The 5′-cap structure is a distinct feature of eukaryotic mRNAs, and eukaryotic viruses generally modify the 5′-end of viral RNAs to mimic cellular mRNA structure, which is important for RNA stability, protein translation and viral immune escape. SARS coronavirus (SARS-CoV) encodes two S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTase) which sequentially methylate the RNA cap at guanosine-N7 and ribose 2′-O positions, catalyzed by nsp14 N7-MTase and nsp16 2′-O-MTase, respectively. A unique feature for SARS-CoV is that nsp16 requires non-structural protein nsp10 as a stimulatory factor to execute its MTase activity. Here we report the biochemical characterization of SARS-CoV 2′-O-MTase and the crystal structure of nsp16/nsp10 complex bound with methyl donor SAM. We found that SARS-CoV nsp16 MTase methylated m7GpppA-RNA but not m7GpppG-RNA, which is in contrast with nsp14 MTase that functions in a sequence-independent manner. We demonstrated that nsp10 is required for nsp16 to bind both m7GpppA-RNA substrate and SAM cofactor. Structural analysis revealed that nsp16 possesses the canonical scaffold of MTase and associates with nsp10 at 1∶1 ratio. The structure of the nsp16/nsp10 interaction interface shows that nsp10 may stabilize the SAM-binding pocket and extend the substrate RNA-binding groove of nsp16, consistent with the findings in biochemical assays. These results suggest that nsp16/nsp10 interface may represent a better drug target than the viral MTase active site for developing highly specific anti-coronavirus drugs.

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The dependence of the fluorescence of catalase upon the concentration of added superoxide dismutase (SOD) indicates that SOD binds to saturable sites on catalase. The affinity of SOD for these sites varies with temperature, and with the concentration of each of three nominally inert polymeric additives--dextran 70, Ficoll 70, and polyethylene glycol 2000. At room temperature (25.0 degrees C) and higher, the addition of high concentrations of polymer is found to significantly enhance the affinity of SOD for catalase, but with decreasing temperature the enhancing effect of polymer addition diminishes, and at 8.0 degrees C, addition of polymer has little or no effect on the affinity of SOD for catalase. The results presented here provide the first experimental evidence for the existence of competition between a repulsive excluded volume interaction between protein and polymer, which tends to enhance association of dilute protein, and an attractive interaction between protein and polymer, which tends to inhibit protein association. The net effect of high concentrations of polymer upon protein associations depends upon the relative strength of these two types of interactions at the temperature of measurement, and may vary significantly between different proteins and/or polymers.

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Previously, the role of YidC in the membrane protein biogenesis of the F 0 sector of the Escherichia coli F 1 F 0 ATP synthase was investigated. Whereas subunits a and c of the F 1 F 0 ATP synthase were strictly dependent on YidC for membrane insertion, subunit b required YidC for efficient insertion (Yi, L., Jiang, F., Chen, M., Cain, B., Bolhuis, A., and Dalbey, R. E. (2003) Biochemistry 42, 10537-10544). In this paper, we investigated other protein components and energetics that are required in the membrane protein assembly of the F 0 sector subunits. We show here that the Sec translocase and the signal recognition particle (SRP) pathway are required for membrane insertion of subunits a and b. In contrast, subunit c required neither the Sec machinery nor the SRP pathway for insertion. While the proton motive force was not required for insertion of subunits b and c, it was required for translocation of the negatively charged periplasmic NH 2 -terminal tail of subunit a, whereas periplasmic loop 2 of subunit a could insert in a proton motive force-independent manner. Taken together, the in vivo data suggest that subunits a and b are inserted by the Sec/SRP pathway with the help of YidC, and subunit c is integrated into the membrane by the novel YidC pathway.The insertion of bacterial inner membrane proteins can occur by two pathways, one involving the Sec translocase and the other independent of the Sec translocase. The Sec translocase is comprised of the protein-conducting SecYEG channel (1) and the heterotrimeric SecDFYajC complex (2) that facilitates translocation. Interestingly, the Sec complex is also associated with YidC (3), a key component that plays an important role in membrane protein biogenesis (4, 5). YidC mediates membrane insertion and comes into contact with the hydrophobic regions of Sec-dependent membrane proteins (6, 7). For translocation of large domains of membrane proteins, SecA is also needed (8 -10). Typically, membrane proteins are targeted to the Sec translocase by the SRP 1 pathway (11). Sec-independent membrane proteins use the YidC pathway for insertion. The M13 procoat and Pf3 coat proteins, which insert by a Sec-independent mechanism (12, 13), strictly require YidC for their membrane insertion (14 -16). In addition, photocross-linking studies have shown that YidC plays a direct role as YidC is cross-linked to the Pf3 coat during membrane insertion (16). No contacts were observed between the Pf3 coat and the Sec components. While insertion of the Pf3 coat and M13 procoat proteins is dependent on the proton motive force (pmf) (13, 17), they do not require the SRP components for targeting (16,18).Recently, it has been shown that YidC is involved in the membrane assembly of cytochrome bo3 oxidase and the F 1 F 0 ATP synthase but not NADH-dehydrogenase (19). These multisubunit membrane complexes play vital roles in cellular respiration where they participate in the complex chemistry of electron transport and coupled oxidative phosphorylation. When YidC is depleted, there is a measurabl...