Mechanism of the Maturation Process of SARS-CoV 3CL Protease 论文

摘要

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus. Viral maturation requires a main protease (3CLpro) to cleave the virus-encoded polyproteins. We report here that the 3CLpro containing additional N- and/or C-terminal segments of the polyprotein sequences undergoes autoprocessing and yields the mature protease in vitro. The dimeric three-dimensional structure of the C145A mutant protease shows that the active site of one protomer binds with the C-terminal six amino acids of the protomer from another asymmetric unit, mimicking the product-bound form and suggesting a possible mechanism for maturation. The P1 pocket of the active site binds the Gln side chain specifically, and the P2 and P4 sites are clustered together to accommodate large hydrophobic side chains. The tagged C145A mutant protein served as a substrate for the wild-type protease, and the N terminus was first digested (55-fold faster) at the Gln-1-Ser1 site followed by the C-terminal cleavage at the Gln306-Gly307 site. Analytical ultracentrifuge of the quaternary structures of the tagged and mature proteases reveals the remarkably tighter dimer formation for the mature enzyme (Kd = 0.35 nm) than for the mutant (C145A) containing 10 extra N-terminal (Kd = 17.2 nm) or C-terminal amino acids (Kd = 5.6 nm). The data indicate that immature 3CLpro can form dimer enabling it to undergo autoprocessing to yield the mature enzyme, which further serves as a seed for facilitated maturation. Taken together, this study provides insights into the maturation process of the SARS 3CLpro from the polyprotein and design of new structure-based inhibitors. Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus. Viral maturation requires a main protease (3CLpro) to cleave the virus-encoded polyproteins. We report here that the 3CLpro containing additional N- and/or C-terminal segments of the polyprotein sequences undergoes autoprocessing and yields the mature protease in vitro. The dimeric three-dimensional structure of the C145A mutant protease shows that the active site of one protomer binds with the C-terminal six amino acids of the protomer from another asymmetric unit, mimicking the product-bound form and suggesting a possible mechanism for maturation. The P1 pocket of the active site binds the Gln side chain specifically, and the P2 and P4 sites are clustered together to accommodate large hydrophobic side chains. The tagged C145A mutant protein served as a substrate for the wild-type protease, and the N terminus was first digested (55-fold faster) at the Gln-1-Ser1 site followed by the C-terminal cleavage at the Gln306-Gly307 site. Analytical ultracentrifuge of the quaternary structures of the tagged and mature proteases reveals the remarkably tighter dimer formation for the mature enzyme (Kd = 0.35 nm) than for the mutant (C145A) containing 10 extra N-terminal (Kd = 17.2 nm) or C-terminal amino acids (Kd = 5.6 nm). The data indicate that immature 3CLpro can form dimer enabling it to undergo autoprocessing to yield the mature enzyme, which further serves as a seed for facilitated maturation. Taken together, this study provides insights into the maturation process of the SARS 3CLpro from the polyprotein and design of new structure-based inhibitors. Severe acute respiratory syndrome (SARS) 1The abbreviations used are: SARS, severe acute respiratory syndrome; SARS-CoV, SARS-coronavirus; TGEV, transmissible gastroenteritis virus; 3CLpro, 3C-like protease; Mpro, main protease; Trx, thioredoxin; GST, glutathione S-transferase; Dabcyl, 4-(4-dimethylaminophenylazo)benzoic acid; Edans, 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid; FXa, factor Xa; Ni-NTA, nickel nitrilotriacetic acid; AUC, analytical ultracentrifuge; IC50, 50% inhibitory concentration; Bicine, N,N-bis(2-hydroxyethyl)glycine; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; HCoV, human coronavirus; aa, amino acid(s). is a severe febrile respiratory illness caused by a newly identified coronavirus, SARS-associated coronavirus (SARS-CoV) (1Drosten C. Gunther S. Preiser W. van der Werf S. Brodt H.R. Becker S. Rabenau H. Panning M. Kolesnikova L. Fouchier R.A. Berger A. Burguiere A.M. Cinatl J. Eickmann M. Escriou N. Grywna K. Kramme S. 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These coronaviruses are large, enveloped, positive single-stranded RNA viruses (27-31 kb) that cause respiratory and enteric diseases in humans and other animals. The SARS-CoV genome comprises about 29,700 nucleotides and encodes two overlapping polyproteins, pp1a (486 kDa) and pp1ab (790 kDa) that mediate all the functions required for viral replication and transcription (5Marra M.A. Jones S.J. Astell C.R. Holt R.A. Brooks-Wilson A. Butterfield Y.S. Khattra J. Asano J.K. Barber S.A. Chan S.Y. Cloutier A. Coughlin S.M. Freeman D. Girn N. Griffith O.L. Leach S.R. Mayo M. McDonald H. Montgomery S.B. Pandoh P.K. Petrescu A.S. Robertson A.G. Schein J.E. Siddiqui A. Smailus D.E. Stott J.M. Yang G.S. Plummer F. Andonov A. Artsob H. Bastien N. Bernard K. Booth T.F. Bowness D. Czub M. Drebot M. Fernando L. Flick R. Garbutt M. Gray M. Grolla A. Jones S. Feldmann H. Meyers A. Kabani A. Li Y. Normand S. Stroher U. Tipples G.A. Tyler S. Vogrig R. Ward D. Watson B. Brunham R.C. Krajden M. 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SARS-CoV 3CLpro cleaves the polyproteins at eleven sites involving a conserved Gln at the P1 position and a small amino acid (Ser, Ala, or Gly) at the P1′ position, a process initiated by enzyme's own autolytic cleavage (autoprocessing) (10Fan K. Wei P. Feng Q. Chen S. Huang C. Ma L. Lai B. Pei J. Liu Y. Chen J. Lai L. J. Biol. Chem. 2004; 279: 1637-1642Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). Several crystal structures of coronavirus 3CLpro (apo form or with suicide inhibitors) reported from TGEV, HCoV 229E, and SARS-CoV (11Anand K. Palm G.J. Mesters J.R. Siddell S.G. Ziebuhr J. Hilgenfeld R. EMBO J. 2002; 21: 3213-3224Crossref PubMed Scopus (470) Google Scholar, 12Anand K. Ziebuhr J. Wadhwani P. Mesters J.R. Hilgenfeld R. Science. 2003; 300: 1763-1767Crossref PubMed Scopus (1275) Google Scholar, 13Yang H. Yang M. Ding Y. Liu Y. Lou Z. Zhou Z. Sun L. Mo L. Ye S. Pang H. Gao G.F. Anand K. Bartlam M. Hilgenfeld R. Rao Z. U. S. A. 2003; PubMed Scopus Google a in two and one The active site of SARS-CoV 3CLpro is in the of the and and includes a of and was to mediate dimer because the C-terminal a dimer J. Wei Z. J. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). the of the C-terminal of in the structure H. Yang M. Ding Y. Liu Y. Lou Z. Zhou Z. Sun L. Mo L. Ye S. Pang H. Gao G.F. Anand K. Bartlam M. Hilgenfeld R. Rao Z. U. S. A. 2003; PubMed Scopus Google Scholar). the crystal structures of the wild-type and the C145A mutant the three-dimensional structure of the wild-type protease H. Yang M. Ding Y. Liu Y. Lou Z. Zhou Z. Sun L. Mo L. Ye S. Pang H. Gao G.F. Anand K. Bartlam M. Hilgenfeld R. Rao Z. U. S. A. 2003; PubMed Scopus Google the new crystal structure of C145A shows C-terminal that are into the protomer a product-bound structure that to an for viral its mechanism is further the maturation a wild-type SARS-CoV 3CLpro with 10 additional amino acids that are of the polyprotein at the N and/or and We used analytical to quaternary In and glutathione to autoprocessing by The mutant C145A with the served as a substrate for facilitated by the wild-type 3CLpro is for polyprotein it is a for We used a to the protease and identified and as of SARS-CoV 3CLpro Wang Huang Lin Huang Chen FEBS Lett. 2004; PubMed Scopus Google Scholar, 2004; PubMed Scopus Google Scholar, Ma Cheng Y.S. Huang D. A. Liu J.M. Chen S.T. U. S. A. 2004; PubMed Scopus Google Scholar). small SARS-CoV 3CLpro identified from acids U. J. A. S.A. 2004; PubMed Scopus Google a Lee J.A. Huang L. Chen G. Chen Z. Zhang L. T. Chan K.H. H. A.P. Ng L.W. H.W. Yang D. D.D. Yuen K.Y. Chem. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google a J.E. C. Chem. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google and Zhang J. C. J.C. J.C. J. Med. Chem. 2004; PubMed Scopus Google Scholar). These are all active site inhibitors. The new structures here the of design protease maturation. study provides insights to substrate protein and for SARS substrate was as 2004; PubMed Scopus Google Scholar). The and from and the protein the and and from The was from and of the and of SARS-CoV and C145A with and extra amino acids in N and of the SARS proteases in N-terminal Trx, and C-terminal or N-terminal The the wild-type 3CLpro was used as a and the mutant for C145A was used in a chain to the mutant mutant containing 10 extra amino acids from the pp1a polyprotein to the N the was for extra amino acids from the pp1a polyprotein to its the was and for the and The of the and the of the are for into the These used to the wild-type SARS protease containing extra N- and C-terminal amino acids and In a of a with the at for at for and at for The was to in and the was with The with the was and the was a The was to the by for at of the N-terminal and C-terminal protease the in the with the and was used as a This containing the and the of used with the of and the of to into The protease was used to that a containing from the and in of containing at The SARS protease of the from the was The was into for protein The protein followed reported 2004; PubMed Scopus Google Scholar). the protease and for the N-terminal and by protease and the was another to the the to used as the substrate for facilitated and protein SARS-CoV 3CLpro and mutant C145A in with a site the N-terminal of the protein was a and the cleavage by FXa, the was a and with the and The containing 3CLpro and for 3CLpro was in a containing 10 and The protein was the by of the wild-type 3CLpro protein with of the and a with of the The at in the for C145A was by and a and a containing 10 and The protein was the by of the C145A with of the Bicine, and and of a with of the was at in the for and for data with the and in The was with an with a data for and C145A at in and in and by the Z. W. of in Scholar). and wild-type structure was by one of HCoV K. Ziebuhr J. Wadhwani P. Mesters J.R. Hilgenfeld R. Science. 2003; 300: 1763-1767Crossref PubMed Scopus (1275) Google as the and with the Biol. PubMed Scopus Google Scholar). The with D.E. J. Biol. PubMed Scopus Google the of The and P. R.W. J.S. J. M. T. Biol. PubMed Scopus Google was used for structure and in The of a was used to of containing The to = and with the by at for The enzyme was at The was to yield of which was in of containing and in the of was used to the at The was and the was The was a which was with the the with the the was with the The was to for to the of the protease protease by autoprocessing of by at the N and of the tagged C145A mutant followed by to the of the substrate and the formation of the with The containing and the active protease was at for or in and the was by SARS 3CLpro and to which the of substrate and Analytical and C145A mutant SARS-CoV 3CLpro with extra N- or C-terminal amino acids at a of of used for the of with the and by a analytical ultracentrifuge with an as J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). was at at with The of the was for with the The was used to and The was used to the of all of the SARS-CoV and for the wild-type and C145A 3CLpro are in The wild-type crystal belongs to the with one in an asymmetric that the two in the dimer are the 3CLpro structures from HCoV 229E, TGEV, and SARS-CoV (11Anand K. Palm G.J. Mesters J.R. Siddell S.G. Ziebuhr J. Hilgenfeld R. EMBO J. 2002; 21: 3213-3224Crossref PubMed Scopus (470) Google Scholar, 12Anand K. Ziebuhr J. Wadhwani P. Mesters J.R. Hilgenfeld R. Science. 2003; 300: 1763-1767Crossref PubMed Scopus (1275) Google Scholar, 13Yang H. Yang M. Ding Y. Liu Y. Lou Z. Zhou Z. Sun L. Mo L. Ye S. Pang H. Gao G.F. Anand K. Bartlam M. Hilgenfeld R. Rao Z. U. S. A. 2003; PubMed Scopus Google SARS-CoV 3CLpro structure is of a for and for The active site is and The six C-terminal in the wild-type protease are in to those of the C145A The sequences the N- and C-terminal sites of 3CLpro in coronaviruses are in and for wild-type and C145A = in to the is the of a and are and structure from of in to the is the of a and are and structure in a new of SARS-CoV C145A 3CLpro (C145A) crystal belongs to the with one dimer in the asymmetric unit, the two in a dimer are The two of the dimeric and are to and protomer as in the novel here is that the active site of protomer is with the C-terminal of protomer as a in and as a in from the dimer in another asymmetric The N-terminal of protomer as a in and as a in are the active site of protomer B. This structure reveals the in which the is in the active site the maturation and the six amino acids at the terminus of protomer the to P1 sites of the The and of SARS 3CLpro dimer are in and of SARS 3CLpro of N- terminus from protomer of N- terminus from protomer of terminus from protomer in a new of SARS-CoV 3CLpro further the six 3CLpro in the of here to the protein autoprocessing to yield mature SARS protease by the 10 amino acids the N terminus of mature The protease to to the because the was by The shows at for this The was for which cleavage sites in N and because it was In the cleavage site in the N terminus was in the and of mutant C145A all a in because the C145A the protein with to its by active The can from the substrate in the N-terminal was followed by cleavage of the C-terminal The to the in the The kDa) to the 3CLpro in in the which The data in that the for the formation of and is and the N terminus is than the C-terminal of and to the quaternary structures of and the wild-type 3CLpro and dimer The data for the wild-type SARS protease as in indicate that the is that of a dimer and the dimeric wild-type protein a of 0.35 In and which N- or C-terminal 10 extra amino shows and and 5.6 with 10 extra amino acids in the N- or dimer formation is with the N-terminal a The of the tagged protease that immature 3CLpro can form a small of dimer enabling it to undergo autoprocessing to yield the mature enzyme, which further serves as a seed for facilitated maturation. C145A from protomer in the the active site of C145A protomer The of the with the active site amino acids are in In the the of a with side chain of The of an to the of the at the of with the N of of with and by hydrophobic is by in to the active the of with the P1 and form the of the site. The site of C145A is by the of and as as the of and suggesting that the P2 site a side chain as or The N in the main chain of with the of and the side chain with and hydrophobic the with of The side chain of is The of the a from the N of and the which a hydrophobic side The of a from the of and the N of the a to the of The side chain of with and hydrophobic The is of of and is in with the The site is at the of the the and of with the N and of of 3CLpro of the form and the product-bound form of the C145A active sites are in We the crystal structure of chain of SARS 3CLpro with a in which the and in and chain in The crystal structures of 3CLpro of HCoV and with SARS 3CLpro, the of the of C145A protomer and those of chain are C145A protomer in which is from the active site that the pocket is than the product-bound In with the active site pocket of 3CLpro of other the is SARS-CoV 3CLpro crystal structures in the structure of the six C-terminal of SARS 3CLpro in that of the in of 3CLpro reported K. Ziebuhr J. Wadhwani P. Mesters J.R. Hilgenfeld R. Science. 2003; 300: 1763-1767Crossref PubMed Scopus (1275) Google for and and in in the active site of protomer the P1 site. an of 3C protease S.A. T.F. R.A. Zhou R. J. J.W. R.A. S.L. M.A. D.M. S.T. U. S. A. PubMed Scopus Google to a for SARS-CoV 3CLpro K. Ziebuhr J. Wadhwani P. Mesters J.R. Hilgenfeld R. Science. 2003; 300: 1763-1767Crossref PubMed Scopus (1275) Google Scholar). in which the with the six C-terminal of protomer the is In at the SARS-CoV 3CLpro report for the C-terminal six amino acids of SARS 3CLpro protomer which into the active site of the protomer B. The product-bound structure provides the first of an SARS viral protease maturation. SARS 3CLpro its N and to the active for a novel to the viral maturation the mature dimer is In a the N-terminal amino acids called the with and of the and of the other the crystal structure of Mpro, it in the N terminus of one the active site of the other and a position at the of the active site K. Ziebuhr J. Wadhwani P. Mesters J.R. Hilgenfeld R. Science. 2003; 300: 1763-1767Crossref PubMed Scopus (1275) Google Scholar). report that the replication to the in an and the to at the protease by the data the maturation process to is likely that the main protease a dimer that the site to other cleavage sites in the data that wild-type SARS 3CLpro a remarkably small dimer and that the extra amino acids in the N or terminus cause a in the This is with the study that the SARS 3CLpro containing an C-terminal that a = at Lin Lin 2004; PubMed Scopus Google Scholar). the wild-type enzyme containing the N- or C-terminal a small of the active which undergoes as This for the mutant C145A protease with extra amino acids of the N and The of autoprocessing from shows that the N-terminal cleavage the C-terminal This is with that a with the N-terminal cleavage is a substrate than one with the C-terminal cleavage sites an (10Fan K. Wei P. Feng Q. Chen S. Huang C. Ma L. Lai B. Pei J. Liu Y. Chen J. Lai L. J. Biol. Chem. 2004; 279: 1637-1642Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). the newly active dimeric SARS main protease can as a seed for further chain to the protease and the other In for a of proteases as and M.S. K. C.R. D. S.A. C. J. Jones C.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. M. Y. M. D.M. R. J.P. Becker J.W. Biol. PubMed Scopus Google Scholar). protomer can the as by the crystal structure of C145A in a product-bound form and the at the N terminus is than that at the terminus at the mature 3CLpro dimer the N terminus of one protomer is to the active site of the other protomer These to a of SARS 3CLpro maturation. in with in the protease as and and with at the N terminus and at the the maturation can into The N-terminal of one polyprotein the active site of the other polyprotein to the protease is (10Fan K. Wei P. Feng Q. Chen S. Huang C. Ma L. Lai B. Pei J. Liu Y. Chen J. Lai L. J. Biol. Chem. 2004; 279: 1637-1642Abstract Full Text Full Text PDF PubMed Scopus (262) Google the for maturation is The N-terminal of the polyprotein is by the polyprotein N-terminal the polyprotein can to its position in the immature of of the N terminus is from the the P1 in the C145A structure and the N terminus of the mature as in The C-terminal cleavage by the C-terminal into the immature dimer the product-bound form of and the mature dimer is The crystal structure of C145A in a product-bound form here the mechanism for the structure-based protease SARS protease because the of the active site pocket of SARS 3CLpro from those of of 3CLpro is from SARS 3CLpro, that with the at P1 site. of the N and are to the active site of the SARS-CoV 3CLpro to the new structure of C145A in a product-bound the dimer the protease maturation. In the viral protease in of 2002; PubMed Scopus Google Scholar). new structure of the C145A mutant provides the for design of the to the maturation which is in with