The macro domain is an ADP-ribose (ADPR) binding module, which is considered to act as a sensor to recognize nicotinamide adenine dinucleotide (NAD) metabolites, including poly ADPR (PAR) and other small molecules. The recognition of macro domains with various ligands is important for a variety of biological functions involved in NAD metabolism, including DNA repair, chromatin remodeling, maintenance of genomic stability, and response to viral infection. Nevertheless, how the macro domain binds to moieties with such structural obstacles using a simple cleft remains a puzzle. We systematically investigated the Middle East respiratory syndrome-coronavirus (MERS-CoV) macro domain for its ligand selectivity and binding properties by structural and biophysical approaches. Of interest, NAD, which is considered not to interact with macro domains, was co-crystallized with the MERS-CoV macro domain. Further studies at physiological temperature revealed that NAD has similar binding ability with ADPR because of the accommodation of the thermal-tunable binding pocket. This study provides the biochemical and structural bases of the detailed ligand-binding mode of the MERS-CoV macro domain. In addition, our observation of enhanced binding affinity of the MERS-CoV macro domain to NAD at physiological temperature highlights the need for further study to reveal the biological functions. Meng-Hsuan Lin et al. investigate MERS-CoV macro domain binding selectivity with NAD and NAD metabolites under various conditions. At physiological temperature, NAD is observed to have enhanced binding affinity to the MERS-CoV macro domain, shedding light on a new possible role of the MERS-CoV macro domain in viral replication. The cellular pool of free NAD is balanced between synthesis and degradation. The metabolic product, nicotinic acid mononucleotide, can be recycled for NAD synthesis. Adenine nucleotides, including adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP), are the side products of the NAD metabolism pathway2. NAD-consuming enzymes such as poly-ADP-ribosyl polymerases, also called Diphtheria toxin-like ADP-ribosyltransferases (ARTDs), use NAD as a group donor. ARTDs produce PAR by polymerizing the ADPR moieties and releasing the nicotinamide groups from NAD. PAR, as one of NAD metabolites, constructs the backbone of ADP-ribosylation, a kind of reversible chemical modification into protein3, DNA4, and RNA5.Nicotinamide adenine dinucleotide (NAD), a dinucleotide consisting of adenine-ribose and nicotinamide-ribose joined by a pair of bridging phosphate groups, is a coenzyme found in all living cells9. However, activation of ADP-ribosylation by viral infection often resulted in apoptosis of the host cell, considered a host immune defense12. The first discovered antiviral ADP-ribosylation enzyme was ARTD13, which was found to bind viral RNA, inhibit viral replication, induce transcription of viral defensing interferons, activate proteasome degradation, and promote apoptosis16. Also, the antiviral effects of mono-ADP-ribosylation enzymes have been reported17. By transcriptome and protein interactome analyses, several mono-ADP-ribosyltransferase, diphtheria toxin-like enzymes  were also found to inhibit viral replication19. Positive feedback loops of the expression of these three ARTDs are activated by interferons (IFNs) during infection with the Venezuelan equine encephalitis virus (VEEV)20. Human sirtuins are NAD-dependent deacylases/mono-ADP ribosyltransferases. By siRNA knockdown and sirtuins inhibitors, all sirtuins were found to have broad-range antiviral properties for both DNA and RNA viruses21. In addition, ARTD11 was found upregulated after virus infection and thus promoted IFN-I antiviral response by mono-ADP-ribosylating ubiquitin E3 ligase22. So viruses might have developed mechanisms to regulate the ADP-ribosylation-related conflict between viral replication and host immunity.ADP-ribosylation has been suspected of having a dual pro-antiviral role in regulating the virulence of diverse viral families. Several viruses requiring ARTD activity for efficient replication have been discovered by using an ARTD1 inhibitor such as vaccinia virus, herpes simplex virus, JC virus, and porcine reproductive and respiratory syndrome virus24. The macro domain is highly evolutionarily conserved and has been found widely distributed throughout all kingdoms of life, including in some positive-strand RNA viruses   harbors a macro domain in its non-structural protein 3 inside the open reading frame 1a (ORF1a)32. Proteins in ORF1a are related to viral replication of MERS-CoV, and polymorphism in ORF1a is considered to influence cross-host transmission and evolution of this virus31. The MERS-CoV macro domain is considered to possess the enzyme activity of ADPR-1”-monophosphatase34, similar to several viral macro domains36. Some studies indicated that the viral macro domains could also catalyze ADPR modifications, including severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus 229E (HCoV229E), chikungunya virus (CHIKV), and VEEV39. Mutagenesis analyses of several viral macro domains suggested the role of viral defense toward host cellular immunity because the mutated macro domain reduced viral titers in cultured host cells42. In addition, the N1040A mutation of the nsp3 macro domain from SARS-CoV and HCoV229E showed increased sensitivity of antiviral IFN from host cells43. However, the critical piece of the puzzle for the molecular function of viral macro domains is still missing.As a human pathogen first identified in Saudi Arabia in 201245. The crystal structure of the MERS-CoV macro domain binding with ADPR was solved and showed an α/β globular structure with the hydrophobic cleft able to accommodate a ligand46. In the ligand binding site of MERS-CoV macro domain, conserved D20 made a critical recognition to the nitrogen of the adenine group of ADPR. G46, I47, S126, G128, I129, and F130 of MERS-CoV macro domain formed hydrogen bonds with the diphosphate groups of ADPR. Furthermore, A37, N38, K42, G44, A48, V93, and G95 formed hydrogen bonds with the distal ribose of ADPR. The snapshot of the ADPR-complex structure threw light on the interaction between the MERS-CoV macro domain and single free ADPR. However, how the MERS-CoV macro domain interacts with NAD metabolites and ADPR moieties buried inside PAR is still unclear. In addition, some viral macro domains possess RNA-binding ability47. The existence of NAD-capping RNA has been discovered in prokaryotes, yeast, and eukaryotes51. Although canonical capping modification of viral RNA involves methylation for efficient replication53, NAD-capping RNA may have potential in the virus shell.We have knowledge of macro domains interacting with NAD-derived metabolites, such as ADPR and PAR, from various speciesHere, we used a variety of structural tools and additional biophysical experiments to systematically probe the possible ligand binding mechanism of the MERS-CoV macro domain with NAD metabolites. The MERS-CoV macro domain demonstrated a ligand-binding mechanism by regulating the flexible loop motifs surrounding the ligand-binding site (loop β3-α2 and β6-α5). Furthermore, the binding behavior of the MERS-CoV macro domain was altered at physiological temperature especially when binding to NAD metabolites with larger chemical structure than ADPR, which suggests possible thermal activation for viral infection.To elucidate the impact of individual chemical moieties of ligand, differential scanning fluorometry (DSF), a fluorescence-based thermal shift assay, was first used to monitor the binding ability of the protein with various NAD metabolites, including NAD, ADPR, ATP, ADP, and AMP Fig. . The MER46, presenting an α/β globular structure with a hydrophobic cleft for ligand binding  and distinguished loop 2, β6-α5 (126SAGIF130)) of the ligand-binding sites tes Fig. . As expetes Fig. . NotablyTo unveil details about the selective modes of ligand recognition, we analyzed the crystal structures of a series of NAD metabolites bound to the MERS-CoV macro domain  experiments were monitored by the acquisition of 46. However, the bindings of other NAD metabolites showed less favorable enthalpy and opposite entropy contribution, resulting in a 30-fold to ≥100-fold decrease in the binding constant . We then checked the effect of continuous temperature change on protein by CD. The sigmoidal thermal denaturation profiles of the macro domain with various NAD metabolites reflected a cooperative protein unfolding pathway. In the presence of AMP, ADP, and ATP, the MERS-CoV macro domain showed a slight loss of CD signal before reaching the Tm value, which suggests a compact losing but still folding structure Fig. , whereasTo test the hypothesis, NMR CSP and ITC experiments performed at 298 K were re-executed at physiological human body temperature (308 K). The results showed almost no chemical shift perturbation of the macro domain with the addition of AMP or ADP, and a fast-exchange on the NMR timescale of the protein with ATP titration  and human body temperature (308 K). Various amounts of MERS-CoV macro domain (from 2.5 μM to 10 μM) were incubated with autoMARylated human ARTD10 catalytic domain (HsARTD10-CatD) at different temperature. The result showed that the mono-ADP-ribosylation (MAR) signals were decreased with the increase of the macro domain  activity of the chikungunya virus, Hepatitis E virus, and SARS-CoV-1 has been estimated beforeain Fig. A, B. Andain Fig. C, D. WhiThe ADPR ITC titration results between 298 and 308 K, conformational changes with less contribution of hydrogen bonding  at 308 K. Secondary structure near distinguished loop 1 (*), β3, and α2, presented high transverse relaxation (R2) values. In addition, the signal decrease of NOE on the residues of distinguished loops 1 and 2 in the apo form became more pronounced, and even fell into a negative level at higher temperature. Such a result indicated these regions were undergoing dynamic motions on the nanosecond to picosecond timescale and suitable for larger ligand entry. The NMR relaxation experiments indicated that the MERS-CoV macro domain became rigid upon ADPR binding at both temperatures. Moreover, R1, R2 values of the distinguished loops 1 and 2 of macro domain in complex with ADPR exhibited much lower values than that of the apo form at 308 K, expect R2 of G44. Remarkably, G44 of the ADPR-bound MERS-CoV macro domain presented an extremely high J(0) value  of G44 in the ADPR-complexed form at 308 K might reflect the deMAR enzyme activity, since there was a suspicion that the coordination of triple-glycines at the relative position of distinguished loop 1 of VEEV regulating the deMAR ability25.NMR relaxation parameters of the MERS-CoV macro domain in complex with ADPR or NAD were further measured at the magnetic field 600 MHz at 298 and 308 K  and the region1 (N154 and D157) compared with that of the ADPR complex and apo form. R2 values of the NAD complex showed many large variations. However, NOE values for the NAD complex showed lower variations than the apo form at distinguished loops 1 and 2. In general, the ligand-binding decreased the flexibility of protein; however, the binding of NAD leaded high flexibility, especially near distinguished loop 2 at 308 K. Indeed, this relaxation quality of NAD-bound form reflects what correlation between the function of the MERS-CoV macro domain still needs more study.Furthermore, we observed a striking scattering pattern of relaxation parameters for the macro domain upon NAD binding at 308 K. In addition, By using molecular dynamics (MD) simulation at 298 and 308 K, we calculated the root mean square fluctuation (RMSF) for each residue in the macro domain over the 100-ns MD trajectory for the overall flexibility of the systems  of the viral macro domain remains a mystery. A model for this arms race has been proposed: ARTDs might trigger antiviral effects by recognizing the ADP-ribosylated viral or host protein via the primate macro domain. However, the effects could be blocked by removing ADPR or competition by the viral macro domain57. A susceptive ADPR modification cleavage model of the macro domain from VEEV was previously reported25. The strong dynamic pattern found in NAD binding the MERS-CoV macro domain  cells were transformed with the plasmid of PET-28a (+) containing the inserted sequence. The cells were grown at 37 °C up to OD600 1.0 with antibiotic kanamycin 50 μg/ml. The recombinant MERS-CoV macro domain with an N-terminal His-tag was induced by the addition of 1 mM IPTG, then grown for 20 h at 16 °C. The recombinant protein was purified using a Ni-NTA column and eluted by elution buffer . After removing His-tag by using thrombin. The protein sample was further purified by gel filtration chromatography with a Superdex75 XK 16/60 column  in 20 mM Tris-HCl buffer (pH 7.0), 100 mM NaCl.Protein expression and purification of the MERS-CoV macro domain were as described previously67.The MERS-CoV macro domain (10 mg/mL) was mixed with various NAD metabolites in a molar ratio of 1:16. Protein crystallization trials were performed at 283 K by the sitting-drop vapor diffusion method with commercial crystallization screen kits, 96-well Intelli-plates (Art Robbins Instruments) and a Crystal Phoenix robot (Art Robbins Instruments). The crystals for data collection were grown for 1 week at 283 K with 1.6 mM sodium citrate acid, pH 7.5. Before data collection, crystals were soaked in mother liquor with cryoprotected 20% glycerol and were flash-frozen in liquid nitrogen at 100 K. The diffraction images were collected at 100 K by using a nitrogen gas stream of BL15A1, BL13B1, or BL13C1 beamlines at the National Synchrotron Radiation Research Center, Taiwan, and processed by using HKL2000 software68 in the PHENIX package69, with coordinates of the macro domain/ADPR structure from the PDB entry 5DUS46. The initial structure was refined with iterative cycles of simulated annealing, energy minimization, and manual rebuilding by using PHENIX refinement69 and COOT70. Molecular visualizations were generated with PyMOL71. Data collection and refinement statistics are summarized in Table The crystal structures of the NAD metabolite-bound MERS-CoV macro domain were solved by molecular replacement by using PhaserCD spectra were measured with 10 μM protein samples in buffer 20 mM phosphate buffer, pH 2.5–11.0, which was placed into a 1-mm pathlength cuvette and recorded on a JASCO J-810 spectrometer. The thermal transition of protein samples with the amount of 1 mM ADPR, NAD, ATP, ADP, and AMP were monitored at 208 nm from 10 to 95 °C at a scan rate of 1 °C/min. The melting temperature (Tm) was determined with the first derivative of the CD signal.Thermal shift assay with DSF involved the use of a CFX48 Real-Time PCR Detection System  and StepOne Real-Time PCR Detection System (Thermo Fisher). Twenty-five microliter of mixture containing 2.5 μL SYPRO Orange (Sigma-Aldrich), 10–200 μM protein sample, and at most 100-fold concentrations of NAD metabolites were mixed in 8-well PCR tube. For DSF titration assays, various concentrations of NAD metabolites were used. Fluorescent signals were measured from 10 to 95 °C . Data evaluation, Tm determination, and data fitting for dissociation constant (Kd) calculation were performed using Prism 8.n) and the dissociation constant (Kd) were derived by fitting with an independent binding model with the use of Launch NanoAnalyze v2.3.6.Binding of various NAD metabolites to the MERS-CoV macro domain was measured by ITC with the Nano Isothermal Titration Calorimeter (TA Instruments). Aliquots of 4 μL of 7.5 mM ATP, 12 mM ADP, or 15 mM AMP were titrated by injection into protein  in 20 mM Tris-HCl buffer, pH 7.0 with 100 mM NaCl at 25 °C (298 K) with 250 rpm stirring. For experiments at physiological temperature, aliquots of 4 μL of 1 mM ADPR, 2 mM NAD, 6 mM ATP, 8 mM ADP, or 6 mM AMP were titrated by injection into protein (0.2 mM in 0.98 mL) in 20 mM Tris-HCl buffer, pH 7.0 with 100 mM NaCl at 25 °C (298 K). Background heats from ligand to buffer titrations were subtracted. The stoichiometry of the binding  and remained stable. Upon completion of the two equilibration phases, followed by 100-ns production of MD simulations with time step 2 fs at constant pressure (1 atm) and target temperatures. The PME algorithm calculated the electrostatic interactions, and all bonds were constrained by using the LINCS algorithm. A cut-off distance used for long-range interactions was 1 nm for van der Waals and 1 nm for electrostatic interactions73. In this method, short-range interactions are calculated at each step of the simulation, whereas interactions at a longer distance are calculated only at each update of the non-bonded pair list and kept constant up to the next update. The reference temperature for coupling was controlled via the v-rescale coupling algorithm, and a the Parrinello–Rahman algorithm maintained a pressure of 1 atm. Convergence was checked by monitoring the RMSD deviation during the final 50 ns. Snapshots were collected every 2 ps and stored for analysis of MD simulations.The MERS-CoV macro domain crystal structure underwent MD simulations to analyze the thermal-induced conformational change of the ligand-binding pocket at 298–308 K with the use of GROMACS 5.1.32O/10% D2O. All NMR experiments were recorded at 298 or 308 K on a Bruker AVANCE 600 spectrometer equipped with 5 mm triple resonance cryoprobe and Z-gradient. The ligand titrations were collected until the saturation of chemical shift differences. The final protein: ADPR ratios at 298 and 308 K were 1:1.76 and 1:4.5, respectively. The final protein: NAD at 298 and 308 K were 1:2.3 and 1:5.6. The final ratios of protein: ATP at 298 and 308 K were 1:72 and 1:73. The final ratios protein: ADP at 298 and 308 K were 1:36 and 1:1.2. The final protein: AMP at 298 and 308 K were 1:80 and 1:2.4. Chemical shift differences between the backbone amide 1H and 15N resonances of the MERS-CoV macro domain in apo form versus bound form were calculated by the equation74 below.NMR protein samples were prepared in 25 mM phosphate buffer, pH 6.5 with 150 mM NaCl, in 90% H1H-15N HSQC, CBCA(CO)NH and HNCACB spectra with the software CARA75. The chemical shift details of the MERS-CoV macro domain saturatedly interacting with ADRP, NAD, ATP, ADP, and AMP at 298 and 308 K were deposited to biological magnetic resonance bank with BMRB codes as 50393, 50394, 50395, 50396, and 50397, respectively.Backbone assignment of ADPR complex the MERS-CoV macro domain involved using 2D 1H-15N HSQC spectra for 15N R1, 15N R2, and 1H-15N NOE measurements were acquired. R1 values were measured in a series of spectra with relaxation delays of 10, 600, 200, 70, 140, 400, 500, 1000, 300, and 800 ms. R2 measurements were taken with relaxation delays of 16.96, 84.8, 169.6, 0, 203.52, 50.88, 118.72, 152.64, 254.4, and 237.43 ms. For evaluating 1H-15N steady-state NOE values, two different datasets with and without an initial proton saturation were measured. The proton saturation period was 4 s. All NMR data were processed with the software Topsipin3.2 and analyzed by using Dynamicscenter2.5.1.All NMR relaxation experiments were recorded at 298 and 308 K on a Bruker AVANCE 600 MHz spectrometer. 2D 76. Fresh 10 mM ANS was prepared by dissolving in methanol and added to the protein solution to a final concentration of 40 μM. The protein solutions with a final concentration of 0.2 mM macro domain were prepared with or without 0.15 mM guanidine hydrochloride. A sample without protein and guanidine hydrochloride was a control. The excitation wavelength was 360 nm, and spectra were respectively recorded in a region of 400–600 nm by temperature 298 or 308 K.8-Anilinonaphthalene-1-sulfonic acid (ANS) is a fluorescence probe to detect the exposure of protein hydrophobic core+ in 20 mM Tris-HCl pH 8.0, 100 mM NaCl, and 0.5 mM DTT was incubated at 30 °C for 30 min. Varying concentration of MERS-CoV-MD (from 2.5 to 10 μM) was then added into the reaction followed by incubation at 25 or 35 °C for another 30 min. The reaction was terminated by adding 1% SDS and proteins were resolved in 12% acrylamide gel. The gel was transferred onto a PVDF membrane (Bio-Rad) and probed with an antibiotin polyclonal antibody (Bethyl Laboratories Inc.) (1/5000). For competition assays, the indicated concentration of NAD or ADPR (from 0.5 to 2 mM) was added into the reaction mixture as described above with 20 μM MERS-CoV-MD by incubation at 25 or 35 °C for 30 min. The reaction was terminated by adding 1% SDS and immunoblotted with an anti-biotin polyclonal antibody.To perform mono-ADP-ribosylation (MAR), reaction mixture containing 10 μM hARTD10-CatD and 100 μM Biotin-NADFor DSF thermal shift assay, deMARylation activity assays, and isothermal titrations, the experiments were performed at least three times.Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Streptococcus pneumoniae TrpA, which is the α subunit of tryptophan synthase, reveals 3D domain swapping, which has never before been observed for a TIM-barrel protein.The structure of  trpA and trpB genes, that function as an αββα complex. Structural and functional features of tryptophan synthase have been extensively studied, explaining the roles of individual residues in the two active sites in catalysis and allosteric regulation. TrpA serves as a model for protein-folding studies. In 1969, Jackson and Yanofsky observed that the typically monomeric TrpA forms a small population of dimers. Dimerization was postulated to take place through an exchange of structural elements of the monomeric chains, a phenomenon later termed 3D domain swapping. The structural details of the TrpA dimer have remained unknown. Here, the crystal structure of the Streptococcus pneumoniae TrpA homodimer is reported, demonstrating 3D domain swapping in a TIM-barrel fold for the first time. The N-terminal domain comprising the H0–S1–H1–S2 elements is exchanged, while the hinge region corresponds to loop L2 linking strand S2 to helix H2′. The structural elements S2 and L2 carry the catalytic residues Glu52 and Asp63. As the S2 element is part of the swapped domain, the architecture of the catalytic apparatus in the dimer is recreated from two protein chains. The homodimer interface overlaps with the α–β interface of the tryptophan synthase αββα heterotetramer, suggesting that the 3D domain-swapped dimer cannot form a complex with the β subunit. In the crystal, the dimers assemble into a decamer comprising two pentameric rings.Tryptophan synthase catalyzes the last two steps of tryptophan biosynthesis in plants, fungi and bacteria. It consists of two protein chains, designated α and β, encoded by  The enzyme is composed of two catalytic units, TrpA (α subunit) and TrpB (β subunit), that associate into a functional heterotetrameric αββα hydrolyase complex (TrpAB) glycerol 3-phosphate (IGP) to indole and d-glyceraldehyde 3-phosphate, while the β subunit condenses indole with l-serine to give l-tryptophan, with pyridoxal 5′-phosphate (PLP) functioning as a cofactor  studied in the present work is a 258-residue protein which in its monomeric form assumes the canonical (β/α)8 TIM-barrel fold with three additional helices  and C-terminal (189–268) fragments can be isolated independently and that mixing the two restores the activity of the α subunit. This is consistent with observations that TIM-barrel enzymes subjected to fragment complementation and circular permutation tolerate such modifications, and further confirms that this fold is composed of two separate N- and C-terminal modules  could be converted to a dimeric form in vitro by treating the monomer with urea and refolding. Dimers were also observed for some missense and nonsense mutants and peptide complementation  of tryptophan synthase with the following primers in the reaction mixture: 5′-TACTTCCAATCCAATGCCATGCCTAAGACACTAACAGAAAAATTAAATGCTATTAAA-3′ and 5′-TTATCCACTTCCAATGTTATTTTTGGTAAGCTACTGCTTGTCTGATAAAATC-3′, as designed using an online tool  for ligation-independent cloning  cells were transformed with the amplified DNA/pMCSG68 mixture and were grown in the presence of ampicillin (150 µg ml−1) and kanamycin (25 µg ml−1). A single colony of each transformation was picked, grown and induced with isopropyl β-d-1-thiogalactopyranoside (IPTG). The construct was tested in small-scale expression and the sequence was verified.Gene cloning was performed according to Kim  al. 2011. BrieflySpTrpA protein, starter cultures were grown overnight at 37°C and 200 rev min−1 in LB medium supplemented with 100 µg ml−1 ampicillin, 30 µg ml−1 kanamycin and 40 mM K2HPO4. The starter culture was used to inoculate 1 l enriched M9 medium for large-scale production of SeMet-labeled protein. The bacterial culture was grown at 37°C and 190 rev min−1 until it reached an OD600 of 1.0. After air-cooling to 4°C for 60 min, inhibitory amino acids  and 90 mg l−1 SeMet (Medicilon) were added. Protein expression was induced by 0.5 mM IPTG. The cells were incubated overnight at 18°C and harvested. Approximately 8 g of cell pellet was resuspended in lysis buffer  and stored at −80°C. SpTrpA was purified using the procedure described previously  and the His6-tagged protein was released with elution buffer . This was followed by buffer exchange on a Sephadex G-25 Fine XK 26/20  desalting column equilibrated with buffer consisting of 20 mM Tris–HCl pH 7.5, 500 mM NaCl, 2 mM DTT. All of these steps were performed using an ÄKTAxpress chromatographic system . The fusion tag was removed by treatment with recombinant His7-tagged Tobacco etch virus (TEV) protease. Nickel-affinity chromatography was used to remove the His6 tag, uncut SpTrpA protein and His7-tagged TEV protease . The protein concentration was determined spectrophotometrically at 280 nm with a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific) using a calculated extinction coefficient of 8940 M−1 cm−1.To express selenomethionine (SeMet)-labeled 2.2.SpTrpA was subjected to an additional purification step via SEC on a Superdex 200 HiLoad 26/60 column  in crystallization buffer A directly following the second affinity-chromatography step. The sample was used for analytical experiments and follow-up crystallization trials. The column was calibrated with the following molecular-weight standards: ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), carbonic anhydrase (29 kDa) and aprotinin (6.5 kDa).A batch of 2.3.SpTrpA protein (46.6 mg ml−1) purified by affinity chromatography was screened for crystallization conditions using a sitting-drop vapor-diffusion setup at 4 and 24°C in CrystalQuick 96-well round-bottom plates (Greiner Bio-One). A 400 nl droplet of protein solution was mixed with 400 nl crystallization reagent and equilibrated over 135 µl crystallization reagent. The protein was screened with the MCSG1–3 screens (Anatrace). Nanopipetting was performed using a Mosquito nanolitre liquid-handling system (TTP Labtech). The plates were incubated in a RoboIncubator automated plate-storage system equipped with a Minstrel III automated crystal visualization system (Rigaku). The best crystals were obtained at 4°C in 31.7 mM sodium acetate, 5.56 mM Tris–HCl pH 8.5, 25% PEG 4K, 15% glycerol.The SeMet 2.4.XDS package  data were collected near the selenium edge on Structural Biology Center beamline 19-ID at the Advanced Photon Source (APS). 180 frames were collected with 1° oscillation and 2 s exposure per frame. Data processing was carried out in the lson 1978 and were2.5.SpTrpA was solved at 2.54 Å resolution in space group P21 by SAD phasing. The data collected near the selenium absorption peak energy (λ = 0.9793 Å) were fed into the CRANK2 pipeline . Only the first 26 sites were considered for phasing, as the subsequent sites had occupancies below 0.15. An initial atomic model of SpTrpA was built using the CCP4 implementation of Buccaneer and was refined using REFMAC5, with additional manual corrections in Coot  was used for structure visualizations and analysis.The structure was refined with uber 1991 was used3.3.1.EcTrpA dimers and mutational studies of StTrpA suggested that the TrpA protein might form stable dimers in some bacterial species . The monomer and dimer fractions could also be separated on native PAGE, with one protein band observed for the monomer and two protein bands (monomer and dimer) observed for the dimer fraction . This suggested that the homodimers generated during the expression of the α subunit in E. coli represent an abundant form. Purified monomer equilibrated into a monomer–dimer mixture and vice versa: purified dimer equilibrated into a similar mixture. Incubation of the monomer or the dimer with 1 M urea resulted in the formation of monomers only .The reports by Jackson and Yanofsky on the observation of P21 with ten chains of SpTrpA in the asymmetric unit. The structure of SpTrpA was solved by single-wavelength anomalous X-ray diffraction (SAD) phasing and refined to 2.54 Å resolution  could also be traced.The dimeric fraction of the monomer–dimer mixture was crystallized in the monoclinic space group n Table 1. A nearl3.2.2 dimers of SpTrpA formed via 3D domain swapping  forming the internal β-barrel, which are separated and flanked on the outside of the barrel by helices H1–H8 from the β–α–β motifs . Helix H2′ is not part of the barrel but is located on top of it and leads directly to the domain-swapping hinge . The β–α–β motif involving strands S3 and S4 is formed with the participation of two helices (H3′–H3). The progression of the β–α–β motifs is preceded by the N-terminal helix H0 located at the bottom of the TIM barrel and is followed by a tandem of helices, H8′–H8, of which helix H8′ is again located at the top of the barrel. In addition, SpTrpA has a small insertion in loop L1 that appears to render the 3D domain-swapped dimers more stable . This feature is only present in TrpA sequences from Streptococcus, Lactoccocus and Floricoccus species.The asymmetric unit comprises five αg Figs. 1 and 2 ▸.α atoms, which vary from 0.43 Å (for superposition of AB with CD) to 1.61 Å (GH with IJ) (Supplementary Table S1). The molecules assemble into a doughnut-like decamer or a pentamer of dimers  as described below.The five dimers in the asymmetric unit have a similar structure Fig. 2, as illu3.3.A) and residues Pro60–Lys258 from the other chain (B) of the dimer, has a very similar fold to the α subunit from the αββα assembly of the TrpAB structure  . The r.m.s.d. for Cα superposition of the TIM barrel of SpTrpA formed by one half of the CD dimer  with the α-subunit chain A of the SpTrpAB complex is 0.45 Å. As is usually the case, domain swapping does not change the overall fold of the folding unit (the TIM barrel in this case). The only visible change is in the conformation of the short linker (Ile55–Asp64) connecting the swapped domains. The active site in each TIM barrel is restored and two α chains now contribute residues to each active site in the dimer . Additionally, in the StTrpAB structure  in ligand binding. We can only speculate that it could either move towards the active site and increase the affinity for the phosphate moiety of a ligand, or it could remain disordered when the ligand is present in the active site, leaving the active site fully open. The active site of SpTrpA is also very similar to that of the Mycobacterium tuberculosis TrpAB (MtTrpAB) structure , which consists of residues Mse1–Asp59 from one chain (for example B) Fig. 2b. The rer Fig. 3. In factel Fig. 3. Based o3.4.et al., 1962et al., 1998et al., 1994et al., 2004http://caps.ncbs.res.in/3dswap/; Shameer et al., 20111pym; Huang et al., 19991l6w; Thorell et al., 2002SpTrpA that this kind of structural rearrangement is indeed possible. Moreover, many of the previous examples with accidental TIM-barrel involvement in oligomerization are not bona fide 3D domain-swapping cases because the monomeric form does not exist. In the domain-swapping of SpTrpA, the N-terminal structural elements (N)–H0–S1–H1–S2 (with the sequence Mse1–Asp59) are contributed by the complementary subunit . Despite the domain swap, the TIM-barrel topology is fully preserved and is identical to that found in monomeric SpTrpA , satisfying the strictest definition of 3D domain swapping. The linker or hinge region comprises the mostly hydrophobic Ile55–Gly64 decapeptide. In the quaternary structure of SpTrpA, the two linker chains form an antiparallel structure, which is the new open interface of the dimer  leaves no doubt that 3D domain swapping has occurred. Surprisingly, at variance with other similar cases, for example the 3D domain-swapped dimers of human cystatin C  and in general correspond to an extended conformation, which is however quite distant from the typical β structure . The two C-terminal residues of the linkers are no longer classified in the Ramachandran β region. Asp63 with its negative ψ angle is found in the α conformation. Gly64 with its positive φ angle lies in a disallowed Ramachandran region that is however possible for glycine residues.3D domain swapping is a mechanism of protein oligomerization in which two (or more) protein chains mutually exchange structural elements in a circular or linear runaway fashion. In the simplest case, two monomers exchange a fragment of their structures. This fragment may consist of a single secondary-structure element, for example an α-helix, or a globular domain. If the exchange is reciprocal between two protomers, symmetric dimers are formed. Domain swapping was first proposed for, and was later observed in, RNase A (Crestfield The TrpA chains in the domain-swapped dimers are (pseudo)twofold-symmetric, as illustrated by the ∼180° rotation required to superpose the subunits, which is practically the same for all of the dimers. The local dyads pass between the two linker chains of the open interface, close to the Asp59–Pro60 peptides.Supplementary Fig. S4). Frequently, proline residues are found in the hinge region, suggesting their possible importance, as prolines can create strain and thereby favor domain swapping  that are quite conserved among TrpA homologs . Pro60 is located in the Ramachandran α region. Interestingly, it has been shown that a cis-prolyl peptide-bond isomerization at this position dominates the folding of the α subunit of tryptophan synthase . The S2–H2′ linker is quite long, and in the structure of the SpTrpA monomer (PDB entry 5kin) has a manifold structure with three sharp kinks: first moving away from helix H2, then turning ‘down’ and finally turning again to join helix H2′ . In its domain-swapped (open) conformation the linker runs straight ahead to reach helix H2′ in the complementary folding unit directly. As illustrated in Supplementary Fig. S6, in the hypothetical situation of an absent helix H2′ the switch from β S2 to helix H2 would require the two linkers to cross, a maneuver preventing 3D domain swapping . A comparison of a ‘simple’ 3D domain-swapping event with a more convoluted event (as in TrpA), in which the hinge has to undergo a significant conformational change in order to reach the complementary folding unit and avoid clashes with its own copy, is illustrated in Supplementary Fig. S6.It has long been recognized that hinge elements that are too short, and also those that are too long, may be an obstacle to productive 3D domain swapping. This might be the reason why the robust TIM-barrel fold is resistant to oligomerization via 3D domain swapping; the tight loops of the β–α–β crossovers may be too short to connect two barrels without steric clashes, while the extended loops at the business side of the barrel may be too long. The presence of the surface-protrusion helix H2′ in the N-terminal part of the TrpA fold is a topological novelty that makes the S2–H2′ loop a suitable hinge for 3D domain swapping  exists in the C-terminal part of the SpTrpA for 3D domain swapping? It appears that the N- and C-terminal regions of TrpA have rather different properties. Folding studies of StTrpA indicated that the very N-terminal region of the protein corresponding to the H0–S1–H1–S2 secondary-structure elements folds rapidly and is tightly packed. In contrast, the C-terminus appears to behave as a molten globule whose folding is strongly coupled to that of the H0–S1–H1–S2 domain. In asymmetric deletion experiments, when the N-terminal fragment terminates at residue 96, the C-terminal fragments tend to start before residue 73. Similarly, when the C-terminal fragment starts at residues 57 or 73, all complementary N-terminal fragments terminate after residue 68 , other mutants can support growth, including substitutions at positions 18, 48 and 66. These results are also consistent with earlier EcTrpA peptide and fragment complementation and circular-permutation results  at the top of this aggregate, whereas the pentamer created by chains B, D, F, H and J forms the bottom ring, with an ∼36° rotation relative to the top pentamer. It is possible to superpose the two rings with a 180° rotation around a twofold axis (i.e. one of the dyads of the domain-swapped dimers) perpendicular to the main fivefold axis of the doughnut . According to the PISA server  conformation. The interface areas between pairs of adjacent chains from the two pentamers not involved in 3D domain swapping vary from 516 Å2 (pair F–G) to 1285 Å2 (pair A–J). The interface area between consecutive chains within one pentamer is smaller and varies from 98 Å2 (D–F) to 469 Å2 (B–D) .The asymmetric part of the unit cell comprises a doughnut-like decamer or pentamer of dimers (D) Fig. 4b.1l6w). In this case, however, the 3D domain-swapping pattern is circular, connecting the protomers of the pentameric rings, while in SpTrpA 3D domain swapping links subunits from both rings.It is interesting to note that FSA, which is a TIM barrel with a C-terminal helix swap, also forms a decameric assembly composed of a stack of two pentameric rings  around the decameric structure are inclined at ∼90° to the principal fivefold axis of the decamer. Despite these interesting geometrical features, we believe that in the light of the small decamer-specific interface areas (as compared with the surface buried on 3D domain-swapping dimerization), the observed SpTrpA decamer is likely to be an artifact of crystallization.The main axis of the decameric doughnut is inclined at ∼43.5° to the crystallographic 2l-tryptophan biosynthesis? Analysis of TrpA genes suggests that only a fraction of TrpA proteins may have a strong propensity to form homodimers. If the presence of the L1 insertion is a reliable indicator, 3D domain swapping may be prominent in TrpA from Gram-positive cocci strains, although mutagenesis experiments with StTrpA showed that dimers can also form in the absence of L1 loop insertion and that domain-swapped dimers should be more stable because of a larger interaction surface.Are the dimerization properties of TrpA of functional significance, or is this just a transitional byproduct of some mutation(s) with little impact on 4.S. pneumoniae TrpA, the α subunit of tryptophan synthase, a key enzyme in tryptophan biosynthesis, and have discovered that it exists in solution and in the crystal as a very unusual dimer formed by swapping the N-terminal H0–S1–H1–S2 portion of the TIM-barrel core. Previous folding studies (Wintrode et al., 20052 dimer is recreated from two protein chains. In solution the α2 dimer can be separated from the monomer by SEC, but it is in equilibrium with the monomer, which is then available for interaction with the β subunit to form the functional tryptophan synthase αββα complex. In the crystal, the 3D domain-swapped α2 dimers assemble into a decamer comprising two superposed pentameric rings. Our work unequivocally demonstrates that TIM-barrel proteins are also capable of exchanging portions of their core structure to form stable dimers and perhaps even higher-order oligomers. The dimerization may increase the enzyme stability and serve as a reservoir of α subunits and as an alternative source of indole from IGP for metabolic pathways that have still to be discovered.We have determined the crystal structure of domain-swapped TrpA, 6qkyPDB reference: 10.1107/S2059798320000212/wa5122sup1.pdfSupplementary Tables and Figures. DOI:


The continuous fine dynamic recrystallization (CDRX) grains occurred in the optimum deformation zone. The predicted instability domains was identified as T = 200–300 °C, −2–1 s−1, which corresponded to the microstructure of deformation twinning and micro cracks at the intersection of grain boundaries.In this work, the Mg-3Sn-2Al-1Zn  alloy with excellent high temperature resistance was compressed using a Gleeble-3500 thermo-mechanical simulator at a wide temperature and the strain rate range. The kinetics analyses showed that the dominant deformation mechanism was likely caused by the cross slipping of dislocations. A constitutive equation which expressed the relationship between the flow stress, deformation temperature, and strain rate was established, and the average activation energy Q was calculated to be 172.1 kJ/mol. In order to delineate the stability and instability working domains, as well as obtain the optimum hot working parameters of the alloy, the hot processing maps in accordance with Prassad’s criterion are constructed at the true strain of 0.2, 0.4, 0.6, and 0.8, respectively. Based on the hot processing map and microstructure observation, the optimum hot working parameter was determined to be 350 °C/1 s Achanisms , but alschanisms . HoweverCompared with the Mg-3Sn-1Ca alloy adopting Ca, the minor addition of Al or Zn as the solid solution strengthening element could enhance the strength , age har6 + 99.5 vol. % CO2). The melt was held at 720 °C for 20 min and mechanically stirred for 2 min to ensure a homogeneous composition, then the pouring was accomplished into a steel die preheated up to 150 °C. After casting, the cast billets were homogenized at 400 °C for 24 h. The actual composition of as-cast and as-homogenized TAZ321 alloy measured using energy dispersive X-ray spectroscopy  is listed in −3–1 s−1. Mo plates covered with boron nitride powder were used as lubrication for minimizing the effect of friction. Before deformed, the samples were heated at a heating rate of 5 K/s, and followed by a holding time of 10 min for ensuring there were uniform temperature throughout the samples. In order to freeze the microstructure, all post-deformation samples were cooled by water-quench up to a true strain of 0.8. The microstructural examinations specimens were sliced in the center parallel to the compression axis.The alloy ingots with the nominal composition of Mg-3Sn-2Al-1Zn (wt. %) were prepared using an electric furnace with a covering flux under a protective atmosphere  X-ray diffraction (XRD) diffractometer with Cu radiation. The microstructure of the as-homogenized and post-deformation samples were examined using an AxioImager A2m  optical microscope (OM), a MALA3 Triglay  scanning electron microscope (SEM) equipped with EDS, and a 2100 F  transmission electron microscope (TEM). Additionally, the electron backscatter diffraction (EBSD) micro orientation analysis of the as-homogenized and post-deformation samples was conducted using an Oxford Instruments-NordlysNano EBSD detector  equipped on the SEM operating at 20 kV. The samples prepared for EBSD analysis were additionally developed by ion etching in a Leica RES101 ion etching apparatus  after mechanical polishing. The orientation imaging microscopy software used for post-EBSD data analysis was provided by HKL Channel 5.11.20405.0, Oxford Instruments NanoAnalysis, Abingdon, UK.2Sn phases can also be observed. Based on the SEM and EDS results (2Sn phases distribute homogeneously into the α-Mg matrix. An EBSD inverse pole map (IPF)  c and graap (IPF) d show thThe true stress-strain curves of the homogenized TAZ321 alloy under different temperature and strain rates at a true strain of 0.8 are indicated in 2Sn particles, as illustrated in Furthermore, it was interestingly found that obvious serration behaviors can be observed in the true stress-strain curves. The type of serrations was associated with the deformation temperature and strain rate, as well as the extent of deformation, which can be attributed to the interaction effect between the dislocation and precipitation and/or solute atoms, i.e., the dynamic strain aging (DSA) effect ,14. It i1, A2, n1, and β are the material constants. −1), and R is the gas constant (8.314 J mol−1 K−1). Taking natural logarithm simultaneously on both sides of Equations (1)–(3), the following equations were obtained, namely:For the metal materials, there are three different constitutive models  can be used to express the relationship among the strain rate (ectively :(1)ε˙=A1In general, the power law was adopted for the low stress level , the activation energy Q associated with diffusion and the precipitation of particles could be conveyed as:2Sn particles, which can provide back stress to hinder the movement of dislocation, resulting in higher Q values.The value of S was equal to the slope of the plots of 2 = 0.9892) was almost 1, validating that the hyperbolic sine model matched well with the experiment data. Finally, substituting the corresponding parameters into the Equation (1), the constitutive equation of the homogenized TAZ321 alloy was obtained as follows:Moreover, the corresponding value of A and n could be determined as Materials engineers usually get the optimum working parameters of the alloys by utilizing the hot processing map on the basis of the dynamic materials model (DMM), i.e., Prassad’s criterion . It was The hot processing maps were overlapped by the power dissipation map and instability map based on the DMM model, and the overall total power P was considered as the sum of G and J. G is the power dissipation value caused by plastic deformation, and J is power dissipation related to the microstructural evolution, such as during phase transformation, DRV, and DRX. The allocation of P for the G and J was determined by a strain rate sensitivity parameter m, which can be expressed by the formula as :(9)m = ∂In addition, for a non-linear power dissipater, the capacity of power dissipation was expressed by means of a power dissipation (η) equation given by :(10)η=2mion (11) :(11)ξ signifies the occurrence of continuous DRX (CDRX) in the alloy. Furthermore, the subgrain evolution can be evaluated by the changes of local misorientaion (point to point) and cumulative misorientaion (point to origin) [For the DRX, it is commonly believed that discontinuous DRX (DDRX) and continuous DRX (CDRX) are the main two mechanisms. Bulging original grain boundaries are the domain nucleation sites of DDRX. Meanwhile, the CDRX mechanism is characterized as the continuous absorption of dislocations in low-angle grain boundaries (subgrain boundaries) and followed by the progressive rotation of subgrains, which leads to the formation of high-angle grain boundaries (HAGBs) ,6. Figur origin) . Figure −2–1 s−1. −2 s−1 and 250 °C/10−1 s−1 at a true strain of 0.8, respectively, corresponding to the microstructure of instability domains in the processing map. The microstructure of the samples deformed at the instability regions was characterized by deformation twining at the interior of grains and micro cracks along the intersection of grain boundaries, which were considered to be induced by flow localization. This was because deformation twining can induce local stress concentration and micro cracks can lead directly to fracture, which should be avoided during hot deformation [The instability regions predicted by the processing map are mainly located at the median temperature regions, which were identified as T = 200–300 °C, (1)2Sn particles.The flow curves exhibited initially increase to a peak value and then decrease and finally reach a steady state. The serrations morphology of the flow curves is indicative for type B for the TAZ321 alloy, which was attributed to the interaction between the dislocations and the dynamic precipitation of Mg(2)The relationship between the flow stress, deformation temperature, and strain rate of homogenized TAZ321 alloy could be described by the constitutive equation as follows: (3)−1–1 s−1), and the other was denoted as domain B (T = 310–350 °C and −3–10−2 s−1). The optimum hot working domains were determined as being part of domain A (T = 310–350 °C and −1–1 s−1) in combination with the hot processing map and microstructure observation, which corresponded to the microstructure of fine CDRX grains.According to the processing map, there were two stability domains with relatively high power dissipation efficiency: one was representing domain A −2–1 s−1. Deformation twinning or micro-cracks at the intersection of grain boundaries were observed in the instability domains.The instability regions predicted by the processing map were identified as T = 200–300 °C, (5)−1 exhibited more a homogeneous and finer grain size, and therefore were viewed strongly as a candidate for structural application.The TAZ321 alloy deformed at 350 °C/1 s


Folding of individual domains in large proteins during translation helps to avoid otherwise prevalent inter-domain misfolding. How folding intermediates observed in vitro for the majority of proteins relate to co-translational folding remains unclear. Combining in vivo and single-molecule experiments, we followed the co-translational folding of the G-domain, encompassing the first 293 amino acids of elongation factor G. Surprisingly, the domain remains unfolded until it is fully synthesized, without collapsing into molten globule-like states or forming stable intermediates. Upon fully emerging from the ribosome, the G-domain transitions to its stable native structure via folding intermediates. Our results suggest a strictly sequential folding pathway initiating from the C-terminus. Folding and synthesis thus proceed in opposite directions. The folding mechanism is likely imposed by the final structure and might have evolved to ensure efficient, timely folding of a highly abundant and essential protein. In vivo experiments and optical tweezers force-spectroscopy measurements assessing the co-translational folding of the G-domain from bacterial elongation factor G reveal a sequential folding pathway initiating from the C-terminus. These results suggest that protein folding and synthesis proceed in opposite directions. However, experimental and computational studies have detected partially folded intermediates for a multitude of proteins, and proteins larger than ~100 amino acids are generally thought to populate folding intermediates before reaching their native structure3.How proteins fold into the native structures that enable their cellular functions remains a central question in biology. Folding of small proteins or single domains often follows single-exponential kinetics, suggesting a highly cooperative process4. Distinguishing these two scenarios experimentally is often challenging, although single-molecule approaches have demonstrated their potential to provide this information6.While the presence of intermediates in most protein folding pathways is universally recognized, their functional roles are less clear. Partially structured states can represent on-pathway intermediates along a multistep folding pathway, or off-pathway misfolded states that must dissolve before productive folding can be achieved8. The foldon hypothesis posits that small, cooperative units acquire structure in a strictly sequential order, resulting in a single folding pathway9. Considerations based on energy landscape theory propose that a folding protein can reach its native structure through several accessible routes10, although specific pathways may be energetically favored. Either theory is supported by evidence from both simulations and experiments. It thus seems possible that proteins might fall into distinct categories based on their folding mechanism. Determining the relationship between amino acid sequence, folding pathway and final structure is important for designing sequences that can adopt novel structures and for understanding how natural proteins robustly reach their functional conformations.How intermediates are linked together into a folding pathway remains a subject of debate11. The vast majority of mechanistic folding studies has been carried out with isolated proteins or domains12. In the cell, proteins begin to fold while they gradually emerge from the ribosome as it translates the information in the messenger RNA into a polypeptide sequence. Biophysical experiments have shown that interactions of the ribosome with the proximal part of the nascent polypeptide can reduce the stability of native domains14, stabilize secondary structure inside the ribosome exit tunnel17, modulate nascent chain folding kinetics18, and prevent misfolding20. Co-translational folding is particularly important for large multi-domain proteins, because it prevents the accumulation of extended unfolded regions during synthesis that have a high propensity for misfolding21. Several examples show that the ribosome does not necessarily change the folding pathway23, but the connection between vectorial emergence and folding of the nascent chain remains poorly defined.Folding pathways are not only shaped by protein-specific properties, but also depend strongly on environmental conditions, many of which differ greatly between in vitro refolding experiments and folding in living cellsEscherichia coli elongation factor G (EF-G). Following nascent chain folding in bacterial cells with an arrest peptide-based reporter assay24, we find that productive folding is not initiated until the full domain has emerged from the ribosome. Force-spectroscopy experiments with optical tweezers confirmed the absence of stable structure at shorter chain lengths. These single-molecule measurements also showed that folding of the full domain proceeds through productive folding intermediates both on the ribosome and in isolation. Our studies thus reveal a strictly ordered folding pathway in which the first step requires the extreme C-terminus of the domain. Folding and synthesis of the domain therefore proceed in opposite directions.Here, we have investigated folding of the N-terminal G-domain of E. coli SecM protein25 (termed SecM17 here). While the residue at position 17 is important for arrest function, it is not incorporated into the nascent polypeptide, and SecM17 causes elongation arrest when its N-terminal 16 amino acids have been synthesized26. Folding-mediated release of elongation arrest results in translation of the coding sequence downstream of SecM17 and production of the full-length encoded protein24. The arrest peptide can thus be utilized to detect nascent chain folding that occurs in close proximity to the ribosome . At this length, offset from the maximum of the peak by 23 amino acids , unfolding of the native G-domain results in a characteristic transition  that exposes only a few EF-G residues outside the exit tunnel does not exhibit any transitions in these experiments. Surprisingly, however, substantially longer nascent chains (252RNC and 316RNC) do not exhibit defined unfolding transitions, either , which manifests as a large peak in the SecM reporter assay  might represent secondary structure formations, especially α-helices, which have previously been shown to accelerate SecM arrest release40. The identity of nascent chain sequences inside the ribosome exit tunnel has been described to also affect arrest release32. Interactions of nascent chain residues with the ribosome could prevent formation of the SecM secondary structure that is required to cause stalling and could thus in principle account for the observed signal. Regardless of their origin, the amplitudes of these fluctuations are small compared to the main peak, suggesting the absence of stably folded structures until the G-domain is fully synthesized.Consistent with previous resultssay Fig. . NonrandL = 316  or collapsed, molten globule-like structures . The contact order43 of the G-domain is moderately low (relative contact order: 0.07), and its overall hydrophobicity44 is similar to that of other proteins  in the endoplasmic reticulum is delayed by the formation of intermediates that are stabilized by non-native disulfide bonds, which slowly rearrange into the native configuration49. Thus, the protein completes its folding post-translationally. LDL-R folds in the oxidizing environment of the endoplasmic reticulum, whereas the G-domain emerges from the ribosome into the cytosol, remaining unfolded. Once the full G-domain has emerged from the ribosome, folding occurs in several steps that appear similar on the ribosome and in isolation  that fulfills an essential cellular function. The efficiency of EF-G synthesis and folding may have been under evolutionary pressure. The coding sequence contains very few rare codons  . All plasmids used in this study are based on a backbone with a pUC origin, Lac-operator-controlled T7 promoter, and Ampicillin resistance gene35. The NanoLuc coding sequence was obtained as a synthetic DNA fragment (Integrated DNA Technologies). The SecM coding sequence was introduced through synthetic DNA fragments that served as primers for PCR amplification. Vector backbone and PCR products were assembled with Gibson Assembly Master Mix (NEB E2611), yielding plasmid pWP3. All primers used for cloning are listed with their sequences in Supplementary Information (Tables In vivo folding experiments were carried out in  Lemo21DE (New Eng® Buffer (NEB) supplemented with 3-mM ATP and 10-mM DTT. The reaction was provided with 4U of each NheI, SpeI, T4PNK and T4 DNA ligase at 10 μl final volume. After 2 h at 37 and 16 °C overnight, the product was transformed and verified by colony PCR with Taq DNA polymerase. Plasmid DNA from colonies showing the correct insert sizes was isolated, and its correct sequences were verified by Sanger sequencing.EF-G fragments of defined lengths were individually PCR-amplified with a NheI tailed universal forward primer (WP3-EF-G-uni-fwd) and reverse primers at designated positions along the EF-G open reading frame  for each plate and sprayed evenly onto the plate with an airbrush (Neo Iwata HP-CN N4500). Images were taken in a shaded home-made imaging box equipped with a Canon Rebel T3 camera, operated in raw image acquisition mode to avoid complications from camera-internal image processing. Camera settings were at Neutral between 5 and 15 s exposure and ISO800 for optimal contrast. Images were recorded with 8-bit color depth. To identify nascent chains that fold into stable structures, colony luminescence was quantified using custom Matlab scripts for image analysis. Circular areas of identical size . At OD600 = 0.2–0.4, cultures were induced with 500 μM L-rhamnose and 500 μM IPTG for 1 h. Cell densities were measured and 100 μl cultures were put onto a white round-bottom 96-well plate for luminescence measurement. All NanoLuc assays in this study were carried out with Nano-Glo® Live Cell Assay from Promega (N2011) according to manufacturer instructions. Luminescence was measured on a GloMax® Navigator Microplate Luminometer (Promega). Signals were linearly normalized to OD600 = 0.4. Each data set was acquired using identical instrument settings to allow comparison between samples. The integration time chosen such that the highest signal did not exceed 1E9 RLU to avoid saturating the detector. For visualization of translation products by Western blotting, Streptavidin-HRP was used to detect biotinylated proteins in whole-cell lysates after ribonuclease treatment. Uncropped images of the Western blots shown in Fig. Cells transformed with individual plasmids were spread on LB agar with antibiotics and allowed to form colonies overnight. LB supplemented with ampicillin and chloramphenicol was inoculated with a single colony to grow an overnight culture. Overnight cultures were diluted into fresh LB containing antibiotics to OD35. Nonstop mRNA templates were generated by in vitro transcription of PCR products , and stored in small aliquots after flash-freezing. Single-molecule experiments were carried out using a custom home-built instrument54. The experiments were carried out in HKMβ buffer. For force ramp experiments, the trap was moved at a constant speed of 100 nm/s to apply continuously increasing forces on the nascent chain in the range from 2 to 50 pN. The force was ramped down at the same loading rate, and the molecule was held at 2 pN for 10 s before being pulled again. Data were collected at a sampling frequency of 1000 Hz and averaged to 33 Hz for plotting. The extension changes were determined using custom MATLAB scripts as described in detail previously18. For constant force experiments, the molecule was first subjected to force ramp cycles to ensure it exhibited the characteristic unfolding transitions. After fully unfolding the molecule at 30 pN, the force was reduced to 3.5 pN to initiate refolding. The change in molecular extension was recorded at a sampling frequency of 1000 Hz. The measurement does not yield the absolute extension of the molecule. For measurements with 328RNC and with the isolated G-domain, the extension of the folded state was defined as 0, all other extensions are relative to this reference state. For measurements with the unfolded 316RNC, the relative extension is reported.Stalled RNCs were generated as described previouslyFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


C. oremlandii are reported. Monothiol glutaredoxin-1 formed domain-swapped structures, one of which was in a hetero-oligomer complex.Structures of monothiol and dithiol glutaredoxin-1 from  Clostridium oremlandii (cGrx1) was used as a model to explore the formation of multiple domain-swapped conformations, which were made possible by modulating several hinge-loop residues that can form a pivot for domain swapping. Specifically, two alternative domain-swapped structures were generated and analyzed using nuclear magnetic resonance (NMR), X-ray crystallography, circular-dichroism spectroscopy and hydrogen/deuterium-exchange (HDX) mass spectrometry. The first domain-swapped structure (β3-swap) was formed by the hexameric cGrx1–cMsrA complex. The second domain-swapped structure (β1-swap) was formed by monothiol cGrx1 (C16S) alone. In summary, the first domain-swapped structure of an oxidoreductase in a hetero-oligomeric complex is presented. In particular, a single point mutation of a key cysteine residue to serine led to the formation of an intramolecular disulfide bond, as opposed to an intermolecular disulfide bond, and resulted in modulation of the underlying free-energy landscape of protein oligomerization.Protein dimerization or oligomerization resulting from swapping part of the protein between neighboring polypeptide chains is known to play a key role in the regulation of protein function and in the formation of protein aggregates. Glutaredoxin-1 from  Specifically, we determined the structures of both dithiol and monothiol versions of cGrx1  and the complex of m-cGrx1 with cMsrA, in which the structures of m-cGrx1 displayed two alternative domain-swapped configurations. Both cGrx1 and cMsrA are native selenoproteins that contain a catalytic selenocysteine  domain swapping is a term for oligomerization in which two or more identical protein monomers exchange their structural domains  after incubation with 5 mM methionine sulfoxide at 4°C for 2 h. The column-purified protein complex was concentrated to a final concentration of approximately 20 mg ml−1.cMsrA was expressed and purified as described previously /1.6 M sodium phosphate (monobasic), respectively.The sitting-drop vapor-diffusion method was used for initial crystallization screening at 20°C by applying various screening kits from Hampton Research  and Anatrace . Optimization of the crystallization conditions was then carried out sequentially by the hanging-drop vapor-diffusion method using 24-well VDX plates (Hampton Research). Each drop was set up by mixing 1 µl concentrated protein solution with an equal volume of reservoir solution and was equilibrated against 500 µl reservoir solution. Crystals of d-cGrx1, domain-swapped m-cGrx1 and the cGrx1–cMsrA complex suitable for X-ray diffraction data collection were obtained using reservoir solutions consisting of 0.1 2.3.HKL-2000 package  at 20°C and incubated for various time points: 0.33, 10, 60 and 240 min. The exchange was quenched with an equal volume of a prechilled quench buffer . The protein was digested on an Enzymate immobilized pepsin column  and the peptides were trapped on a pre-column  and separated using a C18 column  with a linear gradient of acetonitrile (5–95%) supplemented with 0.1% formic acid. Peptides were analyzed using a SYNAPT G2-Si mass spectrometer with IMS . The peptic peptides were identified in undeuterated samples with ProteinLynx Global SERVER 3.0 . To process the HDX-MS data, the amount of deuterium in each peptide was determined by measuring the centroid of the isotopic distribution using DynamX 3.0 .Hydrogen–deuterium exchange was performed using an HDX Manager  equipped with a LEAP PAL autosampler . 40 µ2.5.−1 in buffer  prior to loading. The samples were loaded and then heated from 25 to 85°C at 0.5°C min−1. The circular-dichroism absorbance at 222 nm was recorded using a circular-dichroism spectrophotometer  and normalized to calculate the melting temperature of each protein.The d-cGrx1 and m-cGrx1 proteins were diluted to 0.5 mg ml3.a), Table 1a) and 1d), Table 1et al., 2014et al., 2015et al., 2015b) and 1c), Table 1AB and CD), in which subunits A and C retained their normal tertiary structure, while the other two subunits (i.e. subunits B and D) formed a domain-swapped structure  and 2c)]. Nevertheless, the two domain-swapped m-cGrx1 molecules retained the same overall structure as the other two m-cGrx1 molecules, as well as that of d-cGrx1, except for the hinge loop and the catalytic cysteine (Cys13). The catalytic Cys13, in particular, had a significantly different orientation between d-cGrx1 and the β3-swap structure. Cys13 of d-cGrx1 formed a disulfide bond with its resolving cysteine, which pointed inwards, while Cys13 of the β3-swap structure was directed outwards to form a disulfide bond to another cGrx1 [Fig. 2d)].We have determined three crystal structures: d-cGrx1 and domain-swapped structures (named β1-swap and β3-swap) of m-cGrx1 and the m-cGrx1–cMsrA complex -HSQC spectra , the corresponding spectra for 15N-d-cGrx1 exhibited a multiple peak shift in spectra with and without DTT  and 3d)]. This suggests that the intramolecular disulfide bond formed in d-cGrx1 induced a different conformation compared with the reduced cGrx1. Next, both 15N-labeled cGrx1s were incubated with cMsrA and methionine sulfoxide (Met-O) for 1 h at room temperature and the HSQC spectra were collected for each mixture. The overall peaks of 15N-d-cGrx1 were unchanged . However, unlike the β3-swap structure, the β1-swap structure was formed by exchanging the β1 strands, which was made possible by partially unfolding the α1 helices in the respective domain-swapping partner [Figs. 4b) and 4c)]. Because of this domain swap, the monomers interacted with neighboring monomers through interfaces that differed from those formed in the β3-swap structure. Based on our structures, there are at least two domain-swapped configurations of m-cGrx1 that contain disulfide bonds. How can a single protein, i.e. m-cGrx1, adopt two alternative domain-swapped conformations? We expect that the answer pertains to the movement of the α1 helix. In the cMsrA–cGrx1 hexamer, m-cGrx1 interacts with cMsrA via two different interfaces , both of which involve the α1 helix , indicating that that the reduction of the disulfide bond in the CPHC motif of d-cGrx1 loosens the tertiary structure. However, the deuterium uptake was decreased in m-cGrx1 after treatment with TCEP [Fig. 5c) and Supplementary Fig. S5]. It is possible that disulfide dimerization of m-cGrx1 reduced its dynamic structure, including the domain swapping. Structural changes between β1 and β3 were frequently observed in both of the crystal structures of domain-swapped m-cGrx1, β3-swap and β1-swap, suggesting that the disulfide bond is crucial to the domain-swapped configuration of cGrx1 [Figs. 5b) and 5c)]. The deuterium uptake of the 5–17 peptide in m-cGrx1 in the oxidized form was lower than that in other conditions, suggesting that protein–protein interactions were modulated by domain swapping. The domain-swapped hexameric cMsrA–m-cGrx1 is the first reported structure with hetero-oligomeric domain swapping. To date, only two domain-swapped structures of thiol oxidoreductases have been reported, namely thioredoxin (Trx) from Staphylococcus aureus . CD spectroscopy data for the thermal unfolding transition showed that d-cGrx1 was more stable than m-cGrx1 (Supplementary Table S1). The thermal unfolding of d-cGrx1 is characterized by an enthalpy change of 47.35 kcal mol−1 at the melting temperature (69.12°C), whereas for m-cGrx1 it is 50.34 kcal mol−1 at the melting temperature (53.42°C). In summary, using HDX we demonstrated that the intermolecular disulfide bond is crucial for domain swapping and maintenance of the tertiary structure of cGrx1. The domain swapping was validated in solution by 2D -HSQC NMR spectroscopy. In conclusion, this study has shown that multiple domain-swapping conformations can be induced by a single mutation of m-cGrx1, allowing the formation of alternative intermolecular disulfide bridges. We believe that these findings provide a better understanding of the domain-swapping mechanism and act as novel examples of protein engineering.We carried out HDX experiments on d-cGrx1 and m-cGrx1 under reducing and oxidizing conditions. We obtained greater than 95% sequence coverage for both d-cGrx1 and m-cGrx1 in the HDX experiment, with a few exceptions , 7c12PDB reference: m-cGrx1–cMsrA complex (β3-swap), 7c13PDB reference: 10.1107/S2052252520011598/lz5041sup1.pdfSupplementary Table and Figures. DOI:


Neurodegenerative diseases (ND) remains to be one of the biggest burdens on healthcare systems and serves as a leading cause of disability and death. Alzheimer’s disease (AD) is among the most common of such disorders, followed by Parkinson’s disease (PD). The basic molecular details of disease initiation and pathology are still under research. Only recently, the role of exosomes has been linked to the initiation and progression of these neurodegenerative diseases. Exosomes are small bilipid layer enclosed extracellular vesicles, which were once considered as a cellular waste and functionless. These nano-vesicles of 30–150 nm in diameter carry specific proteins, lipids, functional mRNAs, and high amounts of non-coding RNAs . As the exosomes content is known to vary as per their originating and recipient cells, these vesicles can be utilized as a diagnostic biomarker for early disease detection. Here we review exosomes, their biogenesis, composition, and role in neurodegenerative diseases. We have also provided details for their characterization through an array of available techniques. Their updated role in neurodegenerative disease pathology is also discussed. Finally, we have shed light on a novel field of salivary exosomes as a potential candidate for early diagnosis in neurodegenerative diseases and compared the biomarkers of salivary exosomes with other blood/cerebrospinal fluid (CSF) based exosomes within these neurological ailments.  There has been an ever-increasing interest in the development of early diagnostics and novel treatment modalities for neurodegenerative diseases. These nervous system-related degenerative diseases have highly burdened the population worldwide due to their associated mortality and morbidity. The irreversible damage, which occurs in neurodegeneration, together with the concomitant increase in the rate of progression throughout the treatment has largely impeded the countermeasures. The neurodegenerative disorders encompass various diseases that include Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS). AD and PD remain the first and second most common neurodegenerative diseases worldwide . The majThe search for efficient biomarkers of these diseases is a vividly explored field nowadays. Till now, extensive studies on biofluids based diagnostics are available in the literature ,3,4,5,6.Recent advances in nuclear magnetic resonance (NMR) and mass spectrometry resulted in a rapidly emerging field of metabolomics-based biomarker development. Metabolites like glycerophosphocholine and d-glucosamide were reportedly present in AD patients in comparison to normal healthy controls . AnotherExosomes based biomarker detection is a novel and rapidly evolving field within neurodegenerative disease diagnostics . The exoThe oligodendrocyte-derived exosome cargo plays a key role in myelin sheath biogenesis as well as having a prime role in tissue regeneration ,23. ExosIn this primer, we provide a broader description of exosomes, their isolation, and characterization methodologies in general as well as from a neuropathologic perspective. The role of exosomes in neurodegenerative pathologies along with their most updated exosomal markers is also discussed. Finally, our current understanding of a relatively newer area of salivary exosomes as a potent biomarker for neurodegenerative diseases is elaborated.There exists a well-established process through which a variety of cells release different hormones or neurotransmitters, likewise, most cells have an evolutionarily conserved mechanism to secrete a myriad of membranous vesicles that are known as extracellular vesicles (EVs) . These EExosomes were first described in 1981  and wereThe composition of exosomes is diverse and may reflect its origin cells or tissues. Their content includes proteins, lipids, enzymes, and nucleic acids, which play an important role in cell to cell communications and are responsible for delivering various signal molecules to both proximal and distant locations. Some of the most common cargoes on exosomes are transmembrane proteins, cytoskeletal proteins, and heat shock proteins; various lipids; and various types of RNAs e.g., mRNA, microRNA (miRNA), non-coding RNA (ncRNA), mitochondrial DNA (mtDNA), and single-stranded and double-stranded DNAs (ssDNA and dsDNA) ,32. SomeThe exosomes are generously enriched in lipids, especially lipid rafts such as ceramides, sphingomyelin, cholesterol, sphingolipids, glycerophospholipids, and glycosphingolipids. The exosomal membrane has lysophosphatidic acid which plays an important role in the formation of ILVs from MVEs. Some lipids are found in lesser quantities which include phosphatidylserines, phosphatidylcholine, and phosphatidylinositols. Exosomes also have prostaglandins and some enzymes such as phosphatases, glycosidases, lipases, and proteases. The enzymes found in exosomes generally represent exosomal origin cell types and their metabolic activity ,56. The exosomes also contain nucleic acids that play an important role in intracellular communications as well as in the pathophysiology of a variety of diseases. Both RNA and DNA are found as exosomal cargo and their concentration and composition are dependent on the origin and the target cells ,58,59. EThe characterization of exosomes can be done through physical and biochemical principles. The properties such as size, concentration, and ultrastructural morphology. can be exploited for physical analyses whereas the content and composition of exosomes  can be studied using biochemical analyses. Physical techniques commonly practiced are nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), electron microscopy, and atomic force microscopy, whereas the biochemical techniques include Western blotting, ELISA, qRT-PCR and flow-cytometry ,62,63. HElectron microscopy: Electron microscopy (EM) utilizes a focused beam of accelerated electrons, instead of light, under a vacuum that interacts with the specimen to produces images with superior resolution than that of light microscopy . ScanninAtomic force microscopy (AFM): AFM is a type of scanning probe microscopy, based on Hooke’s law, which uses a small mechanical cantilever that scans the surface of the sample resulting in details of surface topography and presence of substructures . There aNanoparticle tracking analysis: NTA is a particle tracking method that measures the size (hydrodynamic diameter) and concentration of particles in a suspension based on the Brownian motion of the particles ,72. The Dynamic light scattering: DLS is similar to NTA as it is also based on Brownian motion and Stokes–Einstein equation however it provides information only based on intensity-based distribution therefore the fluctuation in intensity over time is used to determine the size of the particle . DLS is Tunable resistive pulse sensing (TRPS): TRPS is based on the Coulter principle that measures transient changes in electric current, generated by particles while passing through a size-tunable pore in membrane to determine the size of the particles ,79. TRPSWestern blotting (WB) and enzyme-linked immunosorbent assay (ELISA) are conventional methods based on antigen–antibody reaction, and the most commonly used techniques to characterize exosomes to demonstrate the presence of targeted exosomal proteins . In WB, Flow cytometry (FCM) is a sensitive technique for characterizing small particles like exosomes by measuring scattered light or fluorescence activation. The samples, labeled with fluorescent tags or antibodies, are passed through the laser of different wavelengths producing scattered light in forward scatter (FSC) and side scatters (SCC) profiles. FSC denotes the size of the particles whereas SCC indicates internal structure details and is more sensitive than FSC ,91. For Briefly, we have tabulated the general techniques used to characterize exosomes along with their pros and cons in Dementia is an umbrella term that is broadly used for the loss of cognitive functioning and memory. Medically, it can be referred to as chronic brain dysfunction. According to the recent statistical reports, the prevalence of dementia is thought to increase from 3% (age group 70–75 years) to 20–25% among those with the age approaching 85 years. It is predicted, the number of individuals currently suffering will be doubled every 20 years and may account for 81.1 million diseased people by 2040. The developing countries are among the highest sufferers  . AlthougThe role of exosomes in the cell to cell communication and pathology of AD is beginning to unfold . The exoThe exosomal cargo content including proteins, lipids, and nucleic acids are gaining importance as prospective biomarkers. In the case of diseases like AD, where behavioral symptoms occur much later in life than the actual events of disease progression, the search for identification of early screening markers has been of utmost importance. Within CSF derived exosomes from severe AD patients, the decreased expression levels of Aβ were reported . In anotParkinson’s disease is the second most common neurodegenerative disease after AD . It is aThe genetically linked familial form of PD is associated with the point mutation in the SNCA gene, encoding the α-syn protein . The perThe role of exosomes in PD results in the seeding of intraneuronal α-syn in a prion-like manner. Exosomal machinery serves as an auxiliary mechanism for dissipating early molecular changes of PD pathology to other cells . The genIn recent years, the search for CSF and plasma biomarkers in PD has been of prime importance for the diagnosis of disease pathology or the associated neuropathological change in an early stage ,179. TheThe two additional, but less prevalent neurodegenerative diseases are Huntington’s disease and amyotrophic lateral sclerosis. HD is linked primarily to the expanded trinucleotide repeat in the huntingtin gene (HTT) which is the pathological carrier-a mutant form of the multi-functional protein huntingtin. ALS is linked to the copper-zinc superoxide dismutase 1 (SOD1) gene mutation and protein inclusions are ubiquitinated and enriched in tar DNA binding protein-43 (TDP-43) and has concomitant behavioral symptoms like FTD. Huntington is another category of prion-based neurodegenerative disease which is autosomal dominant. It is known to panoply the motor, cognitive and behavioral defects. The abnormal amplification of CAG repeats in the Huntington gene is the accountable cause of this disease ,223. TheAmyotrophic lateral sclerosis comes under the umbrella of prion-based neurodegenerative disorders . ALS is Salivary exosomes have recently occupied the center stage of biomarker research. The potential of salivary exosomal cargo content as biomarker molecules have gained enormous importance in a very short time. Their role in systemic diseases, cancer, and neurodegeneration is a widely exploring field ,246. TheThere are two significant studies on salivary exosomes in Parkinson’s disease. One amongst them reported the content of α-synOlig and α-synOlig/α-synTotal is high in extracellular vesicles of salivary origin in PD patients than healthy control . AnotherThe degenerative disorders of the nervous systems are known to cause chronic brain-dysfunction. The major neurodegenerative disorders are caused by the aggregation of misfolded proteins and have a multitude of genetic mutations associated with each one of them. The four major neurodegenerative diseases are AD, PD, HD, and ALS. To minimize the burden of these diseases there is a dire need for biomarkers, which can be used to screen these diseases early and in a non-invasive way. Exosomes are nano-sized extracellular vesicles, which are in high abundance within biofluids and have a tendency to cross the blood–brain barrier (BBB). Recently, the role of exosomes is associated with the seeding of these disease pathologies through a prion-based manner. Since these vesicles are able to cross BBB, they can give a fine imprint of the CNS associated disease pathologies. The richness of the peripheral system in these vesicles and their secretion from all cell-types makes them a potent biomarker candidate in many diseases including neurodegenerative diseases. Numerous studies have examined the association of CSF and blood-derived exosomes with neurodegenerative diseases. The usage of another non-invasive biofluid, saliva, and the vesicles derived from saliva has recently gained prime importance as potent biomarker candidates. Here, in this article, we have elucidated the emerging role of salivary exosomal biomarkers their isolation methodologies, and characterization techniques in neurodegenerative diseases. A brief description of the disease pathologies and the role of extracellular vesicles in each of these neurodegenerative disorders is also explained. In the foreseeable future, we hope this current work will serve as a primer for any further studies targeting this emerging area of salivary exosomes as a promising biomarker for various neurodegenerative diseases.