Characterization of a small tRNA-binding protein that interacts with the archaeal proteasome complex.

The proteasome system allows the elimination of functional or structurally impaired proteins. This includes the degradation of nascent peptides. In Archaea, how the proteasome complex interacts with the translational machinery remains to be described. Here, we characterized a small orphan protein, Q9UZY3 (UniProt ID), conserved in Thermococcales. The protein was identified in native pull-down experiments using the proteasome regulatory complex (proteasome-activating nucleotidase [PAN]) as bait. X-ray crystallography and small-angle X-ray scattering experiments revealed that the protein is monomeric and adopts a ß-barrel core structure with an oligonucleotide/oligosaccharide-binding (OB)-fold, typically found in translation elongation factors. Mobility shift experiment showed that Q9UZY3 displays transfer ribonucleic acid (tRNA)-binding properties. Pull-downs, co-immunoprecipitation and isothermal titration calorimetry (ITC) studies revealed that Q9UZY3 interacts in vitro with PAN. Native pull-downs and proteomic analysis using different versions of Q9UZY3 showed that the protein interacts with the assembled PAN-20S proteasome machinery in Pyrococcus abyssi (Pa) cellular extracts. The protein was therefore named Pbp11, for Proteasome-Binding Protein of 11 kDa. Interestingly, the interaction network of Pbp11 also includes ribosomal proteins, tRNA-processing enzymes and exosome subunits dependent on Pbp11's N-terminal domain that was found to be essential for tRNA binding. Together these data suggest that Pbp11 participates in an interface between the proteasome and the translational machinery.

Effects of chaotropic salts on global proteome stability in halophilic archaea: Implications for life signatures on Mars.

Halophilic archaea thriving in hypersaline environments, such as salt lakes, offer models for putative life in extraterrestrial brines such as those found on Mars. However, little is known about the effect of the chaotropic salts that could be found in such brines, such as MgCl2 , CaCl2 and (per)chlorate salts, on complex biological samples like cell lysates which could be expected to be more representative of biomarkers left behind putative extraterrestrial life forms. We used intrinsic fluorescence to study the salt dependence of proteomes extracted from five halophilic strains: Haloarcula marismortui, Halobacterium salinarum, Haloferax mediterranei, Halorubrum sodomense and Haloferax volcanii. These strains were isolated from Earth environments with different salt compositions. Among the five strains that were analysed, H. mediterranei stood out as a results of its high dependency on NaCl for its proteome stabilization. Interestingly, the results showed contrasting denaturation responses of the proteomes to chaotropic salts. In particular, the proteomes of strains that are most dependent or tolerant on MgCl2 for growth exhibited higher tolerance towards chaotropic salts that are abundant in terrestrial and Martian brines. These experiments bridge together global protein properties and environmental adaptation and help guide the search for protein-like biomarkers in extraterrestrial briny environments.

Resurrection of Ancestral Malate Dehydrogenases Reveals the Evolutionary History of Halobacterial Proteins: Deciphering Gene Trajectories and Changes in Biochemical Properties.

Extreme halophilic Archaea thrive in high salt, where, through proteomic adaptation, they cope with the strong osmolarity and extreme ionic conditions of their environment. In spite of wide fundamental interest, however, studies providing insights into this adaptation are scarce, because of practical difficulties inherent to the purification and characterization of halophilic enzymes. In this work, we describe the evolutionary history of malate dehydrogenases (MalDH) within Halobacteria (a class of the Euryarchaeota phylum). We resurrected nine ancestors along the inferred halobacterial MalDH phylogeny, including the Last Common Ancestral MalDH of Halobacteria (LCAHa) and compared their biochemical properties with those of five modern halobacterial MalDHs. We monitored the stability of these various MalDHs, their oligomeric states and enzymatic properties, as a function of concentration for different salts in the solvent. We found that a variety of evolutionary processes, such as amino acid replacement, gene duplication, loss of MalDH gene and replacement owing to horizontal transfer resulted in significant differences in solubility, stability and catalytic properties between these enzymes in the three Halobacteriales, Haloferacales, and Natrialbales orders since the LCAHa MalDH. We also showed how a stability trade-off might favor the emergence of new properties during adaptation to diverse environmental conditions. Altogether, our results suggest a new view of halophilic protein adaptation in Archaea.

Experimental study of proteome halophilicity using nanoDSF: a proof of concept.

Adaption to environmental conditions is reflected by protein adaptation. In particular, proteins of extremophiles display distinctive traits ensuring functional, structural and dynamical properties under permanently extreme physical and chemical conditions. While it has mostly been studied with approaches focusing on specific proteins, biophysical approaches have also confirmed this link between environmental and protein adaptation at the more complex and diverse scale of the proteome. However, studies of this type remain challenging and often require large amounts of biological material. We report here the use of nanoDSF as a tool to study proteome stability and solubility in cell lysates of the model halophilic archaeon Haloarcula marismortui. Notably, our results show that, as with single halophilic protein studies, proteome stability was correlated to the concentration of NaCl or KCl under which the cells were lysed and hence the proteome exposed. This work highlights that adaptation to environmental conditions can be experimentally observed at the scale of the proteome. Still, we show that the biochemical properties of single halophilic proteins can only be partially extrapolated to the whole proteome.

Observing Protein Degradation by the PAN-20S Proteasome by Time-Resolved Neutron Scattering.

The proteasome is a key player of regulated protein degradation in all kingdoms of life. Although recent atomic structures have provided snapshots on a number of conformations, data on substrate states and populations during the active degradation process in solution remain scarce. Here, we use time-resolved small-angle neutron scattering of a deuterium-labeled GFPssrA substrate and an unlabeled archaeal PAN-20S system to obtain direct structural information on substrate states during ATP-driven unfolding and subsequent proteolysis in solution. We find that native GFPssrA structures are degraded in a biexponential process, which correlates strongly with ATP hydrolysis, the loss of fluorescence, and the buildup of small oligopeptide products. Our solution structural data support a model in which the substrate is directly translocated from PAN into the 20S proteolytic chamber, after a first, to our knowledge, successful unfolding process that represents a point of no return and thus prevents dissociation of the complex and the release of harmful, aggregation-prone products.

Characterization of a Glycyl-Specific TET Aminopeptidase Complex from Pyrococcus horikoshii.

The TET peptidases are large self-compartmentalized complexes that form dodecameric particles. These metallopeptidases, members of the M42 family, are widely distributed in prokaryotes. Three different versions of TET complexes, with different substrate specificities, were found to coexist in the cytosol of the hyperthermophilic archaeon Pyrococcus horikoshii In the present work, we identified a novel type of TET complex that we named PhTET4. The recombinant PhTET4 enzyme was found to self-assemble as a tetrahedral edifice similar to other TET complexes. We determined PhTET4 substrate specificity using a broad range of monoacyl chromogenic and fluorogenic compounds. High-performance liquid chromatographic peptide degradation assays were also performed. These experiments demonstrated that PhTET4 is a strict glycyl aminopeptidase, devoid of amidolytic activity toward other types of amino acids. The catalytic efficiency of PhTET4 was studied under various conditions. The protein was found to be a hyperthermophilic alkaline aminopeptidase. Interestingly, unlike other peptidases from the same family, it was activated only by nickel ions.IMPORTANCE We describe here the first known peptidase displaying exclusive activity toward N-terminal glycine residues. This work indicates a specific role for intracellular glycyl peptidases in deep sea hyperthermophilic archaeal metabolism. These observations also provide critical evidence for the use of these archaeal extremozymes for biotechnological applications.

Unveiling the Binding Modes of the Crystallophore, a Terbium-based Nucleating and Phasing Molecular Agent for Protein Crystallography.

Crystallophores are lanthanide complexes that act as powerful auxiliary for protein crystallography due to their strong nucleating and phasing effects. To get first insights on the mechanisms behind nucleation induced by Crystallophore, we systematically identified various elaborated networks of supramolecular interactions between Tb-Xo4 and subset of 6 protein structures determined by X-ray diffraction in complex with terbium-Crystallophore (Tb-Xo4). Such interaction mapping analyses demonstrate the versatile binding behavior of the Crystallophore and pave the way to a better understanding of its unique properties.

SAXS/SANS on Supercharged Proteins Reveals Residue-Specific Modifications of the Hydration Shell.

Water molecules in the immediate vicinity of biomacromolecules, including proteins, constitute a hydration layer characterized by physicochemical properties different from those of bulk water and play a vital role in the activity and stability of these structures, as well as in intermolecular interactions. Previous studies using solution scattering, crystallography, and molecular dynamics simulations have provided valuable information about the properties of these hydration shells, including modifications in density and ionic concentration. Small-angle scattering of x-rays (SAXS) and neutrons (SANS) are particularly useful and complementary techniques to study biomacromolecular hydration shells due to their sensitivity to electronic and nuclear scattering-length density fluctuations, respectively. Although several sophisticated SAXS/SANS programs have been developed recently, the impact of physicochemical surface properties on the hydration layer remains controversial, and systematic experimental data from individual biomacromolecular systems are scarce. Here, we address the impact of physicochemical surface properties on the hydration shell by a systematic SAXS/SANS study using three mutants of a single protein, green fluorescent protein (GFP), with highly variable net charge (+36, -6, and -29). The combined analysis of our data shows that the hydration shell is locally denser in the vicinity of acidic surface residues, whereas basic and hydrophilic/hydrophobic residues only mildly modify its density. Moreover, the data demonstrate that the density modifications result from the combined effect of residue-specific recruitment of ions from the bulk in combination with water structural rearrangements in their vicinity. Finally, we find that the specific surface-charge distributions of the different GFP mutants modulate the conformational space of flexible parts of the protein.

The TET2 and TET3 aminopeptidases from Pyrococcus horikoshii form a hetero-subunit peptidasome with enhanced peptide destruction properties.

TET aminopeptidases assemble as large homo-dodecameric complexes. The reason why prokaryotic genomes often encode a diverse set of TET peptidases homologues remains unclear. In the archaeon Pyrococcus horikoshii, PhTET1, PhTET2 and PhTET3 homo-oligomeric particles have been proposed to work in concert to breakdown intracellular polypeptides. When coexpressed in Escherichia coli, the PhTET2 and PhTET3 proteins were found to assemble efficiently as heteromeric complexes. Biophysical analysis demonstrated that these particles possess the same quaternary structure as the homomeric TET dodecamers. The same hetero-oligomeric complexes were immunodetected in P. horikoshii cell extracts analysed by sucrose gradient fractionation and ion exchange chromatography. The biochemical activity of a purified hetero-oligomeric TET particle, assessed on chromogenic substrates and on a complex mixture of peptides, reveals that it displays higher efficiency than an equivalent combination of homo-oligomeric TET particles. Interestingly, phylogenetic analysis shows that PhTET2 and PhTET3 are paralogous proteins that arose from gene duplication in the ancestor of Thermococcales. Together, these results establish that the PhTET2 and PhTET3 proteins are two subunits of the same enzymatic complex aimed at the destruction of polypeptidic chains of very different composition. This is the first report for such a mechanism intended to improve multi-enzymatic complex efficiency among exopeptidases.

Microbial diversity and adaptation to high hydrostatic pressure in deep-sea hydrothermal vents prokaryotes.

Prokaryotes inhabiting in the deep sea vent ecosystem will thus experience harsh conditions of temperature, pH, salinity or high hydrostatic pressure (HHP) stress. Among the fifty-two piezophilic and piezotolerant prokaryotes isolated so far from different deep-sea environments, only fifteen (four Bacteria and eleven Archaea) that are true hyper/thermophiles and piezophiles have been isolated from deep-sea hydrothermal vents; these belong mainly to the Thermococcales order. Different strategies are used by microorganisms to thrive in deep-sea hydrothermal vents in which "extreme" physico-chemical conditions prevail and where non-adapted organisms cannot live, or even survive. HHP is known to impact the structure of several cellular components and functions, such as membrane fluidity, protein activity and structure. Physically the impact of pressure resembles a lowering of temperature, since it reinforces the structure of certain molecules, such as membrane lipids, and an increase in temperature, since it will also destabilize other structures, such as proteins. However, universal molecular signatures of HHP adaptation are not yet known and are still to be deciphered.

Molecular adaptation and salt stress response of Halobacterium salinarum cells revealed by neutron spectroscopy.

Halobacterium salinarum is an extreme halophile archaeon with an absolute requirement for a multimolar salt environment. It accumulates molar concentrations of KCl in the cytosol to counterbalance the external osmotic pressure imposed by the molar NaCl. As a consequence, cytosolic proteins are permanently exposed to low water activity and highly ionic conditions. In non-adapted systems, such conditions would promote protein aggregation, precipitation, and denaturation. In contrast, in vitro studies showed that proteins from extreme halophilic cells are themselves obligate halophiles. In this paper, adaptation via dynamics to low-salt stress in H. salinarum cells was measured by neutron scattering experiments coupled with microbiological characterization. The molecular dynamic properties of a proteome represent a good indicator for environmental adaptation and the neutron/microbiology approach has been shown to be well tailored to characterize these modifications. In their natural setting, halophilic organisms often have to face important variations in environmental salt concentration. The results showed deleterious effects already occur in the H. salinarum proteome, even when the external salt concentration is still relatively high, suggesting the onset of survival mechanisms quite early when the environmental salt concentration decreases.

Pyrococcus horikoshii TET2 peptidase assembling process and associated functional regulation.

Tetrahedral (TET) aminopeptidases are large polypeptide destruction machines present in prokaryotes and eukaryotes. Here, the rules governing their assembly into hollow 12-subunit tetrahedrons are addressed by using TET2 from Pyrococcus horikoshii (PhTET2) as a model. Point mutations allowed the capture of a stable, catalytically active precursor. Small angle x-ray scattering revealed that it is a dimer whose architecture in solution is identical to that determined by x-ray crystallography within the fully assembled TET particle. Small angle x-ray scattering also showed that the reconstituted PhTET2 dodecameric particle displayed the same quaternary structure and thermal stability as the wild-type complex. The PhTET2 assembly intermediates were characterized by analytical ultracentrifugation, native gel electrophoresis, and electron microscopy. They revealed that PhTET2 assembling is a highly ordered process in which hexamers represent the main intermediate. Peptide degradation assays demonstrated that oligomerization triggers the activity of the TET enzyme toward large polypeptidic substrates. Fractionation experiments in Pyrococcus and Halobacterium cells revealed that, in vivo, the dimeric precursor co-exists together with assembled TET complexes. Taken together, our observations explain the biological significance of TET oligomerization and suggest the existence of a functional regulation of the dimer-dodecamer equilibrium in vivo.

Small-angle neutron scattering reveals the assembly mode and oligomeric architecture of TET, a large, dodecameric aminopeptidase.

The specific self-association of proteins into oligomeric complexes is a common phenomenon in biological systems to optimize and regulate their function. However, de novo structure determination of these important complexes is often very challenging for atomic-resolution techniques. Furthermore, in the case of homo-oligomeric complexes, or complexes with very similar building blocks, the respective positions of subunits and their assembly pathways are difficult to determine using many structural biology techniques. Here, an elegant and powerful approach based on small-angle neutron scattering is applied, in combination with deuterium labelling and contrast variation, to elucidate the oligomeric organization of the quaternary structure and the assembly pathways of 468 kDa, hetero-oligomeric and symmetric Pyrococcus horikoshii TET2-TET3 aminopeptidase complexes. The results reveal that the topology of the PhTET2 and PhTET3 dimeric building blocks within the complexes is not casual but rather suggests that their quaternary arrangement optimizes the catalytic efficiency towards peptide substrates. This approach bears important potential for the determination of quaternary structures and assembly pathways of large oligomeric and symmetric complexes in biological systems.

DNA Polymerization in Icy Moon Abyssal Pressure Conditions.

Evidence of stable liquid water oceans beneath the ice crust of moons within the Solar System is of great interest for astrobiology. In particular, subglacial oceans may present hydrothermal processes in their abysses, similarly to terrestrial hydrothermal vents. Therefore, terrestrial extremophilic deep life can be considered a model for putative icy moon extraterrestrial life. However, the comparison between putative extraterrestrial abysses and their terrestrial counterparts suffers from a potentially determinant difference. Indeed, some icy moons oceans may be so deep that the hydrostatic pressure would exceed the maximal pressure at which hydrothermal vent organisms have been isolated. While terrestrial microorganisms that are able to survive in such conditions are known, the effect of high pressure on fundamental biochemical processes is still unclear. In this study, the effects of high hydrostatic pressure on DNA synthesis catalyzed by DNA polymerases are investigated for the first time. The effect on both strand displacement and primer extension activities is measured, and pressure tolerance is compared between enzymes of various thermophilic organisms isolated at different depths.

Perspectives in biological physics: the nDDB project for a neutron Dynamics Data Bank for biological macromolecules.

Neutron spectroscopy provides experimental data on time-dependent trajectories, which can be directly compared to molecular dynamics simulations. Its importance in helping us to understand biological macromolecules at a molecular level is demonstrated by the results of a literature survey over the last two to three decades. Around 300 articles in refereed journals relate to neutron scattering studies of biological macromolecular dynamics, and the results of the survey are presented here. The scope of the publications ranges from the general physics of protein and solvent dynamics, to the biologically relevant dynamics-function relationships in live cells. As a result of the survey we are currently setting up a neutron Dynamics Data Bank (nDDB) with the aim to make the neutron data on biological systems widely available. This will benefit, in particular, the MD simulation community to validate and improve their force fields. The aim of the database is to expose and give easy access to a body of experimental data to the scientific community. The database will be populated with as much of the existing data as possible. In the future it will give value, as part of a bigger whole, to high throughput data, as well as more detailed studies. A range and volume of experimental data will be of interest in determining how quantitatively MD simulations can reproduce trends across a range of systems and to what extent such trends may depend on sample preparation and data reduction and analysis methods. In this context, we strongly encourage researchers in the field to deposit their data in the nDDB.

An NMR Study of a 300-kDa AAA+ Unfoldase.

AAA+ ATPases are ubiquitous hexameric unfoldases acting in cellular protein quality control. In complex with proteases, they form protein degradation machinery (the proteasome) in both archaea and eukaryotes. Here, we use solution-state NMR spectroscopy to determine the symmetry properties of the archaeal PAN AAA+ unfoldase and gain insights into its functional mechanism. PAN consists of three folded domains: the coiled-coil (CC), OB and ATPase domains. We find that full-length PAN assembles into a hexamer with C2 symmetry, and that this symmetry extends over the CC, OB and ATPase domains. The NMR data, collected in the absence of substrate, are incompatible with the spiral staircase structure observed in electron-microscopy studies of archaeal PAN in the presence of substrate and in electron-microscopy studies of eukaryotic unfoldases both in the presence and in the absence of substrate. Based on the C2 symmetry revealed by NMR spectroscopy in solution, we propose that archaeal ATPases are flexible enzymes, which can adopt distinct conformations in different conditions. This study reaffirms the importance of studying dynamic systems in solution.

A real-time analysis of GFP unfolding by the AAA+ unfoldase PAN.

Protein quality control systems are essential to maintain a healthy proteome. They often consist of an unfoldase unit, typically an AAA+ ATPase, coupled with a protease unit. In all kingdoms of life, they function to eliminate misfolded proteins, and thus prevent that their aggregates do harm to the cell, and to rapidly regulate protein levels in the presence of environmental changes. Despite the huge progress made in the past two decades in understanding the mechanism of function of protein degradation systems, the fate of the substrate during the unfolding and proteolytic processes remains poorly understood. Here we exploit an NMR-based approach to monitor GFP processing by the archaeal PAN unfoldase and the PAN-20S degradation system in real time. We find that PAN-dependent unfolding of GFP does not involve the release of partially-folded GFP molecules resulting from futile unfolding attempts. In contrast, once stably engaged with PAN, GFP molecules are efficiently transferred to the proteolytic chamber of the 20S subunit, despite the only weak affinity of PAN for the 20S subunit in the absence of substrate. This is essential to guarantee that unfolded but not proteolyzed proteins are not released into solution, where they would form toxic aggregates. The results of our studies are in good agreement with previous results derived from real-time small-angle-neutron-scattering experiments and have the advantage of allowing the investigation of substrates and products at amino-acid resolution.

Studies on the parameters controlling the stability of the TET peptidase superstructure from Pyrococcus horikoshii revealed a crucial role of pH and catalytic metals in the oligomerization process.

The TET proteases from Pyrococcus horikoshii are metallopeptidases that form large dodecameric particles with high thermal stability. The influence of various physico-chemical parameters on PhTET3 quaternary structure was investigated. Analytical ultracentrifugation and biochemical analyses showed that the PhTET3 quaternary structure and enzymatic activity are maintained in high salt and that the complex is stable under extreme acidic conditions. Under basic pH conditions the complex disassembled into a low molecular weight species that was identified as folded dimer. Metal analyses showed that the purified enzyme only contains two equivalent of zinc per monomer, corresponding to the metal ions responsible for catalytic activity. When these metals were removed by EDTA treatment, the complex dissociated into the same dimeric species as those observed at high pH. Dodecameric TET particles were obtained from the metal free dimers when 2mM of divalent ions were added to the protein samples. Most of the dimers remained assembled at high temperature. Thus, we have shown that dimers are the building units in the TET oligomerization pathway and that the active site metals are essential in this process.

Effects of hydrostatic pressure on the quaternary structure and enzymatic activity of a large peptidase complex from Pyrococcus horikoshii.

While molecular adaptation to high temperature has been extensively studied, the effect of hydrostatic pressure on protein structure and enzymatic activity is still poorly understood. We have studied the influence of pressure on both the quaternary structure and enzymatic activity of the dodecameric TET3 peptidase from Pyrococcus horikoshii. Small angle X-ray scattering (SAXS) revealed a high robustness of the oligomer under high pressure of up to 300 MPa at 25°C as well as at 90°C. The enzymatic activity of TET3 was enhanced by pressure up to 180 MPa. From the pressure behavior of the different rate-constants we have determined the volume changes associated with substrate binding and catalysis. Based on these results we propose that a change in the rate-limiting step occurs around 180 MPa.

Relevance of Earth-Bound Extremophiles in the Search for Extraterrestrial Life.

The recent discovery of extrasolar Earth-like planets that orbit in their habitable zone of their system, and the latest clues of the presence of liquid water in the subsurface of Mars and in the subglacial ocean of Jupiter's and Saturn's moons, has reopened debates about habitability and limits of life. Although liquid water, widely accepted as an absolute requirement for terrestrial life, may be present in other bodies of the solar system or elsewhere, physical and chemical conditions, such as temperature, pressure, and salinity, may limit this habitability. However, extremophilic microorganisms found in various extreme terrestrial environments are adapted to thrive in permanently extreme ranges of physicochemical conditions. This review first describes promising environments for life in the Solar System and the microorganisms that inhabit similar environments on the Earth. The effects of extreme temperatures, salt, and hydrostatic pressure conditions on biomolecules will be explained in some detail, and recent advances in understanding biophysical and structural adaptation strategies allowing microorganisms to cope with extreme physicochemical conditions are reviewed to discuss promising environments for life in the Solar System in terms of habitability.