LRET-derived HADDOCK structural models describe the conformational heterogeneity required for DNA cleavage by the Mre11-Rad50 DNA damage repair complex.

The Mre11-Rad50-Nbs1 protein complex is one of the first responders to DNA double-strand breaks. Studies have shown that the catalytic activities of the evolutionarily conserved Mre11-Rad50 (MR) core complex depend on an ATP-dependent global conformational change that takes the macromolecule from an open, extended structure in the absence of ATP to a closed, globular structure when ATP is bound. We have previously identified an additional 'partially open' conformation using luminescence resonance energy transfer (LRET) experiments. Here, a combination of LRET and the molecular docking program HADDOCK was used to further investigate this partially open state and identify three conformations of MR in solution: closed, partially open, and open, which are in addition to the extended, apo conformation. Mutants disrupting specific Mre11-Rad50 interactions within each conformation were used in nuclease activity assays on a variety of DNA substrates to help put the three states into a functional perspective. LRET data collected on MR bound to DNA demonstrate that the three conformations also exist when nuclease substrates are bound. These models were further supported with small-angle X-ray scattering data, which corroborate the presence of multiple states in solution. Together, the data suggest a mechanism for the nuclease activity of the MR complex along the DNA.

Reversible Electron Transfer and Substrate Binding Support [NiFe3S4] Ferredoxin as a Protein-Based Model for [NiFe] Carbon Monoxide Dehydrogenase.

The nickel-iron carbon monoxide dehydrogenase (CODH) enzyme catalyzes the reversible and selective interconversion of carbon dioxide (CO2) to carbon monoxide (CO) with high rates and negligible overpotential. Despite decades of research, many questions remain about this complex metalloenzyme system. A simplified model enzyme could provide substantial insight into biological carbon cycling. Here, we demonstrate reversible electron transfer and binding of both CO and cyanide, a substrate and an inhibitor of CODH, respectively, in a Pyrococcus furiosus (Pf) ferredoxin (Fd) protein that has been reconstituted with a nickel-iron sulfide cluster ([NiFe3S4] Fd). The [NiFe3S4] cluster mimics the core of the native CODH active site and thus serves as a protein-based structural model of the CODH subsite. Notably, despite binding cyanide, no CO binding is observed for the physiological [Fe4S4] clusters in Pf Fd, providing chemical rationale underlying the evolution of a site-differentiated cluster for substrate conversion in native CODH. The demonstration of a substrate-binding metalloprotein model of CODH sets the stage for high-resolution spectroscopic and mechanistic studies correlating the subsite structure and function, ultimately guiding the design of anthropogenic catalysts that harness the advantages of CODH for effective CO2 reduction.

Development of Nonheme {FeNO}7 Complexes Based on the Pyrococcus furiosus Rubredoxin for Red-Light-Controllable Nitric Oxide Release.

Nitric oxide (NO) is an essential biological messenger, contributing a significant role in a diverse range of physiological processes. The light-controllable NO releasers are of great interest because of their potential as agents for NO-related research and therapeutics. Herein, we developed a pair of red-light-controllable NO releasers, pfRd-C9A-{FeNO}7 and pfRd-C42A-{FeNO}7 (pfRd = Pyrococcus furiosus rubredoxin), by constructing a nonheme {FeNO}7 center within the redesigned iron-sulfur protein scaffolds. While shown to be both air and thermally stable, these complexes are highly sensitive to red-light irradiation with temporal precision, which was confirmed by electron paramagnetic resonance spin trapping and Griess assay. The temporally controlled NO release from these complexes was also demonstrated in DNA cleavage assay. Overall, this study demonstrates that such a protein-based nonheme iron nitrosyl system could be a viable chemical tool for precise NO administration.

Streamlined purification and characterization of Pyrococcus furiosus rubredoxins with different N-terminal modifications by reversed-phase HPLC.

Rubredoxins (Rds), like those from Pyrococcus furious (Pf), have largely been found to be expressed in Escherichia coli (E. coli) as a mixture of different N-terminal forms, which may affect the properties of the protein. The typical procedures for the purification of Rds are cumbersome and usually with low yield. We present herein a streamlined purification strategy based on the reversed-phase high performance liquid chromatography (RP-HPLC), which offers high yield and high resolution after simply one-step purification following pre-treatment. We also show that RP-HPLC can be a valuable tool to gain information related to the thermal decomposition pathway of Pf-Rds.

Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life.

Modern day aerobic respiration in mitochondria involving complex I converts redox energy into chemical energy and likely evolved from a simple anaerobic system now represented by hydrogen gas-evolving hydrogenase (MBH) where protons are the terminal electron acceptor. Here we present the cryo-EM structure of an early ancestor in the evolution of complex I, the elemental sulfur (S0)-reducing reductase MBS. Three highly conserved protein loops linking cytoplasmic and membrane domains enable scalable energy conversion in all three complexes. MBS contains two proton pumps compared to one in MBH and likely conserves twice the energy. The structure also reveals evolutionary adaptations of MBH that enabled S0 reduction by MBS catalyzed by a site-differentiated iron-sulfur cluster without participation of protons or amino acid residues. This is the simplest mechanism proposed for reduction of inorganic or organic disulfides. It is of fundamental significance in the iron and sulfur-rich volcanic environments of early earth and possibly the origin of life. MBS provides a new perspective on the evolution of modern-day respiratory complexes and of catalysis by biological iron-sulfur clusters.

Combined prophylactic and therapeutic immune responses against human papillomaviruses induced by a thioredoxin-based L2-E7 nanoparticle vaccine.

Global burden of cervical cancer, the most common cause of mortality caused by human papillomavirus (HPV), is expected to increase during the next decade, mainly because current alternatives for HPV vaccination and cervical cancer screening programs are costly to be established in low-and-middle income countries. Recently, we described the development of the broadly protective, thermostable vaccine antigen Trx-8mer-OVX313 based on the insertion of eight different minor capsid protein L2 neutralization epitopes into a thioredoxin scaffold from the hyperthermophilic archaeon Pyrococcus furiosus and conversion of the resulting antigen into a nanoparticle format (median radius ~9 nm) upon fusion with the heptamerizing OVX313 module. Here we evaluated whether the engineered thioredoxin scaffold, in addition to humoral immune responses, can induce CD8+ T-cell responses upon incorporation of MHC-I-restricted epitopes. By systematically examining the contribution of individual antigen modules, we demonstrated that B-cell and T-cell epitopes can be combined into a single antigen construct without compromising either immunogenicity. While CD8+ T-cell epitopes had no influence on B-cell responses, the L2 polytope (8mer) and OVX313-mediated heptamerization of the final antigen significantly increased CD8+ T-cell responses. In a proof-of-concept experiment, we found that vaccinated mice remained tumor-free even after two consecutive tumor challenges, while unvaccinated mice developed tumors. A cost-effective, broadly protective vaccine with both prophylactic and therapeutic properties represents a promising option to overcome the challenges associated with prevention and treatment of HPV-caused diseases.

Secondary modification of oxidatively-modified proline N-termini for the construction of complex bioconjugates.

A convenient two-step method is reported for the ligation of alkoxyamine- or hydrazine-bearing cargo to proline N-termini. Using this approach, bifunctional proline N-terminal bioconjugates are constructed and proline N-terminal proteins are immobilized.

Application of a protein domain as chaperone for enhancing biological activity and stability of other proteins.

Chaperones are a diverse class of molecules known for increasing thermo-stability of proteins, preventing protein aggregation, favoring disaggregation, increasing solubility and in some cases imparting resistance to proteolysis. These functions can be employed for various biotechnological applications including point of care testing, nano-biotechnology, bio-process engineering, purification technologies and formulation development. Here we report that the N-terminal domain of Pyrococcus furiosusl-asparaginase, (NPfA, a protein chaperone lacking α-crystallin domain) can serve as an efficient, industrially relevant, protein additive. We tested the effect of NPfA on substrate proteins, ascorbate peroxidase (APX), IgG peroxidase antibodies (I-HAbs) and KOD DNA polymerase. Each protein not only displayed increased thermal stability but also increased activity in the presence of NPfA. This increase was either comparable or higher than those obtained by common osmolytes; glycine betaine, sorbitol and trehalose. Most dramatic activity enhancement was seen in the case of KOD polymerase (∼ 40 % increase). NPfA exerts its effect through transient binding to the substrate proteins as discerned through isothermal titration calorimetry, dynamic light scattering and size exclusion chromatography. Mechanistic insights obtained through simulations suggested a remodeled architecture and emergence of H-binding network between NPfA and substrate protein with an effective enhancement in the solvent accessibility at the active site pocket of the latter. Thus, the capability of NPfA to engage in specific manner with other proteins is demonstrated to reduce the concentration of substrate proteins/enzymes required per unit operation. The functional expansion obtained through our finding establishes NPfA as a novel class of ATP-independent molecular chaperone with immense future biotechnological applications.

Pyrococcus furiosus Argonaute-mediated nucleic acid detection.

We demonstrate that short single-stranded DNA generated by Pyrococcus furiosus Argonaute (PfAgo) can initiate a second round of cleavage. Based on this principle, we established a molecular diagnostic method, termed PfAgo-mediated Nucleic acid Detection (PAND). This method could detect DNA at attomolar sensitivities, distinguish single-nucleotide mutants and accomplish multiplexed detection.

The N-terminal domain of an archaeal multidrug and toxin extrusion (MATE) transporter mediates proton coupling required for prokaryotic drug resistance.

As a contributor to multidrug resistance, the family of multidrug and toxin extrusion (MATE) transporters couples the efflux of chemically dissimilar compounds to electrochemical ion gradients. Although divergent transport mechanisms have been proposed for these transporters, previous structural and functional analyses of members of the MATE subfamily DinF suggest that the N-terminal domain (NTD) supports substrate and ion binding. In this report, we investigated the relationship of ligand binding within the NTD to the drug resistance mechanism of the H+-dependent MATE from the hyperthermophilic archaeon Pyrococcus furiosus (PfMATE). To facilitate this study, we developed a cell growth assay in Escherichia coli to characterize the resistance conferred by PfMATE to toxic concentrations of the antimicrobial compound rhodamine 6G. Expression of WT PfMATE promoted cell growth in the presence of drug, but amino acid substitutions of conserved NTD residues compromised drug resistance. Steady-state binding analysis with purified PfMATE indicated that substrate affinity was unperturbed in these NTD variants. However, exploiting Trp fluorescence as an intrinsic reporter of conformational changes, we found that these variants impaired formation of a unique H+-stabilized structural intermediate. These results imply that disruption of H+ coupling is the origin of compromised toxin resistance in PfMATE variants. These findings support a model mechanism wherein the NTD mediates allosteric coupling to ion gradients through conformational changes to drive substrate transport in PfMATE. Furthermore, the results provide evidence for diverging transport mechanisms within a prokaryotic MATE subfamily.

Deciphering the Allosterically Driven Conformational Ensemble in Tryptophan Synthase Evolution.

Multimeric enzyme complexes are ubiquitous in nature and catalyze a broad range of useful biological transformations. They are often characterized by a tight allosteric coupling between subunits, making them highly inefficient when isolated. A good example is Tryptophan synthase (TrpS), an allosteric heterodimeric enzyme in the form of an αßßα complex that catalyzes the biosynthesis of L-tryptophan. In this study, we decipher the allosteric regulation existing in TrpS from Pyrococcus furiosus (PfTrpS), and how the allosteric conformational ensemble is recovered in laboratory-evolved stand-alone ß-subunit variants. We find that recovering the conformational ensemble of a subdomain of TrpS affecting the relative stabilities of open, partially closed, and closed conformations is a prerequisite for enhancing the catalytic efficiency of the ß-subunit in the absence of its binding partner. The distal mutations resuscitate the allosterically driven conformational regulation and alter the populations and rates of exchange between these multiple conformational states, which are essential for the multistep reaction pathway of the enzyme. Interestingly, these distal mutations can be a priori predicted by careful analysis of the conformational ensemble of the TrpS enzyme through computational methods. Our study provides the enzyme design field with a rational approach for evolving allosteric enzymes toward improved stand-alone function for biosynthetic applications.

Conservation of the structural and functional architecture of encapsulated ferritins in bacteria and archaea.

Ferritins are a large family of intracellular proteins that protect the cell from oxidative stress by catalytically converting Fe(II) into less toxic Fe(III) and storing iron minerals within their core. Encapsulated ferritins (EncFtn) are a sub-family of ferritin-like proteins, which are widely distributed in all bacterial and archaeal phyla. The recently characterized Rhodospirillum rubrum EncFtn displays an unusual structure when compared with classical ferritins, with an open decameric structure that is enzymatically active, but unable to store iron. This EncFtn must be associated with an encapsulin nanocage in order to act as an iron store. Given the wide distribution of the EncFtn family in organisms with diverse environmental niches, a question arises as to whether this unusual structure is conserved across the family. Here, we characterize EncFtn proteins from the halophile Haliangium ochraceum and the thermophile Pyrococcus furiosus, which show the conserved annular pentamer of dimers topology. Key structural differences are apparent between the homologues, particularly in the centre of the ring and the secondary metal-binding site, which is not conserved across the homologues. Solution and native mass spectrometry analyses highlight that the stability of the protein quaternary structure differs between EncFtn proteins from different species. The ferroxidase activity of EncFtn proteins was confirmed, and we show that while the quaternary structure around the ferroxidase centre is distinct from classical ferritins, the ferroxidase activity is still inhibited by Zn(II). Our results highlight the common structural organization and activity of EncFtn proteins, despite diverse host environments and contexts within encapsulins.

A Multipronged Method for Unveiling Subtle Structural-Functional Defects of Mutant Chaperone Molecules Causing Human Chaperonopathies.

Chaperonopathies are diseases in which abnormal chaperones play an etiopathogenic role. A chaperone is mutated or otherwise abnormal (e.g., modified by an aberrant posttranslational modification) in structure/function. To understand the pathogenic mechanisms of chaperonopathies, it is necessary to elucidate the impact of the pathogenic mutation or posttranslational modification on the chaperone molecule's properties and functions. This impact is usually subtle because if it were more than subtle the overall effect on the cell and organism would be catastrophic, lethal. This is because most chaperones are essential for life and, if damaged in structure/function too strongly, there would be death of the cell/organism, and no phenotype, i.e., there would be no patients with chaperonopathies. Consequently, diagnostic procedures and analysis of defects of the abnormal chaperones require a multipronged method for assessing the chaperone molecule from various angles. Here, we present such a method that includes assessing the intrinsic properties and the chaperoning functions of chaperone molecules.

Detergent-modified catalytic and enzymomimetic activity of silver and palladium nanoparticles biotemplated by Pyrococcus furiosus ferritin.

Palladium and silver nanoparticles (NPs) anchored at the outer surface of ferritin form stable suspension of non-coated particles that possess several catalytic and enzymomimetic activities. These activities are strongly affected by detergents that significantly influence the reaction efficiency and specificity. Reductive dehalogenation of various azo dye substrates shows strong differences in reactivity for each substrate-detergent pair. Reductive dehalogenation is negatively influenced by cationic detergents while catalytic depropargylation is severely impaired by polyethylene oxide containing detergents that is an important finding in respect to potential biorthogonal applications. Moreover, Suzuki-Miyaura reaction is promoted by polyethylene oxide containing detergents but some of them also facilitate dehalogenation. Enzymomimetic peroxidase activity of silver NPs can be detected only in presence of sodium dodecyl sulfate (SDS) while peroxidase activity of palladium NPs is enhanced by SDS and sodium deoxycholate.

Structural Relaxation Processes and Collective Dynamics of Water in Biomolecular Environments.

In this simulation study, we investigate the influence of biomolecular confinement on dynamical processes in water. We compare water confined in a membrane protein nanopore at room temperature to pure liquid water at low temperatures with respect to structural relaxations, intermolecular vibrations, and the propagation of collective modes. We observe distinct potential energy landscapes experienced by water molecules in the two environments, which nevertheless result in comparable hydrogen bond lifetimes and sound propagation velocities. Hence, we show that a viscoelastic argument that links slow rearrangements of the water-hydrogen bond network to ice-like collective properties applies to both, the pure liquid and biologically confined water, irrespective of differences in the microscopic structure.

Engineered Biosynthesis of ß-Alkyl Tryptophan Analogues.

Noncanonical amino acids (ncAAs) with dual stereocenters at the α and ß positions are valuable precursors to natural products and therapeutics. Despite the potential applications of such bioactive ß-branched ncAAs, their availability is limited due to the inefficiency of the multistep methods used to prepare them. Herein we report a stereoselective biocatalytic synthesis of ß-branched tryptophan analogues using an engineered variant of Pyrococcus furiosus tryptophan synthase (PfTrpB), PfTrpB7E6 . PfTrpB7E6 is the first biocatalyst to synthesize bulky ß-branched tryptophan analogues in a single step, with demonstrated access to 27 ncAAs. The molecular basis for the efficient catalysis and broad substrate tolerance of PfTrpB7E6 was explored through X-ray crystallography and UV/Vis spectroscopy, which revealed that a combination of active-site and remote mutations increase the abundance and persistence of a key reactive intermediate. PfTrpB7E6 provides an operationally simple and environmentally benign platform for the preparation of ß-branched tryptophan building blocks.

Displacement of the transcription factor B reader domain during transcription initiation.

Transcription initiation by archaeal RNA polymerase (RNAP) and eukaryotic RNAP II requires the general transcription factor (TF) B/ IIB. Structural analyses of eukaryotic transcription initiation complexes locate the B-reader domain of TFIIB in close proximity to the active site of RNAP II. Here, we present the first crosslinking mapping data that describe the dynamic transitions of an archaeal TFB to provide evidence for structural rearrangements within the transcription complex during transition from initiation to early elongation phase of transcription. Using a highly specific UV-inducible crosslinking system based on the unnatural amino acid para-benzoyl-phenylalanine allowed us to analyze contacts of the Pyrococcus furiosus TFB B-reader domain with site-specific radiolabeled DNA templates in preinitiation and initially transcribing complexes. Crosslink reactions at different initiation steps demonstrate interactions of TFB with DNA at registers +6 to +14, and reduced contacts at +15, with structural transitions of the B-reader domain detected at register +10. Our data suggest that the B-reader domain of TFB interacts with nascent RNA at register +6 and +8 and it is displaced from the transcribed-strand during the transition from +9 to +10, followed by the collapse of the transcription bubble and release of TFB from register +15 onwards.

The trimeric Hef-associated nuclease HAN is a 3'→5' exonuclease and is probably involved in DNA repair.

Nucleases play important roles in nucleic acid metabolism. Some archaea encode a conserved protein known as Hef-associated nuclease (HAN). In addition to its C-terminal DHH nuclease domain, HAN also has three N-terminal domains, including a DnaJ-Zinc-finger, ribosomal protein S1-like, and oligonucleotide/oligosaccharide-binding fold. To further understand HAN's function, we biochemically characterized the enzymatic properties of HAN from Pyrococcus furiosus (PfuHAN), solved the crystal structure of its DHH nuclease domain, and examined its role in DNA repair. Our results show that PfuHAN is a Mn2+-dependent 3'-exonuclease specific to ssDNA and ssRNA with no activity on blunt and 3'-recessive double-stranded DNA. Domain truncation confirmed that the intrinsic nuclease activity is dependent on the C-terminal DHH nuclease domain. The crystal structure of the DHH nuclease domain adopts a trimeric topology, with each subunit adopting a classical DHH phosphoesterase fold. Yeast two hybrid assay confirmed that the DHH domain interacts with the IDR peptide of Hef nuclease. Knockout of the han gene or its C-terminal DHH nuclease domain in Haloferax volcanii resulted in increased sensitivity to the DNA damage reagent MMS. Our results imply that HAN nuclease might be involved in repairing stalled replication forks in archaea.

The Protein Microenvironment Governs the Suitability of Labeling Sites for Single-Molecule Spectroscopy of RNP Complexes.

Single-molecule techniques allow unique insights into biological systems as they provide unrivaled access to structural dynamics and conformational heterogeneity. One major bottleneck for reliable single-molecule Förster resonance energy transfer (smFRET) analysis is the identification of suitable fluorophore labeling sites that neither impair the function of the biological system nor cause photophysical artifacts of the fluorophore. To address this issue, we identified the contribution of virtually all individual parameters that affect Förster resonance energy transfer between two fluorophores attached to a ribonucleoprotein complex consisting of the RNA-binding protein L7Ae and a cognate kink turn containing RNA. A non-natural amino acid was incorporated at various positions of the protein using an amber suppression system (pEVOL) to label the protein via copper(I)-catalyzed alkyne-azide cycloaddition. On the basis of simulations followed by functional, structural, and multiparameter fluorescence analysis of five different smFRET RNPs, new insights into the design of smFRET RNPs were obtained. From this, a correlation between the photophysical properties of fluorophores attached to the protein and the predictability of the corresponding smFRET construct was established. Additionally, we identify a straightforward experimental method for characterizing selected labeling sites. Overall, this protocol allows fast generation and assessment of functional RNPs for accurate single-molecule experiments.

Rapid access to RNA resonances by proton-detected solid-state NMR at >100 kHz MAS.

Fast (>100 kHz) magic angle spinning solid-state NMR allows combining high-sensitive proton detection with the absence of an intrinsic molecular weight limit. Using this technique we observe for the first time narrow 1H RNA resonances and assign nucleotide spin systems with only 200 µg of uniformly 13C,15N-labelled RNA.