Structural Basis of Redox-Sensing Transcriptional Repressor Rex with Cofactor NAD+ and Operator DNA.

The transcriptional repressor Rex plays important roles in regulating the expression of respiratory genes by sensing the reduction-oxidation (redox) state according to the intracellular NAD+/NADH balance. Previously, we reported on crystal structures of apo, NAD+-bound, and NADH-bound forms of Rex from Thermotoga maritima to analyze the structural basis of transcriptional regulation depending on either NAD+ or NADH binding. In this study, the crystal structure of Rex in ternary complex with NAD+ and operator DNA revealed that the N-terminal domain of Rex, including the helix-turn-helix motif, forms extensive contacts with DNA in addition to DNA sequence specificity. Structural comparison of the Rex in apo, NAD+-bound, NADH-bound, and ternary complex forms provides a comprehensive picture of transcriptional regulation in the Rex. These data demonstrate that the conformational change in Rex when binding with the reduced NADH or oxidized NAD+ determines operator DNA binding. The movement of the N-terminal domains toward the operator DNA was blocked upon binding of NADH ligand molecules. The structural results provide insights into the molecular mechanism of Rex binding with operator DNA and cofactor NAD+/NADH, which is conserved among Rex family repressors. Structural analysis of Rex from T. maritima also supports the previous hypothesis about the NAD+/NADH-specific transcriptional regulation mechanism of Rex homologues.

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.

Structural determinants and distribution of phosphate specificity in ribonucleotide reductases.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, the building blocks of DNA. RNRs are specific for either ribonucleoside diphosphates or triphosphates as substrates. As far as is known, oxygen-dependent class I RNRs (NrdAB) all reduce ribonucleoside diphosphates, and oxygen-sensitive class III RNRs (NrdD) are all ribonucleoside triphosphate reducers, whereas the adenosylcobalamin-dependent class II (NrdJ) contains both ribonucleoside diphosphate and triphosphate reducers. However, it is unknown how this specificity is conveyed by the active site of the enzymes and how this feature developed in RNR evolution. By structural comparison of the active sites in different RNRs, we identified the apical loop of the phosphate-binding site as a potential structural determinant of substrate specificity. Grafting two residues from this loop from a diphosphate- to a triphosphate-specific RNR caused a change in preference from ribonucleoside triphosphate to diphosphate substrates in a class II model enzyme, confirming them as the structural determinants of phosphate specificity. The investigation of the phylogenetic distribution of this motif in class II RNRs yielded a likely monophyletic clade with the diphosphate-defining motif. This indicates a single evolutionary-split event early in NrdJ evolution in which diphosphate specificity developed from the earlier triphosphate specificity. For those interesting cases where organisms contain more than one nrdJ gene, we observed a preference for encoding enzymes with diverse phosphate specificities, suggesting that this varying phosphate specificity confers a selective advantage.

Structures and kinetics of Thermotoga maritima MetY reveal new insights into the predominant sulfurylation enzyme of bacterial methionine biosynthesis.

Bacterial methionine biosynthesis can take place by either the trans-sulfurylation route or direct sulfurylation. The enzymes responsible for trans-sulfurylation have been characterized extensively because they occur in model organisms such as Escherichia coli. However, direct sulfurylation is actually the predominant route for methionine biosynthesis across the phylogenetic tree. In this pathway, most bacteria use an O-acetylhomoserine aminocarboxypropyltransferase (MetY) to catalyze the formation of homocysteine from O-acetylhomoserine and bisulfide. Despite the widespread distribution of MetY, this pyridoxal 5'-phosphate-dependent enzyme remains comparatively understudied. To address this knowledge gap, we have characterized the MetY from Thermotoga maritima (TmMetY). At its optimal temperature of 70 °C, TmMetY has a turnover number (apparent kcat = 900 s-1) that is 10- to 700-fold higher than the three other MetY enzymes for which data are available. We also present crystal structures of TmMetY in the internal aldimine form and, fortuitously, with a ß,γ-unsaturated ketimine reaction intermediate. This intermediate is identical to that found in the catalytic cycle of cystathionine γ-synthase (MetB), which is a homologous enzyme from the trans-sulfurylation pathway. By comparing the TmMetY and MetB structures, we have identified Arg270 as a critical determinant of specificity. It helps to wall off the active site of TmMetY, disfavoring the binding of the first MetB substrate, O-succinylhomoserine. It also ensures a strict specificity for bisulfide as the second substrate of MetY by occluding the larger MetB substrate, cysteine. Overall, this work illuminates the subtle structural mechanisms by which homologous pyridoxal 5'-phosphate-dependent enzymes can effect different catalytic, and therefore metabolic, outcomes.

Exploring targeting peptide-shell interactions in encapsulin nanocompartments.

Encapsulins are recently discovered protein compartments able to specifically encapsulate cargo proteins in vivo. Encapsulation is dependent on C-terminal targeting peptides (TPs). Here, we characterize and engineer TP-shell interactions in the Thermotoga maritima and Myxococcus xanthus encapsulin systems. Using force-field modeling and particle fluorescence measurements we show that TPs vary in native specificity and binding strength, and that TP-shell interactions are determined by hydrophobic and ionic interactions as well as TP flexibility. We design a set of TPs with a variety of predicted binding strengths and experimentally characterize these designs. This yields a set of TPs with novel binding characteristics representing a potentially useful toolbox for future nanoreactor engineering aimed at controlling cargo loading efficiency and the relative stoichiometry of multiple concurrently loaded cargo proteins.

A conserved π-helix plays a key role in thermoadaptation of catalysis in the glycoside hydrolase family 4.

Here, we characterize the role of a π-helix in the molecular mechanisms underlying thermoadaptation in the glycoside hydrolase family 4 (GH4). The interspersed π-helix present in a subgroup is evolutionarily related to a conserved α-helix in other orthologs by a single residue insertion/deletion event. The insertional residue, Phe407, in a hyperthermophilic α-glucuronidase, makes specific interactions across the inter-subunit interface. In order to establish the sequence-structure-stability implications of the π-helix, the wild-type and the deletion variant (Δ407) were characterized. The variant showed a significant lowering of melting temperature and optimum temperature for the highest activity. Crystal structures of the proteins show a transformation of the π-helix to a continuous α-helix in the variant, identical to that in orthologs lacking this insertion. Thermodynamic parameters were determined from stability curves representing the temperature dependence of unfolding free energy. Though the proteins display maximum stabilities at similar temperatures, a higher melting temperature in the wild-type is achieved by a combination of higher enthalpy and lower heat capacity of unfolding. Comparisons of the structural changes, and the activity and thermodynamic profiles allow us to infer that specific non-covalent interactions, and the existence of residual structure in the unfolded state, are crucial determinants of its thermostability. These features permit the enzyme to balance the preservation of structure at a higher temperature with the thermodynamic stability required for optimum catalysis.

Community Network Analysis of Allosteric Proteins.

Community network analysis (CNA) of correlated protein motions allows modeling of signals propagation in allosteric proteic systems. From standard classical molecular dynamics (MD) simulations, protein motions can be analysed by means of mutual information between pairs of amino acid residues, providing dynamical weighted networks that contains fundamental information of the communication among amino acids. The CNA method has been successfully applied to a variety of allosteric systems including an enzyme, a nuclear receptor and a bacterial adaptive immune system, providing characterization of the allosteric pathways. This method is complementary to network analyses based on different metrics and it is particularly powerful for studying large proteic systems, as it provides a coarse-grained view of the communication flows within large and complex networks.

Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities.

Enzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales. Using a continuous directed evolution platform called OrthoRep, we rapidly evolve the Thermotoga maritima tryptophan synthase ß-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only on TmTrpB's primary activity of synthesizing L-tryptophan from indole and L-serine. We find that the resulting sequence-diverse TmTrpB variants span a range of substrate profiles useful in industrial biocatalysis and suggest that the depth and scale of evolution that OrthoRep affords will be generally valuable in enzyme engineering and the evolution of biomolecular functions.

RNA and DNA Binding Epitopes of the Cold Shock Protein TmCsp from the Hyperthermophile Thermotoga maritima.

Prokaryotic cold shock proteins (CSPs) are considered to play an important role in the transcriptional and translational regulation of gene expression, possibly by acting as transcription anti-terminators and "RNA chaperones". They bind with high affinity to single-stranded nucleic acids. Here we report the binding epitope of TmCsp from Thermotoga maritima for both single-stranded DNA and RNA, using heteronuclear 2D NMR spectroscopy. At "physiological" growth temperatures of TmCsp (≥ 343 K), all oligonucleotides studied have dissociation constants between 1.6 ((dT)7) and 25.2 ((dA)7) µM as determined by tryptophan fluorescence quenching. Reduction of the temperature to 303 K leads to a pronounced increase of affinity for thymidylate (dT)7 and uridylate (rU)7 heptamers with dissociation constants of 4.0 and 10.8 nM, respectively, whereas the weak binding of TmCsp to cytidylate, adenylate, and guanylate heptamers (dC)7, (dA)7, and (dT)7 is almost unaffected by temperature. The change of affinities of TmCsp for (dT)7 and (rU)7 by approximately 3 orders of magnitude shows that it represents a cold chock sensor that switches on the cold shock reaction of the cell. A temperature dependent conformational switch of the protein is required for this action. The binding epitope on TmCsp for the ssDNA and RNA heptamers is very similar and comprises ß-strands 1 and 2, the loop ß1-ß2 as well as the loops connecting ß3 with ß4 and ß4 with ß5. Besides the loop regions, surprisingly, mainly the RNA-binding motif RNP1 is involved in ssDNA and RNA binding, while only two amino acids, H28 and W29, of the postulated RNA-binding motif RNP2 interact with the uridylate and thymidylate homonucleotides, although a high affinity in the nanomolar range is achieved. This is in contrast to the binding properties of other CSPs or cold shock domains, where RNP1 as well as RNP2 are involved in binding. TmCsp takes up a unique position since it is the only one which possesses a tryptophan residue instead of a usually highly conserved phenylalanine or tyrosine residue at the end of RNP2. NMR titrations suggest that neither (dT)7 nor (rU)7 represent the full binding motif and that non-optimal intercalation of W29 into these oligonucleotides blocks the access of the RNP2 site to the DNA or RNA. NMR-experiments with (dA)7 suggest an interaction of W29 with the adenine ring. Full binding seems to require at least one single purine base well-positioned within a thymine- or uracil-rich stretch of nucleic acids.

RNA-hydrolyzing activity of metallo-ß-lactamase IMP-1.

Metallo-ß-lactamases (MBLs) hydrolyze a wide range of ß-lactam antibiotics. While all MBLs share a common αß/ßα-fold, there are many other proteins with the same folding pattern that exhibit different enzymatic activities. These enzymes, together with MBLs, form the MBL superfamily. Thermotoga maritima tRNase Z, a tRNA 3' processing endoribonuclease of MBL-superfamily, and IMP-1, a clinically isolated MBL, showed a striking similarity in tertiary structure, despite low sequence homology. IMP-1 hydrolyzed both total cellular RNA and synthetic small unstructured RNAs. IMP-1 also hydrolyzed pre-tRNA, but its cleavage site was different from those of T. maritima tRNase Z and human tRNase Z long form, indicating a key difference in substrate recognition. Single-turnover kinetic assays suggested that substrate-binding affinity of T. maritima tRNase Z is much higher than that of IMP-1.

A suite of 19F based relaxation dispersion experiments to assess biomolecular motions.

Proteins and nucleic acids are highly dynamic bio-molecules that can populate a variety of conformational states. NMR relaxation dispersion (RD) methods are uniquely suited to quantify the associated kinetic and thermodynamic parameters. Here, we present a consistent suite of 19F-based CPMG, on-resonance R1ρ and off-resonance R1ρ RD experiments. We validate these experiments by studying the unfolding transition of a 7.5 kDa cold shock protein. Furthermore we show that the 19F RD experiments are applicable to very large molecular machines by quantifying dynamics in the 360 kDa half-proteasome. Our approach significantly extends the timescale of chemical exchange that can be studied with 19F RD, adds robustness to the extraction of exchange parameters and can determine the absolute chemical shifts of excited states. Importantly, due to the simplicity of 19F NMR spectra, it is possible to record complete datasets within hours on samples that are of very low costs. This makes the presented experiments ideally suited to complement static structural information from cryo-EM and X-ray crystallography with insights into functionally relevant motions.

Guanidinium binding to proteins: The intriguing effects on the D1 and D2 domains of Thermotoga maritima Arginine Binding Protein and a comprehensive analysis of the Protein Data Bank.

Thermotoga maritima Arginine Binding Protein has been extensively characterized because of its peculiar features and its possible use as a biosensor. In this characterization, deletion of the C-terminal helix to obtain the monomeric protein TmArgBP20-233 and dissection of the monomer in its two domains, D1 and D2, have been performed. In the present study the stability of these three forms against guanidinium chloride is investigated by means of circular dichroism and differential scanning calorimetry measurements. All three proteins show a high conformational stability; moreover, D1 shows an unusual behavior in the presence of low concentrations of guanidinium chloride. This finding has led us to investigate a possible binding interaction by means of isothermal titration calorimetry and X-ray crystallography; the results indicate that D1 is able to bind the guanidinium ion (GuH+), due to its similarity with the arginine terminal moiety. The analysis of the structural and dynamic properties of the D1-GuH+ complex indicates that the protein binds the ligand through multiple and diversified interactions. An exhaustive survey of the binding modes of GuH+ to proteins indicates that this is a rather common feature. These observations provide interesting insights into the effects that GuH+ is able to induce in protein structures.

Significance of the Protein Interface Configuration for Allostery in Imidazole Glycerol Phosphate Synthase.

Imidazole glycerol phosphate synthase (ImGPS) from Thermotoga maritima is a model enzyme for studying allostery. The ImGPS complex consists of the cyclase subunit HisF and the glutaminase subunit HisH whose activity is stimulated by substrate binding to HisF in a V-type manner. To investigate the significance of a putative closing hinge motion at the cyclase:glutaminase interface for HisH activity, we replaced residue W123 in HisH with the light-switchable unnatural amino acid phenylalanine-4'-azobenzene (AzoF). Crystal structure analysis employing angle, buried surface area, and distance measurements showed that incorporation of AzoF at this position causes a closing of the interface by ∼18 ± 3%. This slightly different interface configuration results in a much higher catalytic efficiency in unstimulated HisH due to an elevated turnover number. Moreover, the catalytic efficiency of HisH when stimulated by binding of a substrate to HisF was also significantly increased by AzoF incorporation. This was caused by a K-type stimulation that led to a decrease in the apparent dissociation constant for its substrate, glutamine. In addition, AzoF improved the apparent binding of a substrate analogue at the HisF active site. Remarkably, light-induced isomerization of AzoF considerably enhanced these effects. In conclusion, our findings confirm that signal transduction from HisF to HisH in ImGPS involves the closing of the cyclase:glutaminase subunit interface and that incorporation of AzoF at a hinge position reinforces this catalytically relevant conformational change.

The maturase HydF enables [FeFe] hydrogenase assembly via transient, cofactor-dependent interactions.

[FeFe] hydrogenases have attracted extensive attention in the field of renewable energy research because of their remarkable efficiency for H2 gas production. H2 formation is catalyzed by a biologically unique hexanuclear iron cofactor denoted the H-cluster. The assembly of this cofactor requires a dedicated maturation machinery including HydF, a multidomain [4Fe4S] cluster protein with GTPase activity. HydF is responsible for harboring and delivering a precatalyst to the apo-hydrogenase, but the details of this process are not well understood. Here, we utilize gas-phase electrophoretic macromolecule analysis to show that a HydF dimer forms a transient interaction complex with the hydrogenase and that the formation of this complex depends on the cofactor content on HydF. Moreover, Fourier transform infrared, electron paramagnetic resonance, and UV-visible spectroscopy studies of mutants of HydF show that the isolated iron-sulfur cluster domain retains the capacity for binding the precatalyst in a reversible fashion and is capable of activating apo-hydrogenase in in vitro assays. These results demonstrate the central role of the iron-sulfur cluster domain of HydF in the final stages of H-cluster assembly, i.e. in binding and delivering the precatalyst.

Capturing Substrate Translocation in an ABC Exporter at the Atomic Level.

ATP-binding cassette (ABC) transporters chemomechanically couple ATP binding and hydrolysis to large-scale conformational changes, ultimately leading to substrate translocation across biological membranes. Despite recent progress in the structure determination of substrate-bound ABC exporters, the inherently dynamic mechanism of substrate transport remains unclear at the atomic level. In this work, we capture substrate translocation in the heterodimeric ABC exporter TM287/288 from the hyperthermophilic bacterium Thermotoga maritima using all-atom molecular dynamics (MD) simulations. Unguided multimicrosecond simulations at 375 K show how the drugs daunorubicin and verapamil, which were initially docked into the ABC transporter, get translocated through the exporter by following its large-scale alternating access conformational transitions between an inward-facing (IF) and an outward-facing (OF) conformation. Triggered by the affinity difference due to differential solvation of the binding cavity in the IF and OF conformations, the substrates unbind from the OF transporter and partition into the lipid bilayer. While daunorubicin is stably inserted into the outer leaflet of the bilayer, verapamil dynamically flip flops between the bilayer leaflets, possibly rendering its net transport futile.

A ß-hairpin is a Minimal Latch that Supports Positive Supercoiling by Reverse Gyrase.

Reverse gyrase is a unique type I topoisomerase that catalyzes the introduction of positive supercoils into DNA in an ATP-dependent reaction. Supercoiling is the result of a functional cooperation of the N-terminal helicase domain with the C-terminal topoisomerase domain. The helicase domain is a nucleotide-dependent conformational switch that alternates between open and closed states with different affinities for single- and double-stranded DNA. The isolated helicase domain as well as full-length reverse gyrase can transiently unwind double-stranded regions in an ATP-dependent reaction. The latch region of reverse gyrase, an insertion into the helicase domain with little conservation in sequence and length, has been proposed to coordinate events in the helicase domain with strand passage by the topoisomerase domain. Latch deletions lead to a reduction in or complete loss of supercoiling activity. Here we show that the latch consists of two functional parts, a globular domain that is dispensable for DNA supercoiling and a ß-hairpin that connects the globular domain to the helicase domain and is required for supercoiling activity. The ß-hairpin thus constitutes a minimal latch that couples ATP-dependent processes in the helicase domain to DNA processing by the topoisomerase domain.

On the extraordinary pressure stability of the Thermotoga maritima arginine binding protein and its folded fragments - a high-pressure FTIR spectroscopy study.

The arginine binding protein from T. maritima (ArgBP) exhibits several distinctive biophysical and structural properties. Here we show that ArgBP is also endowed with a ramarkable pressure stability as it undergoes minor structural changes only, even at 10 kbar. A similar stability is also observed for its folded fragments (truncated monomer and individual domains). A survey of literature data on the pressure stability of proteins highlights the uncommon behavior of ArgBP.

Rational modulation of the enzymatic intermediates for tuning the phosphatase activity of histidine kinase HK853.

Histidine kinase (HK) of two-component signal transduction system (TCS) is a potential drug target for treating bacterial infections, and most HKs are bifunctional. We have previously identified the HXXXT motif of HK in HisKA subfamily to perform the phosphatase activity, but the specific working mechanism of the threonine is not well understood. In this paper, we use the phosphate group analog BeF3- to capture the enzymatic intermediates between HK853 and RR468 from Thermotoga maritima during dephosphorylation, and demonstrate that the T264 site is essential for populating capable near attack conformers (NAC) between enzyme and substrate to facilitate catalysis. Importantly, mutations at this site can modulate the phosphatase activity of HK. Our results help to understand the TCS signal transduction mechanisms and provide a reference for drug design.

Three-Dimensional Protein Cage Array Capable of Active Enzyme Capture and Artificial Chaperone Activity.

Development of protein cages for encapsulation of active enzyme cargoes and their subsequent arrangement into a controllable three-dimensional array is highly desirable. However, cargo capture is typically challenging because of difficulties in achieving reversible assembly/disassembly of protein cages in mild conditions. Herein we show that by using an unusual ferritin cage protein that undergoes triggerable assembly under mild conditions, we can achieve reversible filling with protein cargoes including an active enzyme. We demonstrate that these filled cages can be arrayed in three-dimensional crystal lattices and have an additional chaperone-like effect, increasing both thermostability and enzymatic activity of the encapsulated enzyme.

The ATPase mechanism of UvrA2 reveals the distinct roles of proximal and distal ATPase sites in nucleotide excision repair.

The UvrA2 dimer finds lesions in DNA and initiates nucleotide excision repair. Each UvrA monomer contains two essential ATPase sites: proximal (P) and distal (D). The manner whereby their activities enable UvrA2 damage sensing and response remains to be clarified. We report three key findings from the first pre-steady state kinetic analysis of each site. Absent DNA, a P2ATP-D2ADP species accumulates when the low-affinity proximal sites bind ATP and enable rapid ATP hydrolysis and phosphate release by the high-affinity distal sites, and ADP release limits catalytic turnover. Native DNA stimulates ATP hydrolysis by all four sites, causing UvrA2 to transition through a different species, P2ADP-D2ADP. Lesion-containing DNA changes the mechanism again, suppressing ATP hydrolysis by the proximal sites while distal sites cycle through hydrolysis and ADP release, to populate proximal ATP-bound species, P2ATP-Dempty and P2ATP-D2ATP. Thus, damaged and native DNA trigger distinct ATPase site activities, which could explain why UvrA2 forms stable complexes with UvrB on damaged DNA compared with weaker, more dynamic complexes on native DNA. Such specific coupling between the DNA substrate and the ATPase mechanism of each site provides new insights into how UvrA2 utilizes ATP for lesion search, recognition and repair.