disordered regions undergoes disorder-to-order transition (DOT) upon complex formation with tRNA whereas the other remains disordered (DR). In this work, we have explored the importance of these disordered regions using molecular dynamics simulations of both free and RNA-complexed states. We observed that the DOT and DR regions of the first subunit acts as a flap and interact with the acceptor arm of the tRNA. The DOT-DR flap closes when tyrosine (TyrRSTyr) is present at the active site of the complex and opens in the presence of tyrosine monophosphate (TyrRSYMP). The DOT and DR regions of the second subunit interact with the anticodon stem as well as D-loop of the tRNA, which might be involved in stabilizing the complex. The anticodon loop of the tRNA binds to the structured region present in the C-terminal of the protein, which is observed to be flexible during simulations. Detailed energy calculations also show that TyrRSTyr complex has stronger binding energy between tRNA and protein compared to TyrRSYMP; on the contrary, the anticodon is strongly bound in TyrRSYMP. The results obtained in the present study provide additional insights for understanding catalysis and the involvement of disordered regions in Tyr transfer to cognate tRNA.

Ion Binding and Selectivity of the Na+/H+ Antiporter MjNhaP1 from Experiment and Simulation.

Cells employ membrane-embedded antiporter proteins to control their pH, salt concentration, and volume. The large family of cation/proton antiporters is dominated by Na+/H+ antiporters that exchange sodium ions against protons, but homologous K+/H+ exchangers have recently been characterized. We show experimentally that the electroneutral antiporter NhaP1 of Methanocaldococcus jannaschii (MjNhaP1) is highly selective for Na+ ions. We then characterize the ion selectivity in both the inward-open and outward-open states of MjNhaP1 using classical molecular dynamics simulations, free energy calculations, and hybrid quantum/classical (QM/MM) simulations. We show that MjNhaP1 is highly selective for binding of Na+ over K+ in the inward-open state, yet it is only weakly selective in the outward-open state. These findings are consistent with the function of MjNhaP1 as a sodium-driven deacidifier of the cytosol that maintains a high cytosolic K+ concentration in environments of high salinity. By combining experiment and computation, we gain mechanistic insight into the Na+/H+ transport mechanism and help elucidate the molecular basis for ion selectivity in cation/proton exchangers.

Molecular modeling and inhibitor docking analysis of the Na+/H+ exchanger isoform one 1.

Na+/H+ exchanger isoform one (NHE1) is a mammalian plasma membrane protein that removes intracellular protons, thereby elevating intracellular pH (pHi). NHE1 uses the energy of allowing an extracellular sodium down its gradient into cells to remove one intracellular proton. The ubiquitous protein has several important physiological and pathological influences on mammalian cells as a result of its activity. The three-dimensional structure of human NHE1 (hNHE1) is not known. Here, we modeled NHE1 based on the structure of MjNhaP1 of Methanocaldoccocus jannaschii in combination with biochemical surface accessibility data. hNHE1 contained 12 transmembrane segments including a characteristic Na+/H+ antiporter fold of two transmembrane segments with a helix - extended region - helix conformation crossing each other within the membrane. Amino acids 363-410 mapped principally to the extracellular surface as an extracellular loop (EL5). A large preponderance of amino acids shown to be surface accessible by biochemical experiments mapped near to, or on, the extracellular surface. Docking of Na+/H+ exchanger inhibitors to the extracellular surface suggested that inhibitor binding on an extracellular site is made up from several amino acids of different regions of the protein. The results present a novel testable, three-dimensional model illustrating NHE1 structure and accounting for experimental biochemical data.

Cell-Free Expression of Unnatural Amino Acid Incorporated Aquaporin SS9 with Improved Separation Performance in Biomimetic Membranes.

Aquaporins (AQPs) are widely applied in biomimetic membranes for water recycling and desalination. In this study, a novel aquaporin was isolated from Photobacterium profundum SS9 (AQP SS9), which showed high water permeability and potential for practical water purification applications. To improve the stability of the AQP SS9 embedded biomimetic membranes, a modified AQP SS9 was obtained by incorporation of an unnatural amino acid (p-propargyloxyphenylalanine, pPpa) (P-AQP SS9) in vitro using a mutated Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) and the cell-free expression system. The modified AQP SS9 can covalently link with phospholipids and hence significantly improve the stability of biomimetic membranes. The concentration of Mg2+ and fusion expression with signal peptides were evaluated to enhance the expression level of P-AQP SS9, resulting in a highest yield of 49 mg/L. The modified AQP SS9 was then reconstituted into DOPC liposomes and analyzed by a stopped-flow spectrophotometer. The obtained water permeability coefficient (Pf) of 7.46×10-4 m/s was 5.7 times higher than that of proteoliposomes with the wild-type AQP SS9 (Pf=1.31×10-4 m/s) and 12.1 times higher than that of the DOPC liposomes (Pf=6.15×10-5m/s). This study demonstrates the development of a cell-free system for the expression of membrane proteins with much higher stability and the potential application of the modified aquaporins for water filtration.

Key residues controlling bidirectional ion movements in Na+/Ca2+ exchanger.

Prokaryotic and eukaryotic Na+/Ca2+ exchangers (NCX) control Ca2+ homeostasis. NCX orthologs exhibit up to 104-fold differences in their turnover rates (kcat), whereas the ratios between the cytosolic (cyt) and extracellular (ext) Km values (Kint = KmCyt/KmExt) are highly asymmetric and alike (Kint ≤ 0.1) among NCXs. The structural determinants controlling a huge divergence in kcat at comparable Kint remain unclear, although 11 (out of 12) ion-coordinating residues are highly conserved among NCXs. The crystal structure of the archaeal NCX (NCX_Mj) was explored for testing the mutational effects of pore-allied and loop residues on kcat and Kint. Among 55 tested residues, 26 mutations affect either kcat or Kint, where two major groups can be distinguished. The first group of mutations (14 residues) affect kcat rather than Kint. The majority of these residues (10 out of 14) are located within the extracellular vestibule near the pore center. The second group of mutations (12 residues) affect Kint rather than kcat, whereas the majority of residues (9 out 12) are randomly dispersed within the extracellular vestibule. In conjunction with computational modeling-simulations and hydrogen-deuterium exchange mass-spectrometry (HDX-MS), the present mutational analysis highlights structural elements that differentially govern the intrinsic asymmetry and transport rates. The key residues, located at specific segments, can affect the characteristic features of local backbone dynamics and thus, the conformational flexibility of ion-transporting helices contributing to critical conformational transitions. The underlying mechanisms might have a physiological relevance for matching the response modes of NCX variants to cell-specific Ca2+ and Na+ signaling.

Structural Basis for the Specific Cotranslational Incorporation of p-Boronophenylalanine into Biosynthetic Proteins.

The site-specific incorporation of the non-natural amino acid p-boronophenylalanine (Bpa) into recombinant proteins enables the development of novel carbohydrate-binding functions as well as bioorthogonal chemical modification. To this end, Bpa is genetically encoded by an amber stop codon and cotranslationally inserted into the recombinant polypeptide chain at the ribosome by means of an artificial aminoacyl-tRNA synthetase (aaRS) in combination with a compatible suppressor tRNA. We describe the crystal structure of an aaRS specific for Bpa, which had been engineered on the basis of the TyrRS from Methanocaldococcus jannaschii, in complex with both Bpa and AMP. The substrates are bound in an orientation resembling the aminoacyl-AMP mixed anhydride intermediate and engaged in a network of four hydrogen bonds that allows specific recognition of the boronate moiety by the aaRS. The key determinant of this interaction is the coplanar alignment of its Glu162 carboxylate group with Bpa, which results in a double hydrogen bond with the boronic acid substituent. Our structural study elucidates how a small set of five side chain exchanges within the TyrRS active site can switch its substrate specificity to the hydrophilic amino acid Bpa, thus stimulating the reprogramming of other aaRS to recruit useful non-natural amino acids for next-generation protein engineering.

Characterization of the Dihydroorotase from Methanococcus jannaschii.

The gene that codes for the putative dihydroorotase in the hyperthermophilic archaeon Methanococcus jannaschii was subcloned in pET-21a and expressed in Escherichia coli. A purification protocol was devised. The purity of the protein was evaluated by SDS-PAGE and the protein was confirmed by sequencing using LC-MS. The calculated molecular mass is 48104 Da. SEC-LS suggested that the protein is a monomer in solution. ICP-MS showed that there are two Zn ions per monomer. Kinetic analysis of the recombinant protein gave hyperbolic kinetics with Vmax = 12.2 µmol/min/mg and Km = 0.14 mM at 25 °C. Furthermore the activity of the protein increased with temperature consistent with the hyperthermophilic nature of the organism. A homology model was constructed using the mesophilic Bacillus anthracis protein as the template. Residues known to be critical for Zn and substrate binding were conserved. The activity of the enzyme at 85 and 90 °C was found to be relatively constant over 160 min and this correlates with the temperature of optimal growth of the organism of 85 °C. The amino acid sequences and structures of the two proteins were compared and this gave insight into some of the factors that may confer thermostability-more Lys and Ile, fewer Ala, Thr, Gln and Gly residues, and shorter N- and C-termini. Additional and better insight into the thermostabilization strategies adopted by this enzyme will be provided when its crystal structure is determined.

Single molecule force spectroscopy reveals the effect of BiP chaperone on protein folding.

BiP (Immunoglobulin Binding Protein) is a member of the Hsp70 chaperones that participates in protein folding in the endoplasmic reticulum. The function of BiP relies on cycles of ATP hydrolysis driving the binding and release of its substrate proteins. It still remains unknown how BiP affects the protein folding pathway and there has been no direct demonstration showing which folding state of the substrate protein is bound by BiP, as previous work has used only peptides. Here, we employ optical tweezers for single molecule force spectroscopy experiments to investigate how BiP affects the folding mechanism of a complete protein and how this effect depends on nucleotides. Using the protein MJ0366 as the substrate for BiP, we performed pulling and relaxing cycles at constant velocity to unfold and refold the substrate. In the absence of BiP, MJ0366 unfolded and refolded in every cycle. However, when BiP was added, the frequency of folding events of MJ0366 significantly decreased, and the loss of folding always occurred after a successful unfolding event. This process was dependent on ATP and ADP, since when either ATP was decreased or ADP was added, the duration of periods without folding events increased. Our results show that the affinity of BiP for the substrate protein increased in these conditions, which correlates with previous studies in bulk. Therefore, we conclude that BiP binds to the unfolded state of MJ0366 and prevents its refolding, and that this effect is dependent on both the type and concentration of nucleotides.

Site-Specific Fluorescent Labeling of Argonaute for FRET-Based Bio-Assays.

Deciphering the molecular mechanisms of eukaryotic Argonaute proteins is crucial for the understanding of RNA interference (RNAi), a posttranscriptional gene silencing process. Fluorescence-based single-molecule studies like single-molecule Förster resonance energy transfer (FRET) between a donor and acceptor dye represent a versatile tool to gain a mechanistic understanding of the structural dynamics of a biomolecular complex. Until today it was not possible to site-specifically introduce fluorophores into eukaryotic Argonaute. Using an archaeal Argonaute variant from Methanocaldococcus jannaschii that closely resembles its eukaryotic counterpart, we site-specifically incorporated fluorescent probes into Argonaute. In this chapter, we first describe how to express archaeal Argonaute with the site-specifically engineered unnatural amino acid para-azido-L-phenylalanine (pAzF) and subsequently describe the coupling of a fluorophore exploiting the unique chemistry of the azide group of pAzF. In the second part of the chapter, we present a methodological approach that probes complex formation between acceptor-labeled archaeal Argonaute and guide and target nucleic acids equipped with a donor fluorophore which ultimately allows single-molecule FRET measurements. Furthermore we describe binding and cleavage assays that report on the functionality of Argonaute-nucleic acid complexes.

Characterization of RNA-binding properties of the archaeal Hfq-like protein from Methanococcus jannaschii.

The Sm and Sm-like proteins are widely distributed among bacteria, archaea and eukarya. They participate in many processes related to RNA-processing and regulation of gene expression. While the function of the bacterial Lsm protein Hfq and eukaryotic Sm/Lsm proteins is rather well studied, the role of Lsm proteins in Archaea is investigated poorly. In this work, the RNA-binding ability of an archaeal Hfq-like protein from Methanococcus jannaschii has been studied by X-ray crystallography, anisotropy fluorescence and surface plasmon resonance. It has been found that MjaHfq preserves the proximal RNA-binding site that usually recognizes uridine-rich sequences. Distal adenine-binding and lateral RNA-binding sites show considerable structural changes as compared to bacterial Hfq. MjaHfq did not bind mononucleotides at these sites and would not recognize single-stranded RNA as its bacterial homologues. Nevertheless, MjaHfq possesses affinity to poly(A) RNA that seems to bind at the unstructured positive-charged N-terminal tail of the protein.

A central cavity within the holo-translocon suggests a mechanism for membrane protein insertion.

The conserved SecYEG protein-conducting channel and the accessory proteins SecDF-YajC and YidC constitute the bacterial holo-translocon (HTL), capable of protein-secretion and membrane-protein insertion. By employing an integrative approach combining small-angle neutron scattering (SANS), low-resolution electron microscopy and biophysical analyses we determined the arrangement of the proteins and lipids within the super-complex. The results guided the placement of X-ray structures of individual HTL components and allowed the proposal of a model of the functional translocon. Their arrangement around a central lipid-containing pool conveys an unexpected, but compelling mechanism for membrane-protein insertion. The periplasmic domains of YidC and SecD are poised at the protein-channel exit-site of SecY, presumably to aid the emergence of translocating polypeptides. The SecY lateral gate for membrane-insertion is adjacent to the membrane 'insertase' YidC. Absolute-scale SANS employing a novel contrast-match-point analysis revealed a dynamic complex adopting open and compact configurations around an adaptable central lipid-filled chamber, wherein polytopic membrane-proteins could fold, sheltered from aggregation and proteolysis.

Crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus, Sulfolobus tokodaii and Methanocaldococcus jannaschii.

The crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus, Sulfolobus tokodaii and Methanocaldococcus jannaschii were determined and their structural characteristics were analyzed. For PurS from T. thermophilus, two structures were determined using two crystals that were grown in different conditions. The four structures in the dimeric form were almost identical to one another despite their relatively low sequence identities. This is also true for all PurS structures determined to date. A few residues were conserved among PurSs and these are located at the interaction site with PurL and PurQ, the other subunits of the formylglycinamide ribonucleotide amidotransferase. Molecular-dynamics simulations of the PurS dimer as well as a model of the complex of the PurS dimer, PurL and PurQ suggest that PurS plays some role in the catalysis of the enzyme by its bending motion.

Crystallographic analysis of a subcomplex of the transsulfursome with tRNA for Cys-tRNA(Cys) synthesis.

In most organisms, Cys-tRNA(Cys) is directly synthesized by cysteinyl-tRNA synthetase (CysRS). Many methanogenic archaea, however, use a two-step, indirect pathway to synthesize Cys-tRNA(Cys) owing to a lack of CysRS and cysteine-biosynthesis systems. This reaction is catalyzed by O-phosphoseryl-tRNA synthetase (SepRS), Sep-tRNA:Cys-tRNA synthase (SepCysS) and SepRS/SepCysS pathway enhancer (SepCysE) as the transsulfursome, in which SepCysE connects both SepRS and SepCysS. On the transsulfursome, SepRS first ligates an O-phosphoserine to tRNA(Cys), and the mischarged intermediate Sep-tRNA(Cys) is then transferred to SepCysS, where it is further modified to Cys-tRNA(Cys). In this study, a subcomplex of the transsulfursome with tRNA(Cys) (SepCysS-SepCysE-tRNA(Cys)), which is involved in the second reaction step of the indirect pathway, was constructed and then crystallized. The crystals diffracted X-rays to a resolution of 2.6 Šand belonged to space group P6522, with unit-cell parameters a = b = 107.2, c = 551.1 Å. The structure determined by molecular replacement showed that the complex consists of a SepCysS dimer, a SepCysE dimer and one tRNA(Cys) in the asymmetric unit.

Geometrical assembly of ultrastable protein templates for nanomaterials.

The fabrication of nanoscale devices requires architectural templates on which to position functional molecules in complex arrangements. Protein scaffolds are particularly promising templates for nanomaterials due to inherent molecular recognition and self-assembly capabilities combined with genetically encoded functionalities. However, difficulties in engineering protein quaternary structure into stable and well-ordered shapes have hampered progress. Here we report the development of an ultrastable biomolecular construction kit for the assembly of filamentous proteins into geometrically defined templates of controllable size and symmetry. The strategy combines redesign of protein-protein interaction specificity with the creation of tunable connector proteins that govern the assembly and projection angles of the filaments. The functionality of these nanoarchitectures is illustrated by incorporation of nanoparticles at specific locations and orientations to create hybrid materials such as conductive nanowires. These new structural components facilitate the manufacturing of nanomaterials with diverse shapes and functional properties over a wide range of processing conditions.