The 3C-like protease (3CL pro ) of severe acute respiratory syndrome coronavirus (SARS-CoV) cleaves 11 sites in the polyproteins, including its own N-and C-terminal autoprocessing sites, by recognizing P4-P1 and P1′. In this study, we determined the crystal structure of 3CL pro with the C-terminal prosequence and the catalytic-site C145A mutation, in which the enzyme binds the C-terminal prosequence of another molecule. Surprisingly, Phe at the P3′ position [Phe(P3′)] is snugly accommodated in the S3′ pocket. Mutations of Phe(P3′) impaired the C-terminal autoprocessing, but did not affect N-terminal autoprocessing. This difference was ascribed to the P2 residue, Phe(P2) and Leu(P2), in the C-and N-terminal sites, as follows. The S3′ subsite is formed by Phe(P2)-induced conformational changes of 3CL pro and the direct involvement of Phe(P2) itself. In contrast, the N-terminal prosequence with Leu(P2) does not cause such conformational changes for the S3′ subsite formation. In fact, the mutation of Phe(P2) to Leu in the C-terminal autoprocessing site abolishes the dependence on Phe(P3′). These mechanisms explain why Phe is required at the P3' position when the P2 position is occupied by Phe rather than Leu, which reveals a type of subsite cooperativity. Moreover, the peptide consisting of P4-P1 with Leu(P2) inhibits protease activity, whereas that with Phe (P2) exhibits a much smaller inhibitory effect, because Phe(P3′) is missing. Thus, this subsite cooperativity likely exists to avoid the autoinhibition of the enzyme by its mature C-terminal sequence, and to retain the efficient C-terminal autoprocessing by the use of Phe(P2).SARS | 3CL protease | specificity | subsite cooperativity | crystal structure

SulA protein is known to be one of the physiological substrates of Lon protease, an ATP-dependent protease from Escherichia coli. In this study, we investigated the cleavage speci®city of Lon protease toward SulA protein.The enzyme was shown to cleave 27 peptide bonds in the presence of ATP. Among them, six peptide bonds were cleaved preferentially in the early stage of digestion, which represented an apparently unique cleavage sites with mainly Leu and Ser residues at the P 1 , and P 1 ¢ positions, respectively, and one or two 1Gln residues in positions P 2 ±P 5 . They were located in the central region and partly in the C-terminal region, both of which are known to be important for the function of SulA, such as inhibition of cell growth and interaction with Lon protease, respectively. The other cleavage sites did not represent such consensus sequences, though hydrophobic or noncharged residues appeared to be relatively preferred at the P 1 sites. On the other hand, the cleavage in the absence of ATP was very much slower, especially in the central region, than in the presence of ATP. The central region was predicted to be rich in a helix and b sheet structures, suggesting that the enzyme required ATP for disrupting such structures prior to cleavage. Taken together, SulA is thought to contain such unique cleavage sites in its functionally and structurally important regions whose preferential cleavage accelerates the ATP-dependent degradation of the protein by Lon protease.

Like many other RNA viruses, severe acute respiratory syndrome coronavirus (SARS-CoV) produces polyproteins containing several non-structural proteins, which are then processed by the viral proteases. These proteases often exist within the polyproteins, and are excised by their own proteolytic activity ('autoprocessing'). It is important to investigate the autoprocessing mechanism of these proteases from the point of view of anti-SARS-CoV drug design. In this paper, we describe a new method for investigating the autoprocessing mechanism of the main protease (M pro ), which is also called the 3C-like protease (3CL pro ). Using our method, we measured the activities, under the same conditions, of the mature form and pro-forms with the N-terminal pro-sequence, the C-terminal pro-sequence or both pro-sequences, toward the pro-form with both N-and C-terminal pro-sequences. The data indicate that the pro-forms of the enzyme have proteolytic activity, and are stimulated by the same proteolytic activity. The stimulation occurs in two steps, with approximately eightfold stimulation by N-terminal cleavage, approximately fourfold stimulation by C-terminal cleavage, and 23-fold stimulation by the cleavage of both termini, compared to the pro-form with both the N-and C-terminal pro-sequences. Such cleavage mainly occurs in a trans manner; i.e. the pro-form dimer cleaves the monomeric form. The stimulation by N-terminal pro-sequence removal is due to the cis (intra-dimer and inter-protomer) effect of formation of the new N-terminus, whereas that by C-terminal cleavage is due to removal of its trans (inter-dimer) inhibitory effect. A numerical simulation of the maturation pathway is presented.

The Escherichia coli ATP-dependent protease Lon degrades ribosomal S2 protein in the presence of inorganic polyphosphate (polyP). In this study, the process of the degradation was investigated in detail. During the degradation, 68 peptides with various sizes (4-29 residues) were produced in a processive fashion. Cleavage occurred at 45 sites, whose P 1 and P 3 positions were dominantly occupied by hydrophobic residues. These cleavage sites were located preferentially at the regions with rigid secondary structures and the P 1 residues of the major cleavage sites appeared to be concealed from the surface of the substrate molecule. Furthermore, polyP changed not only the substrate preference but also the oligomeric structure of the enzyme.

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