Covalent linkers bridging the domains of multidomain proteins are considered to be crucial for assembly and function. In this report, an exception in which the linker of a two-domain dimeric L-asparaginase from Pyrococcus furiosus (PfA) was found to be dispensable is presented. Domains of this enzyme assembled without the linker into a conjoined tetrameric form that exhibited higher activity than the parent enzyme. The global shape and quaternary structure of the conjoined PfA were also similar to the wild-type PfA, as observed by their solution scattering profiles and X-ray crystallographic data. Comparison of the crystal structures of substrate-bound and unbound enzymes revealed an altogether new active-site composition and mechanism of action. Thus, conjoined PfA is presented as a unique enzyme obtained through noncovalent, linker-less assembly of constituent domains that is stable enough to function efficiently at elevated temperatures.

The enzyme L-asparaginase of Pyrococcus furiosus (PfA) functions as a dimer with each monomer consisting of distinct N-and C-terminal domains (NPfA and CPfA, respectively), connected by a linker. Here we present data to show that NPfA functions as a non-specific molecular chaperone. Independently expressed NPfA refolded spontaneously whereas CPfA formed insoluble aggregates. However, when mixed and refolded together, NPfA augmented CPfA to fold with~90% recovery. NPfA also protected a variety of substrate proteins from thermal and refolding-mediated aggregation as monitored by a reduction in light scattering. The co-appearance of substrate protein with NPfA in antibody pull-down assays as well as in eluted gel filtration peaks indicated direct protein-protein interaction. These interactions were hydrophobic in nature as determined by 8-anilino-1-naphthalene sulfonic acid fluorescence. NPfA inhibited polyglutamine-mediated amyloid formation and also facilitated disintegration of preformed amyloid fibrils of amyloid-b (1-42) as determined by reverse-phase HPLC-based sedimentation assay and thioflavin T binding assays, respectively. Dynamic light scattering experiments suggested that NPfA readily assembled into polydispersed oligomeric species. With no sequence similarity to a-crystallin or any known molecular chaperone, we present here NPfA as a novel molecular chaperone. Structured digital abstract• Ab amyloid 1-42 and Ab amyloid 1-42 bind by fluorescence technology (View interaction)• alpha-amylase and alpha-amylase bind by light scattering (View interaction)• Ab amyloid 1-42 and Ab amyloid 1-42 bind by transmission electron microscopy (View interaction)• NPfa binds to BCA II by anti tag coimmunoprecipitation (View interaction)• MSG and MSG bind by light scattering (View interaction)

Dietary exposure to aflatoxins is a significant risk factor in the development of hepatocellular carcinomas. Following bioactivation by microsomal P450s, the reaction of aflatoxin B 1 (AFB 1 ) with guanine (Gua) in DNA leads to the formation of stable, imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B 1 (AFB 1 -FapyGua) adducts. In contrast to most base modifications that result in destabilization of the DNA duplex, the AFB 1 -FapyGua adduct increases the thermal stability of DNA via 5′-interface intercalation and base-stacking interactions. Although it was anticipated that this stabilization might make these lesions difficult to repair relative to helix distorting modifications, prior studies have shown that both the nucleotide and base excision repair pathways participate in the removal of the AFB 1 -FapyGua adduct. Specifically for base excision repair, we previously showed that the DNA glycosylase NEIL1 excises AFB 1 -FapyGua and catalyzes strand scission in both synthetic oligodeoxynucleotides and liver DNA of exposed mice. Since it is anticipated that error-prone replication bypass of unrepaired AFB 1 -FapyGua adducts contributes to cellular transformation and carcinogenesis, the structural and thermodynamic parameters that modulate the efficiencies of these repair pathways are of considerable interest. We hypothesized that the DNA sequence context in which the AFB 1 -FapyGua adduct is formed might modulate duplex stability and consequently alter the efficiencies of NEIL1-initiated repair. To address this hypothesis, site-specific AFB 1 -FapyGua adducts were synthesized in three sequence contexts, with the 5′ neighbor nucleotide being varied. DNA structural stability analyses were conducted using UV absorbance-and NMR-based melting experiments. These data revealed differentials in thermal stabilities associated with the 5′neighbor base pair. Single turnover kinetic analyses using the NEIL1 glycosylase demonstrated corresponding sequence-dependent differences in the repair of this adduct, such that there was an inverse correlation between the stabilization of the duplex and the efficiency of NEIL1-mediated catalysis.

The N-(2-deoxy-d-erythro-pentofuranosyl)-urea DNA lesion forms following hydrolytic fragmentation of cis-5R,6S- and trans-5R,6R-dihydroxy-5,6-dihydrothymidine (thymine glycol, Tg) or from oxidation of 7,8-dihydro-8-oxo-deoxyguanosine (8-oxodG) and subsequent hydrolysis. It interconverts between α and β deoxyribose anomers. Synthetic oligodeoxynucleotides containing this adduct are efficiently incised by unedited (K242) and edited (R242) forms of the hNEIL1 glycosylase. The structure of a complex between the active site unedited mutant CΔ100 P2G hNEIL1 (K242) glycosylase and double-stranded (ds) DNA containing a urea lesion reveals a pre-cleavage intermediate, in which the Gly2 N-terminal amine forms a conjugate with the deoxyribose C1′ of the lesion, with the urea moiety remaining intact. This structure supports a proposed catalytic mechanism in which Glu3-mediated protonation of O4′ facilitates attack at deoxyribose C1′. The deoxyribose is in the ring-opened configuration with the O4′ oxygen protonated. The electron density of Lys242 suggests the ‘residue 242-in conformation’ associated with catalysis. This complex likely arises because the proton transfer steps involving Glu6 and Lys242 are hindered due to Glu6-mediated H-bonding with the Gly2 and the urea lesion. Consistent with crystallographic data, biochemical analyses show that the CΔ100 P2G hNEIL1 (K242) glycosylase exhibits a residual activity against urea-containing dsDNA.

It remains undeciphered how thermophilic enzymes display enhanced stability at elevated temperatures. Taking l-asparaginase from P. furiosus (PfA) as an example, we combined scattering shapes deduced from small-angle X-ray scattering (SAXS) data at increased temperatures with symmetry mates from crystallographic structures to find that heating caused end-to-end association. The small contact point of self-binding appeared to be enabled by a terminal short β-strand in N-terminal domain, Leu179-Val-Val-Asn182 (LVVN). Interestingly, deletion of this strand led to a defunct enzyme, whereas suplementation of the peptide LVVN to the defunct enzyme restored structural frameworkwith mesophile-type functionality. Crystal structure of the peptide-bound defunct enzyme showed that one peptide ispresent in the same coordinates as in original enzyme, explaining gain-of lost function. A second peptide was seen bound to the protein at a different location suggesting its possible role in substrate-free molecular-association. Overall, we show that the heating induced self-assembly of native shapes of PfA led to an apparent super-stable assembly.

Here, we report the folding and assembly of a Pyrococcus furiosus-derived protein, L-asparaginase (PfA). PfA functions as a homodimer, with each monomer made of distinct N- and C-terminal domains. The purified individual domains as well as single Trp mutant of each domain were subjected to chemical denaturation/renaturation and probed by combination of spectroscopic, chromatographic, quenching and scattering techniques. We found that the N-domain acts like a folding scaffold and assists the folding of remaining polypeptide. The domains displayed sequential folding with the N-domain having higher thermodynamic stability. We report that the extreme thermal stability of PfA is due to the presence of high intersubunit associative forces supported by extensive H-bonding and ionic interactions network. Our results proved that folding cooperativity in a thermophilic, multisubunit protein is dictated by concomitant folding and association of constituent domains directly into a native quaternary structure. This report gives an account of the factors responsible for folding and stability of a therapeutically and industrially important protein.

Aflatoxin B1 (AFB1) exposure through contaminated food is a primary contributor to hepatocellular carcinogenesis worldwide. Hepatitis B viral infections in livers dramatically increase the carcinogenic potency of AFB1 exposures. Liver cytochrome P450 oxidizes AFB1 to the epoxide, which in turn reacts with N7-guanine in DNA, producing the cationic trans-8,9-dihydro-8-(N7-guanyl)–9-hydroxyaflatoxin B1 adduct (AFB1–N7-Gua). The opening of the imidazole ring of AFB1–N7-Gua under physiological conditions causes the formation of the cis- and trans-diastereomers of 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)–9-hydroxyaflatoxin B1 (AFB1–FapyGua). These adducts primarily lead to G → T mutations, with AFB1–FapyGua being significantly more mutagenic than AFB1–N7-Gua. The unequivocal identification and accurate quantification of these AFB1–Gua adducts as biomarkers are essential for a fundamental understanding and prevention of AFB1-induced hepatocellular carcinogenesis. Among a variety of analytical techniques used for this purpose, liquid chromatography–tandem mass spectrometry, with the use of the stable isotope-labeled analogues of AFB1–FapyGua and AFB1–N7-Gua as internal standards, provides the greatest accuracy and sensitivity. cis-AFB1–FapyGua-15N5, trans-AFB1–FapyGua-15N5, and AFB1–N7-Gua-15N5 have been synthesized and used successfully as internal standards. However, the availability of these standards from either academic institutions or commercial sources ceased to exist. Thus, quantitative genomic data regarding AFB1-induced DNA damage in animal models and humans remain challenging to obtain. Previously, AFB1–N7-Gua-15N5 was prepared by reacting AFB1-exo-8,9-epoxide with the uniformly 15N5-labeled DNA isolated from algae grown in a pure 15N-environment, followed by alkali treatment, resulting in the conversion of AFB1–N7-Gua-15N5 to AFB1–FapyGua-15N5. In the present work, we used a different and simpler approach to synthesize cis-AFB1–FapyGua-15N5, trans-AFB1–FapyGua-15N5, and AFB1–N7-Gua-15N5 from a partial double-stranded 11-mer Gua-15N5-labeled oligodeoxynucleotide, followed by isolation and purification. We also show the validation of these 15N5-labeled standards for the measurement of cis-AFB1–FapyGua, trans-AFB1–FapyGua, and AFB1–N7-Gua in DNA of livers of AFB1-treated mice.

A correction is made to Fig. 7 in the article by Tomaret al.[(2014).Acta Cryst.D70, 3187–3197].