Severe Acute Respiratory Syndrome Coronavirus 3C-like Proteinase N Terminus Is Indispensable for Proteolytic Activity but Not for Enzyme Dimerization

Abstract

Severe acute respiratory syndrome (SARS) coronavirus is a novel human coronavirus and is responsible for SARS infection. SARS coronavirus 3C-like proteinase (SARS 3CLpro) plays key roles in viral replication and transcription and is an attractive target for anti-SARS drug discovery. In this report, we quantitatively characterized the dimerization features of the full-length and N-terminal residues 1–7 deleted SARS 3CLpros by using glutaraldehyde cross-linking SDS-PAGE, size-exclusion chromatography, and isothermal titration calorimeter techniques. Glutaraldehyde cross-linking SDS-PAGE and size-exclusion chromatography results show that, similar to the full-length SARS 3CLpro, the N-terminal deleted SARS 3CLpro still remains a dimer/monomer mixture within a wide range of protein concentrations. Isothermal titration calorimeter determinations indicate that the equilibrium dissociation constant (Kd) of the N-terminal deleted proteinase dimer (262 μm) is very similar to that of the full-length proteinase dimer (227 μm). Enzymatic activity assay using the fluorescence resonance energy transfer method reveals that N-terminal deletion results in almost complete loss of enzymatic activity for SARS 3CLpro. Molecular dynamics and docking simulations demonstrate the N-terminal deleted proteinase dimer adopts a state different from that of the full-length proteinase dimer, which increases the angle between the two protomers and reduces the binding pocket that is not beneficial to the substrate binding. This conclusion is verified by the surface plasmon resonance biosensor determination, indicating that the model substrate cannot bind to the N-terminal deleted proteinase. These results suggest the N terminus is not indispensable for the proteinase dimerization but may fix the dimer at the active state and is therefore vital to enzymatic activity. Severe acute respiratory syndrome (SARS) coronavirus is a novel human coronavirus and is responsible for SARS infection. SARS coronavirus 3C-like proteinase (SARS 3CLpro) plays key roles in viral replication and transcription and is an attractive target for anti-SARS drug discovery. In this report, we quantitatively characterized the dimerization features of the full-length and N-terminal residues 1–7 deleted SARS 3CLpros by using glutaraldehyde cross-linking SDS-PAGE, size-exclusion chromatography, and isothermal titration calorimeter techniques. Glutaraldehyde cross-linking SDS-PAGE and size-exclusion chromatography results show that, similar to the full-length SARS 3CLpro, the N-terminal deleted SARS 3CLpro still remains a dimer/monomer mixture within a wide range of protein concentrations. Isothermal titration calorimeter determinations indicate that the equilibrium dissociation constant (Kd) of the N-terminal deleted proteinase dimer (262 μm) is very similar to that of the full-length proteinase dimer (227 μm). Enzymatic activity assay using the fluorescence resonance energy transfer method reveals that N-terminal deletion results in almost complete loss of enzymatic activity for SARS 3CLpro. Molecular dynamics and docking simulations demonstrate the N-terminal deleted proteinase dimer adopts a state different from that of the full-length proteinase dimer, which increases the angle between the two protomers and reduces the binding pocket that is not beneficial to the substrate binding. This conclusion is verified by the surface plasmon resonance biosensor determination, indicating that the model substrate cannot bind to the N-terminal deleted proteinase. These results suggest the N terminus is not indispensable for the proteinase dimerization but may fix the dimer at the active state and is therefore vital to enzymatic activity. Between the end of year 2002 and June of year 2003, a highly contagious illness called severe acute respiratory syndrome (SARS) 1The abbreviations used are: SARS, severe acute respiratory syndrome; SARS-CoV, severe acute respiratory syndrome coronavirus; 3CLpro, 3C-like proteinase; SEC, size-exclusion chromatography; ITC, isothermal titration calorimeter; FRET, fluorescence resonance energy transfer; MD, molecular dynamics; SPR, surface plasmon resonance; TGEV, transmissible gastroenteritis virus; DTT, dithiothreitol; FPLC, fast protein liquid chromatography; RU, resonance units; HIPs, hydrophobic interaction pairs; EDANS, 5-[(2′-aminoethyl)-amino]naphthelenesulfonic acid; Dabcyl, 4-[[4-(dimethylamino)phenyl]azo]benzoic acid. broke out in China and quickly spread to more than 30 other countries. A novel coronavirus, named SARS coronavirus (SARS-CoV), was soon identified as the etiological agent of the infection (1Kathryn V.H. J. Clin. Investig. 2002; 111: 1605-1609Google Scholar, 2Peiris J.S. Lai S.T. Poon L.L. Guan Y. Yam L.Y. Lim W. Nicholls J. Yee W.K. Yan W.W. Cheung M.T. Cheng V.C. Chan K.H. Tsang D.N. Yung R.W. Ng T.K. Yuen K.Y. Lancet. 2003; 361: 1319-1325Abstract Full Text Full Text PDF PubMed Scopus (2361) Google Scholar, 3Fouchier R.A. Kuiken T. Schutten M. Amerongen VanG. Van Doornum G.J. Van Den Hoogen B.G. Peiris M. Lim W. Stohr K. Osterhaus A.D. Nature. 2003; 423: 240Crossref PubMed Scopus (693) Google Scholar). It was revealed that the open reading frame of the replicase gene in SARS-CoV genome, similar to other coronaviruses (4Herold J. Raabe T. Schelle-Prinz B. Siddell S.G. Virology. 1993; 195: 680-691Crossref PubMed Scopus (98) Google Scholar, 5Lee H.J. Shieh C.K. Gorbalenya A.E. Koonin E.V. La Monica N. Tuler J. Bagdzhadzhyan A. Lai M.M. Virology. 1991; 180: 567-582Crossref PubMed Scopus (278) Google Scholar), features the sequence motifs of both papain-like proteinase and 3C-like proteinase (3CLpro). SARS 3CLpro is fully conserved among all released SARS coronavirus genome sequences and is highly homologous to other coronavirus 3CL proteinases (6Thiel V. Ivanov K.A. Putics A. Hertzig T. Schelle B. Bayer B. Gorbalenya A.E. J. J. 2003; PubMed Scopus Google Scholar, V. J. Poon L.L. Guan Y. M. Gorbalenya A.E. J. 2003; PubMed Scopus Google Scholar). of in the coronavirus SARS 3CLpro an attractive target in anti-SARS two of 3CL proteinases from human coronavirus and transmissible gastroenteritis and in a of for the K. J. 2003; PubMed Scopus Google Scholar). of SARS 3CLpro and an revealed and an model for binding M. Y. Y. K. M. A. 2003; PubMed Scopus Google Scholar). the that coronavirus 3CL proteinases conserved at the SARS 3CLpro the and an a the is in a between two residues a to by a K. J. 2003; PubMed Scopus Google Scholar, M. Y. Y. K. M. A. 2003; PubMed Scopus Google Scholar, K. G.J. Siddell S.G. J. J. 2002; PubMed Scopus Google Scholar). the of 3CL proteinases in different coronaviruses similar K. J. 2003; PubMed Scopus Google Scholar, M. Y. Y. K. M. A. 2003; PubMed Scopus Google and all of 3CLpro in the of the dimer that the dimer the of 3CLpro K. J. 2003; PubMed Scopus Google Scholar). the of coronavirus that a in substrate and responsible for the N terminus of to the active of the other M. Y. Y. K. M. A. 2003; PubMed Scopus Google Scholar, K. G.J. Siddell S.G. J. J. 2002; PubMed Scopus Google Scholar, J. Gorbalenya A.E. J. PubMed Scopus Google Scholar). of in dimerization and enzymatic activity of SARS 3CLpro by a J. J. J. Full Text Full Text PDF PubMed Scopus Google Scholar). In Lai and K. Lai B. J. Y. J. Lai J. Full Text Full Text PDF PubMed Scopus Google that SARS 3CLpro as a mixture at a protein and is at a protein in and the dimer was the active of the proteinase. the and of coronavirus was that the N-terminal residues 1–7 an in both dimerization and enzymatic activity of SARS 3CLpro M. Y. Y. K. M. A. 2003; PubMed Scopus Google Scholar). molecular dynamics simulations revealed that the N terminus was not for the dimer of SARS 3CLpro, the N-terminal deleted SARS 3CLpro may a dimer a state different from the full-length proteinase. of the of the N terminus in dimerization and enzymatic activity of SARS 3CLpro may to the of In the we and of the dimerization and of the full-length and N-terminal residues 1–7 deleted SARS 3CLpros using fluorescence resonance energy transfer glutaraldehyde cross-linking SDS-PAGE, size-exclusion chromatography and isothermal titration calorimeter techniques. results the simulations two It is that the N terminus of SARS 3CLpro is for enzymatic activity but not for N-terminal deleted SARS 3CLpro still a dimer, and the binding between the two protomers is almost to that of the full-length SARS 3CLpro. the full-length SARS 3CLpro, the N-terminal deleted SARS 3CLpro a dimer a different which reduces the activity of SARS 3CLpro. This conclusion is verified by molecular docking and surface plasmon resonance biosensor determination, indicating that the N-terminal deleted SARS 3CLpro almost the binding to a model and used in this from and the and the from and from and from and molecular for SDS-PAGE from other of and from and N-terminal SARS 3CLpros full-length SARS 3CLpro was to method T. J. J. J. Y. Y. K. Y. 2003; PubMed Scopus Google Scholar). proteinase was highly to SDS-PAGE and to and protein was by N-terminal and and by protein used in all was from the at of N-terminal deleted SARS 3CLpro was by a method using 3CLpro as sequences of the used for N-terminal deletion as and 3CLpro the N-terminal deleted of SARS 3CLpro was verified by and and the protein was and in a similar to that of the full-length proteinase. and of the N-terminal deleted proteinase by SDS-PAGE, N-terminal and proteinase was in DTT, at a protein was in at of from to using a and by the for the a range from 30 to was at by of was and the was the of the Enzymatic enzymatic of the full-length and N-terminal deleted SARS 3CLpros by using a that was and characterized by using a by PubMed Scopus Google Scholar, L.L. T. J. PubMed Scopus Google Scholar). of the substrate was in and at using the assay DTT, and the of than in a a at the the μm) was for 30 at in the the assay by the of the substrate to a of in fluorescence was at a a fluorescence a the the was to the substrate in the assay of the substrate as a of was by the fluorescence at a at and the was from the of the fluorescence of the substrate was by the full-length SARS 3CLpro fluorescence at was Enzymatic activity was the of and the activity of the full-length SARS 3CLpro was as Glutaraldehyde the full-length and N-terminal deleted SARS 3CLpros from to in DTT, an of glutaraldehyde was to a of This was at for 30 by the cross-linking by of the was in and at for SDS-PAGE was of the full-length and N-terminal deleted SARS 3CLpros by size-exclusion chromatography a an used was DTT, and was and the was the protein a wide range of from to the and the at a of by of at of by was by using a molecular the A A and by the of to the of molecular for equilibrium out at using a titration calorimeter the was by of the assay and the was out using a the full-length N-terminal deleted SARS 3CLpro and at of protein between and A 30 of of protein the the assay and a between This titration to a of for using and in of a dissociation model to the dissociation constant of the dimer (Kd) and the of dimerization using the Molecular molecular dynamics simulations using the Scopus Google the Scholar). for the simulations the of SARS 3CLpro at a M. Y. Y. K. M. A. 2003; PubMed Scopus Google Scholar). the model of the full-length SARS 3CLpro dimer for the the of the and in using the molecular model of the N-terminal deleted proteinase dimer was by residues 1–7 from model These two in a was by the model J. B. Scholar). by the B. J. Scopus Google Scholar). was a the between the protein and the to that the protein not the of the of used a of for the and the by and in model and by and in model at of the the model protein and in a and model and in a and the protein the of the protein by the and the and to two simulations and the N-terminal sequence of a substrate for SARS 3CLpro K. Lai B. J. Y. J. Lai J. Full Text Full Text PDF PubMed Scopus Google was as a model binding of the full-length and N-terminal deleted SARS 3CLpros to the substrate using the biosensor A. M. A. B. N. 2002; PubMed Scopus Google Scholar). using the full-length N-terminal deleted proteinase was to of the of the surface by using and and a of protein was to the of a by in the by a mixture of and for an of the full-length and N-terminal deleted proteinases to the the protein at the in was the surface a was by for in a of as the substrate was in the and in a of μm). binding in resonance at a of as and as a of by using for equilibrium by the binding model in the and of the N-terminal the similar for the full-length SARS 3CLpro T. J. J. J. Y. Y. K. Y. 2003; PubMed Scopus Google Scholar), the N-terminal deleted SARS 3CLpro was and in of the in the of the at for molecular of the characterized by not of the full-length and N-terminal deleted proteinases and which in the from the protein the of the full-length and N-terminal deleted of the two a at of a and at and of an indicating that the of the N-terminal deleted SARS 3CLpro is similar to that of the full-length and similar to of and 3CLpros K. J. 2003; PubMed Scopus Google Scholar). This the of by N-terminal deletion of the two was by the of at within from 30 to the results in the N-terminal deletion not the of the and the N-terminal deleted proteinase adopts a similar to that of the full-length proteinase. of the N-terminal deleted proteinase was as which is very to that of the full-length results that N-terminal deletion not the and of the of the full-length and N-terminal deleted SARS of the full-length and N-terminal deleted SARS 3CLpros at at a range from 30 to for the full-length and N-terminal deleted SARS used in and all protein in Enzymatic K. G.J. Siddell S.G. J. J. 2002; PubMed Scopus Google that the N-terminal residues deleted of 3CLpro is almost this and the M. Y. Y. K. M. A. 2003; PubMed Scopus Google that the N terminus is indispensable for enzymatic activity of SARS 3CLpro. this we the enzymatic of both the full-length and N-terminal residues 1–7 deleted SARS 3CLpros using the assay is a and method for proteinase activity In this the method used to for proteinases PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, M. 2003; PubMed Scopus Google Scholar). of the substrate an in fluorescence as the and which used to the of the enzymatic a for of the enzymatic activity of SARS 3CLpro PubMed Scopus Google Scholar). In this we used a substrate an to the enzymatic of the full-length and N-terminal deleted SARS 3CLpros L.L. T. J. PubMed Scopus Google Scholar). of the substrate by the full-length SARS 3CLpro results in an in fluorescence at the of fluorescence of the substrate is in a the of the substrate in the assay in the of 3CLpro fluorescence not that the substrate by the full-length SARS 3CLpro. was as from the of the In we the of the enzymatic activity for the full-length and we that SARS 3CLpro a activity at This is in the K. Lai B. J. Y. J. Lai J. Full Text Full Text PDF PubMed Scopus Google Scholar, K. Y. Lai PubMed Scopus Google Scholar), indicating that method for SARS 3CLpro enzymatic activity is the fluorescence of the substrate by the N-terminal deleted SARS 3CLpro is the of the activity assay that the N-terminal deleted 3CLpro is almost This the of the N terminus for enzymatic activity of SARS 3CLpro. of the and N-terminal revealed that SARS 3CLpro, similar to 3CL proteinases of and K. J. 2003; PubMed Scopus Google Scholar), a dimer in the and as a mixture of and dimer in M. Y. Y. K. M. A. 2003; PubMed Scopus Google Scholar, J. J. J. Full Text Full Text PDF PubMed Scopus Google Scholar, K. Lai B. J. Y. J. Lai J. Full Text Full Text PDF PubMed Scopus Google Scholar). the of 3CLpros in different coronaviruses the and all the of 3CLpro in the of the dimer that the of 3CLpro responsible for activity K. J. 2003; PubMed Scopus Google Scholar, M. Y. Y. K. M. A. 2003; PubMed Scopus Google Scholar). the other the of SARS 3CLpro that the N-terminal residues 1–7 between and of the and of the other to a of very the other in the this was that the N-terminal residues 1–7 an in both proteinase dimerization and enzymatic activity M. Y. Y. K. M. A. 2003; PubMed Scopus Google Scholar). dimerization of SARS 3CLpro and the of the N terminus in dimer we SDS-PAGE using a glutaraldehyde cross-linking method K. A. V. 2002; PubMed Scopus Google Scholar). results in the full-length proteinase at a of a of other to the dimer of the proteinase. the of glutaraldehyde the full-length SARS 3CLpro a similar cross-linking indicating that the of glutaraldehyde was and cross-linking protein the and was almost to the of the at and These results suggest that dimerization of the full-length SARS 3CLpro is the results J. J. J. Full Text Full Text PDF PubMed Scopus Google Scholar, K. Lai B. J. Y. J. Lai J. Full Text Full Text PDF PubMed Scopus Google Scholar). similar results the N-terminal deleted proteinase. of the N-terminal deleted SARS 3CLpro within a wide range of protein concentrations. the N-terminal deleted 3CLpro at a of that as and dimer and the was the at a protein the protein to the of the was and the to the of the N-terminal deleted 3CLpro was than that of the dimer and These results indicate that the N-terminal deletion to the dimerization of 3CLpro. this is not fully the conclusion M. Y. Y. K. M. A. 2003; PubMed Scopus Google Scholar), which that the N terminus plays an in both dimerization and of the active of SARS 3CLpro. In to more the equilibrium of SARS 3CLpro in and to the of the N-terminal deletion in 3CLpro a out by using the method A. V. 2002; PubMed Scopus Google Scholar). is in was for of the of the protein In the similar to for cross-linking SDS-PAGE the to and 3CLpros within a wide range of protein the full-length 3CLpro at two at and that the at is between the of and for the two molecular of and similar that this to the state of SARS 3CLpro other at is than the of for the molecular protein of which is to the of 3CLpro. This that the full-length 3CLpros the both of and of the dimer and by the of two to the of the the dimer/monomer for the of and as and indicating that 3CLpro dimerization is This is in the results from glutaraldehyde cross-linking SDS-PAGE determinations and J. J. J. Full Text Full Text PDF PubMed Scopus Google Scholar, K. Lai B. J. Y. J. Lai J. 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In a similar the of the N-terminal deleted 3CLpro was which is in and indicating that dimerization of the N-terminal deleted proteinase is and dimer dissociation and for dimerization of the two proteinases from and the and for dimerization by using in N-terminal deletion not the binding between two protomers of the proteinase and and the binding for dimerization of the full-length and N-terminal deleted proteinases and These results demonstrate that the N terminus is not for the proteinase In the and for dimerization in indicate that, for dimerization of SARS 3CLpro, the N-terminal deletion the and by and for SARS at and of in by a dissociation of in by a dissociation from the from the of in by a dissociation from the in a of the the of the N-terminal deletion dimerization of SARS 3CLpro and to the N terminus is indispensable for activity but not for dimerization at the molecular and simulations both the full-length and N-terminal deleted SARS of the to an of the of the of interaction between the two protomers of and and and in and the full-length proteinase dimer the interaction energy between the two protomers is an the interaction energy the N-terminal deleted proteinase of N-terminal the interaction energy and and between the two protomers at the of the the the two protomers of model may and the dimer to more and more both the and and the interaction energy results indicate that the N-terminal deletion reduces the interaction which is in the for dimerization we the between the two protomers of the two is in between the two protomers for the N-terminal deleted proteinase at the of the but the between the two protomers for the full-length proteinase residues in the at the for both full-length and N-terminal deleted proteinases in which that the not a of the N-terminal the of the N-terminal deletion not the between the two the dynamics of the two SARS we the between two protomers for the two angle between two protomers was as the of two in from the of of to the of of and is in the full-length the angle between two protomers the for the N-terminal deleted this angle increases from to indicating that the two protomers of the N-terminal deleted proteinase a dimer a state different from that of the full-length proteinase. of equilibrium of the N-terminal deleted proteinase dimer that of the full-length proteinase dimer is in which the dimerization state from the full-length proteinase dimer to the N-terminal deleted proteinase and of A and as by the of dimer is an for the of the of the between the two protomers of and which in for the full-length SARS 3CLpro remains the of the N-terminal the to at the of the of the between the two the increases to the that the of the 3CLpro is the dimer of the N-terminal deleted proteinase adopts a different state from the full-length an almost to that of the full-length proteinase. to the the hydrophobic interaction between the two protomers for both the full-length and N-terminal deleted proteinases the and the is in the N-terminal deletion the at the of the for the N-terminal deleted proteinase the and the for the full-length proteinase. the N-terminal deletion not the hydrophobic interaction between the two the of the N-terminal deletion the we the of the of the of and the and the results in and of both the full-length and N-terminal deleted proteinases the binding pocket of the full-length proteinase is that of the N-terminal deleted proteinase is a of demonstrate the of the of the binding pocket for the N-terminal deleted SARS 3CLpro to the substrate we an A model substrate the N-terminal sequence of a substrate for SARS 3CLpro K. Lai B. J. Y. J. Lai J. Full Text Full Text PDF PubMed Scopus Google was as a model substrate to the binding of a of from the of and using the J. Scopus Google Scholar), and the binding energy of the substrate to was using the docking is in the binding energy of the substrate to and the in binding of the substrate to the two proteinases and indicating that the binding of the N-terminal deleted SARS 3CLpro to the substrate is than that of the full-length SARS 3CLpro to the This may of the the N-terminal deleted proteinase is in the substrate cannot bind the binding This is in the determination, as of the to the and N-terminal the from simulations that the N-terminal deletion reduces the of the binding pocket of SARS 3CLpro and the substrate to bind the binding the model substrate was to the binding binding of the to the full-length and N-terminal deleted SARS 3CLpros was by using biosensor the of the for at a of to for binding the proteinase the of the full-length and N-terminal deleted proteinases the in the full-length proteinase the in a and in and fast binding and dissociation indicating a and to a binding model in the for binding dissociation constant between the substrate and the full-length proteinase was as the N-terminal deleted a and was indicating that almost binding between the substrate and the N-terminal deleted proteinase. This is the results of the and docking In this the and dimerization of both the full-length and N-terminal deleted SARS 3CLpros fully by using FRET, glutaraldehyde cross-linking SDS-PAGE, SEC, ITC, and 3CLpros of coronaviruses of roles in replication K. J. 2003; PubMed Scopus Google Scholar, M. Y. Y. K. M. A. 2003; PubMed Scopus Google Scholar, J. Siddell S.G. J. PubMed Google Scholar). SARS-CoV is a novel coronavirus year the of SARS, sequence that SARS 3CLpro the 3CLpros of other coronaviruses as and human K. J. 2003; PubMed Scopus Google Scholar, B. J. L.L. T. 2003; Google Scholar). of the 3CLpros of TGEV, human and SARS revealed that the of 3CL proteinases more conserved than sequences K. J. 2003; PubMed Scopus Google Scholar, M. Y. Y. K. M. A. 2003; PubMed Scopus Google Scholar). that the and and of coronavirus 3CLpros the and of coronavirus 3CLpros human and a for the N terminus of the coronavirus 3CLpros in both the enzymatic activity and the N terminus is indispensable for both the activity and dimerization K. J. 2003; PubMed Scopus Google Scholar, M. Y. Y. K. M. A. 2003; PubMed Scopus Google Scholar). In that the N-terminal residues 1–7 deleted SARS 3CLpro a that is similar to the full-length proteinase This that the N-terminal deletion not the and of the proteinase. the 3CLpro of TGEV, the N-terminal deletion almost the enzymatic activity of SARS 3CLpro This verified the by M. Y. Y. K. M. A. 2003; PubMed Scopus Google Scholar). glutaraldehyde cross-linking SDS-PAGE the that N-terminal residues 1–7 an in dimerization of SARS 3CLpro and 3CLpros of other coronaviruses M. Y. Y. K. M. A. 2003; PubMed Scopus Google Scholar, K. G.J. Siddell S.G. J. J. 2002; PubMed Scopus Google Scholar), SDS-PAGE that the N-terminal deleted SARS 3CLpro dimer and the of dimerization is not by N-terminal deletion revealed that, the full-length equilibrium in the of the N-terminal deleted SARS 3CLpro the cross-linking SDS-PAGE and that the N terminus to of SARS 3CLpro that N-terminal residues 1–7 deletion not dimerization of SARS 3CLpro constant determinations for the proteinase dimer revealed that the binding between the two protomers of the N-terminal deleted proteinase μm) is almost to that between the two protomers of the full-length proteinase μm) simulations indicate that the N-terminal deletion the interaction energy between the two protomers which was by the of the for dimerization of the N-terminal deleted proteinase in that for dimerization of the full-length proteinase the other the N-terminal deletion the of the proteinase and the loss the the two protomers which for the for dimerization of the N-terminal deleted proteinase. This may the the N-terminal deletion almost not the binding of the two simulations that the two protomers of the N-terminal deleted proteinase a dimerization for the N-terminal deleted proteinase a different dimerization state from the between the two which from for the full-length proteinase dimer to for the N-terminal deleted proteinase dimer dimerization state of the N-terminal deleted proteinase the and the interaction between the two by simulations is that the binding pocket of the N-terminal deleted proteinase to which is not beneficial to the substrate binding This may of the the N-terminal deleted proteinase activity. This verified by proteinase substrate docking and by proteinase substrate binding assay of SARS 3CLpro that the N terminus of is between and of M. Y. Y. K. M. A. 2003; PubMed Scopus Google Scholar). that the between the N terminus of and and of to the than the of the N terminus to the of the dimer, but the to an active this verified by the determination, which a