Nuclear Magnetic Resonance Structure Shows that the Severe Acute Respiratory Syndrome Coronavirus-Unique Domain Contains a Macrodomain Fold

The nuclear magnetic resonance (NMR) structure of a central segment of the previously annotated severe acute respiratory syndrome (SARS)-unique domain (SUD-M, for “middle of the SARS-unique domain”) in SARS coronavirus (SARS-CoV) nonstructural protein 3 (nsp3) has been determined. SUD-M(513-651) exhibits a macrodomain fold containing the nsp3 residues 528 to 648, and there is a flexibly extended N-terminal tail with the residues 513 to 527 and a C-terminal flexible tail of residues 649 to 651. As a follow-up to this initial result, we also solved the structure of a construct representing only the globular domain of residues 527 to 651 [SUD-M(527-651)]. NMR chemical shift perturbation experiments showed that SUD-M(527-651) binds single-stranded poly(A) and identified the contact area with this RNA on the protein surface, and electrophoretic mobility shift assays then confirmed that SUD-M has higher affinity for purine bases than for pyrimidine bases. In a further search for clues to the function, we found that SUD-M(527-651) has the closest three-dimensional structure homology with another domain of nsp3, the ADP-ribose-1"-phosphatase nsp3b, although the two proteins share only 5% sequence identity in the homologous sequence regions. SUD-M(527-651) also shows three-dimensional structure homology with several helicases and nucleoside triphosphate-binding proteins, but it does not contain the motifs of catalytic residues found in these structural homologues. The combined results from NMR screening of potential substrates and the structure-based homology studies now form a basis for more focused investigations on the role of the SARS-unique domain in viral infection.     

Nuclear Magnetic Resonance Structure of the Nucleic Acid-Binding Domain of Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein 3

The nuclear magnetic resonance (NMR) structure of a globular domain of residues 1071 to 1178 within the previously annotated nucleic acid-binding region (NAB) of severe acute respiratory syndrome coronavirus nonstructural protein 3 (nsp3) has been determined, and N- and C-terminally adjoining polypeptide segments of 37 and 25 residues, respectively, have been shown to form flexibly extended linkers to the preceding globular domain and to the following, as yet uncharacterized domain. This extension of the structural coverage of nsp3 was obtained from NMR studies with an nsp3 construct comprising residues 1066 to 1181 [nsp3(1066-1181)] and the constructs nsp3(1066-1203) and nsp3(1035-1181). A search of the protein structure database indicates that the globular domain of the NAB represents a new fold, with a parallel four-strand β-sheet holding two α-helices of three and four turns that are oriented antiparallel to the β-strands. Two antiparallel two-strand β-sheets and two 3₁₀-helices are anchored against the surface of this barrel-like molecular core. Chemical shift changes upon the addition of single-stranded RNAs (ssRNAs) identified a group of residues that form a positively charged patch on the protein surface as the binding site responsible for the previously reported affinity for nucleic acids. This binding site is similar to the ssRNA-binding site of the sterile alpha motif domain of the Saccharomyces cerevisiae Vts1p protein, although the two proteins do not share a common globular fold.The coronavirus replication cycle begins with the translation of the 29-kb positive-strand genomic RNA to produce two large polyprotein species (pp1a and pp1ab), which are subsequently cleaved to produce 15 or possibly 16 nonstructural proteins (nsp''s) (11). Among these, nsp3 is the largest nsp and also the largest coronavirus protein. nsp3 is a glycosylated (16, 22), multidomain (36, 51), integral membrane protein (38). All known coronaviruses encode a homologue of severe acute respiratory syndrome coronavirus (SARS-CoV) nsp3, and sequence analysis suggests that at least some functions of nsp3 may be found in all members of the order Nidovirales (11). Hallmarks of the coronavirus nsp3 proteins include one or two papain-like proteinase domains (3, 12, 16, 31, 56, 62), one to three histone H2A-like macrodomains which may bind RNA or RNA-like substrates (5, 9, 48, 54, 55), and a carboxyl-terminal Y domain of unknown function (13). An extensive bioinformatics analysis of the coronavirus replicase proteins by Snijder et al. (51) provided detailed annotations of the then-recently sequenced SARS-CoV genome (35, 47), including the identification of a domain unique to SARS-CoV and the prediction of the ADP-ribose-1″-phosphatase (ADRP) activity of the X domain (since shown to be one of the macrodomains).Only limited information is so far available regarding the ways in which the functions of nsp3 are involved in the coronavirus replication cycle. Some functions of nsp3 appear to be directed toward protein; e.g., the nsp3 proteinase domain cleaves the amino-terminal two or three nsp''s from the polyprotein and has deubiquitinating activity (4, 6, 14, 30, 53, 60). Most homologues of the most conserved macrodomain of nsp3 appear to possess ADRP activity (9, 34, 41-43, 48, 59) and may act on protein-conjugated poly(ADP-ribose); however, this function appears to be dispensable for replication (10, 42) and may not be conserved in all coronaviruses (41). The potential involvement of nsp3 in RNA replication is suggested by the presence of several RNA-binding domains (5, 36, 49, 54, 55). nsp3 has been identified in convoluted membrane structures that are also associated with other replicase proteins and that have been shown to be involved in viral RNA synthesis (16, 24, 52), and nsp3 papain-like proteinase activity is essential for replication (14, 62). Other conserved structural features of nsp3 include two ubiquitin-like domains (UB1 and UB2) (45, 49). We have also recently reported that nsp3 is a structural protein, since it was identified as a minor component of purified SARS-CoV preparations, although it is not known whether nsp3 is directly involved in virogenesis or is incidentally incorporated due to protein-protein or protein-RNA interactions (36).A nucleic acid-binding region (NAB) is located within the polypeptide segment of residues 1035 to 1203 of nsp3. The NAB is expected to be located in the cytoplasm, along with the papain-like protease, ADRP, a region unique to SARS-CoV (the SARS-CoV unique domain [SUD]), and nsp3a, since both the N and C termini of nsp3 were shown previously to be cytoplasmic (38). Two hydrophobic segments are membrane spanning (38), and the NAB is located roughly 200 residues in the N-terminal direction from the first membrane-spanning segment. This paper presents the next step in the structural coverage of nsp3, with the determination of the NAB structure. The structural studies included nuclear magnetic resonance (NMR) characterization of two constructs, an nsp3 construct comprising residues 1035 to 1181 [nsp3(1035-1181)] and nsp3(1066-1203), and complete NMR structure determination for the construct nsp3(1066-1181) (see Fig. ​Fig.8).8). The structural data were then used as a platform from which to investigate the nature of the previously reported single-stranded RNA (ssRNA)-binding activity of the NAB (36). Since no three-dimensional (3D) structures for the corresponding domains in other group II coronaviruses are known and since the SARS-CoV NAB has only very-low-level sequence identity to other proteins, such data could not readily be derived from comparisons with structurally and functionally characterized homologues.Open in a separate windowFIG. 8.Sequence alignment of the polypeptide segment nsp3(1066-1181) that forms the globular domain of the SARS-CoV NAB with homologues from other group II coronaviruses. Protein multiple-sequence alignment was performed using ClustalW2 and included sequences from SARS-CoV Tor2 (accession no. {"type":"entrez-protein","attrs":{"text":"AAP41036","term_id":"30795144","term_text":"AAP41036"}}AAP41036) and representatives of three protein clusters corresponding to three group II coronavirus lineages identified by a BLAST search: bat coronavirus HKU5-5 (BtCoV-HKU5-5; accession no. {"type":"entrez-protein","attrs":{"text":"ABN10901","term_id":"124389468","term_text":"ABN10901"}}ABN10901), BtCoV-HKU9-1 (accession no. {"type":"entrez-protein","attrs":{"text":"P0C6T6","term_id":"190360129","term_text":"P0C6T6"}}P0C6T6), and human coronavirus HKU1-N16 (HCoV-HKU1-N16; accession no. {"type":"entrez-protein","attrs":{"text":"ABD75496","term_id":"89515407","term_text":"ABD75496"}}ABD75496). Above the sequences, the positions in full-length SARS-CoV nsp3, the locations of the regular secondary structures in the presently solved NMR structure of the SARS-CoV NAB globular domain, and the residue numbering in this domain are indicated. Amino acids are colored according to conservation and biochemical properties, following ClustalW conventions. Residues implicated in interactions with ssRNA are marked with inverted black triangles. In the present context, the key features are that there is only one position with conservation of K or R (red) and that there are extended sequences with conservation of hydrophobic residues (blue) (see the text).     

Enhanced decolorization and biodegradation of textile azo dye Scarlet R by using developed microbial consortium-GR

A developed consortium-GR, consisting of Proteus vulgaris NCIM-2027 (PV) and Micrococcus glutamicus NCIM-2168 (MG), completely decolorized an azo dye Scarlet R under static anoxic condition with an average decolorization rate of 16,666 μg h^?1; which is much faster than that of the pure cultures (PV, 3571 μg h^?1; MG, 2500 μg h^?1). Consortium-GR gave best decolorization performance with nearly complete mineralization of Scarlet R (over 90% TOC and COD reduction) within 3 h, much shorter relative to the individual strains. Induction in the riboflavin reductase and NADH–DCIP reductase was observed in the consortium, suggesting the involvement of these enzymes during the fast decolorization process. The FTIR and GC–MS analysis showed that 1,4-benzenediamine was formed during decolorization/degradation of Scarlet R by consortium-GR. Phytotoxicity studies revealed no toxicity of the biodegraded products of Scarlet R by consortium-GR. In addition, consortium-GR applied for mixture of industrial dyes showed 88% decolorization under static condition with significant reduction in TOC (62%) and COD (68%) within 72 h, suggesting potential application of this microbial consortium in bioremediation of dye-containing wastewater.     

Structural characterization of the large soluble oligomers of the GTPase effector domain of dynamin

Dynamin, a protein playing crucial roles in endocytosis, oligomerizes to form spirals around the necks of incipient vesicles and helps their scission from membranes. This oligomerization is known to be mediated by the GTPase effector domain (GED). Here we have characterized the structural features of recombinant GED using a variety of biophysical methods. Gel filtration and dynamic light scattering experiments indicate that in solution, the GED has an intrinsic tendency to oligomerize. It forms large soluble oligomers (molecular mass > 600 kDa). Interestingly, they exist in equilibrium with the monomer, the equilibrium being largely in favour of the oligomers. This equilibrium, observed for the first time for GED, may have regulatory implications for dynamin function. From the circular dichroism measurements the multimers are seen to have a high helical content. From multidimensional NMR analysis we have determined that about 30 residues in the monomeric units constituting the oligomers are flexible, and these include a 17 residue stretch near the N-terminal. This contains two short segments with helical propensities in an otherwise dynamic structure. Negatively charged SDS micelles cause dissociation of the oligomers into monomers, and interestingly, the helical characteristics of the oligomer are completely retained in the individual monomers. The segments along the chain that are likely to form helices have been predicted from five different algorithms, all of which identify two long stretches. Surface electrostatic potential calculation for these helices reveals that there is a distribution of neutral, positive and negative potentials, suggesting that both electrostatic and hydrophobic interactions could be playing important roles in the oligomer core formation. A single point mutation, I697A, in one of the helices inhibited oligomerization quite substantially, indicating firstly, a special role of this residue, and secondly, a decisive, though localized, contribution of hydrophobic interaction in the association process.     

4-Hydroxy-2(E)-nonenal inhibits CNS mitochondrial respiration at multiple sites

A destructive cycle of oxidative stress and mitochondrial dysfunction is proposed in neurodegenerative disease. Lipid peroxidation, one outcome of oxidative challenge, can lead to the formation of 4-hydroxy-2(E)-nonenal (HNE), a lipophilic alkenal that forms stable adducts on mitochondrial proteins. In this study, we characterized the effects of HNE on brain mitochondrial respiration. We used whole rat brain mitochondria and concentrations of HNE comparable to those measured in patients with Alzheimer's disease. Our results showed that HNE inhibited respiration at multiple sites. Complex I-linked and complex II-linked state 3 respirations were inhibited by HNE with IC50 values of approximately 200 microM HNE. Respiration was apparently diminished owing to the inhibition of complex III activity. In addition, complex II activity was reduced slightly. The lipophilicity and adduction characteristics of HNE were responsible for the effects of HNE on respiration. The inhibition of respiration was not prevented by N-acetylcysteine or aminoguanidine. Studies using mitochondria isolated from porcine cerebral cortex also demonstrated an inhibition of complex I- and complex II-linked respiration. Thus, in neurodegenerative disease, oxidative stress may impair mitochondrial respiration through the production of HNE.     

Mitochondrial oxidation of 4-hydroxy-2-nonenal in rat cerebral cortex

4-hydroxy-trans-2-nonenal (HNE) is a neurotoxic product of lipid peroxidation whose levels are elevated in multiple neurodegenerative diseases and CNS trauma. The detoxification of HNE may take the route of glutathione conjugation to the C3 carbon and the oxidation or reduction of the C1 aldehyde. In this work, we examined whether the oxidation of HNE to its corresponding carboxylic acid, 4-hydroxy-trans-2-nonenoate (HNEAcid) was detoxifying event, if it occurred in rat cerebral cortex, and in which subcellular compartments. Our results show that HNEAcid did not form protein adducts and was non-toxic to Neuro 2A cells. HNEAcid formation occurred in rat cerebral cortex slices following exposure to HNE in a time-dependent and dose-dependent fashion. Homogenate studies indicated that HNEAcid formation was NAD+ dependent. Subcellular fractionation demonstrated that mitochondria had the highest specific activity for HNEAcid formation with a KM of 21 micro m HNE. These data indicate that oxidation of HNE to its corresponding acid is a major detoxification pathway of HNE in the CNS and that mitochondria play a role in this process.     

Application of HN(C)N to rapid estimation of 1J(N-C(alpha)) coupling constants correlated to psi torsion angles in proteins: implication to structural genomics

We recently described a triple resonance experiment, HN(C)N, for sequential correlation of H(N) and 15N atoms in (15N, 13C) labeled proteins [J. Biomol. NMR. 20 (2001) 135]. Here, we describe an approach based on this experiment for estimation of one bond N-C(alpha) J-couplings in medium size labeled proteins, which seem to show good correlations with psi torsion angles along the protein backbone. The approach uses the ratio of the intensities of the sequential and diagonal peaks in the F(2)-F(3) planes of the HN(C)N spectrum. The reliability of the approach has been demonstrated using a short peptide wherein the coupling constants have been measured by the present method and also independently from peak splittings in HSQC spectra. The two results agree within 10%. The applicability of the procedure to proteins has been demonstrated using doubly labeled FK506 binding protein (FKBP, molecular mass approximately 12 kDa). Coupling constant estimates have been obtained for 62 out of 100 non-proline residues and they show a correlation with psi torsion angles, as has been reported before. This semi-quantitative application of HN(C)N extends the significance of the experiment especially, in the context of structural genomics, since the single experiment, not only provides a great enhancement in the speed of resonance assignment, but also provides quantitative structural information.     

Macroaffinity ligand-facilitated three-phase partitioning for purification of glucoamylase and pullulanase using alginate

Starch-degrading enzymes glucoamylase (from Aspergillus niger), and pullulanase (from Bacillus acidopullulyticus) were purified using alginates (polysaccharides consisting of mannuronic acids and guluronic acids) by a recently developed technique called macroaffinity ligand-facilitated three-phase partitioning (MLFTPP). In this process, a crude preparation of the enzyme was mixed with alginate. On addition of appropriate amounts of ammonium sulfate and t-butanol, the alginate bound enzyme appeared as an interfacial precipitate between the lower aqueous and the upper t-butanol phase. Enzyme activity from this interfacial precipitate was recovered using 1M maltose. Glucoamylase and pullulanase were purified 20- and 38-fold with 83% and 89% activity recovery, respectively. Both the purified preparations showed a single band on SDS-PAGE.