The severe acute respiratory syndrome coronavirus 3C-like protease has been proposed to be a key target for structurally based drug design against SARS. The enzyme exists as a mixture of dimer and monomer, and only the dimer was considered to be active. In this report, we have investigated, using molecular dynamics simulation and mutational studies, the problems as to why only the dimer is active and whether both of the two protomers in the dimer are active. The molecular dynamics simulations show that the monomers are always inactive, that the two protomers in the dimer are asymmetric, and that only one protomer is active at a time. The enzyme activity of the hybrid severe acute respiratory syndrome coronavirus 3C-like protease of the wild-type protein and the inactive mutant proves that the dimerization is important for enzyme activity and only one active protomer in the dimer is enough for the catalysis. Our simulations also show that the right conformation for catalysis in one protomer can be induced upon dimer formation. These results suggest that the enzyme may follow the association, activation, catalysis, and dissociation mechanism for activity control.In early 2003, a highly epidemic disease named severe acute respiratory syndrome (SARS) 3 first broke out in China and then quickly spread to other circumjacent countries (1). Research proved that the nosogenesis was a novel coronavirus. In the coronavirus life cycle, 3C-like proteinase (3CL pro ) is important and indispensable and is a pivotal target in anti-SARS drug design (2). SARS 3CL pro shares 40 and 44% sequence identity to 3CL pro of human coronaviruses 229E and transmissible gastroenteritis virus, the crystal structures of which have been resolved (2, 3). Several homology models for SARS 3CL pro have been reported (2, 4, 5). More recently, the crystal structures of the enzyme and the inhibitor-enzyme complex have been determined (6 -11). All structures are very similar and consist of three domains. The first two domains form a chymotrypsin fold, and the third domain is an extra helix domain that plays an important role in dimerization and enzyme activity (12). All of the proteins are dimeric in the crystal structures, and there exists an equilibrium between the monomer and dimer in solution. In our previous work, we have observed that the activity increases with the increase of enzyme concentration, indicating the dimer is the active form of the proteinase (13). Other groups have studied the function of the N-finger in dimerization and enzyme activity. The N-terminal residues 1-5 delete transmissible gastroenteritis virus 3CL pro , and the N-terminal residues 1-7 delete SARS 3CL pro ; both have been reported to have no enzyme activities (3, 14 -16). Interestingly, Chen et al. (14) report that the N-finger deletion mutation does not affect the dimerization of SARS 3CLpro . Contrary to this, Hsu et al. (16) has found that the N-4 truncated protease is mainly monomeric and has little enzyme activity, but the N-3 truncated protease is almost the ...

Wood remains the world's most abundant and renewable resource for timber and pulp and is an alternative to fossil fuels. Understanding the molecular regulation of wood formation can advance the engineering of wood for more efficient material and energy productions. We integrated a black cottonwood (Populus trichocarpa) wood-forming cell system with quantitative transcriptomics and chromatin binding assays to construct a transcriptional regulatory network (TRN) directed by a key transcription factor (TF), PtrSND1-B1 (secondary wall-associated NAC-domain protein). The network consists of four layers of TF-target gene interactions with quantitative regulatory effects, describing the specificity of how the regulation is transduced through these interactions to activate cell wall genes (effector genes) for wood formation. PtrSND1-B1 directs 57 TF-DNA interactions through 17 TFs transregulating 27 effector genes. Of the 57 interactions, 55 are novel. We tested 42 of these 57 interactions in 30 genotypes of transgenic P. trichocarpa and verified that ;90% of the tested interactions function in vivo. The TRN reveals common transregulatory targets for distinct TFs, leading to the discovery of nine TF protein complexes (dimers and trimers) implicated in regulating the biosynthesis of specific types of lignin. Our work suggests that wood formation may involve regulatory homeostasis determined by combinations of TF-DNA and TF-TF (protein-protein) regulations.

The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. Accurate determination of the dimer dissociation constant and the role of the N-finger (residues 1-7) will provide more insights into the enzyme catalytic mechanism of SARS 3CL proteinase. The dimer dissociation constant of the wild-type protein was determined to be 14.0microM by analytical ultracentrifugation method. The N-finger fragment of the enzyme plays an important role in enzyme dimerization as shown in the crystal structure. Key residues in the N-finger have been studied by site-directed mutagenesis, enzyme assay, and analytical ultracentrifugation. A single mutation of M6A was found to be critical to maintain the dimer structure of the enzyme. The N-terminal octapeptide N8 and its mutants were also synthesized and tested for their potency as dimerization inhibitors. Peptide cleavage assay confirms that peptide N8 is a dimerization inhibitor with a K(i) of 2.20mM. The comparison of the inhibitory activities of N8 and its mutants indicates that the hydrophobic interaction of Met-6 and the electrostatic interaction of Arg-4 contribute most for inhibitor binding. This study describes the first example of inhibitors targeting the dimeric interface of SARS 3CL proteinase, providing a novel strategy for drug design against SARS and other coronaviruses.

Supplementary data are available at Bioinformatics online.