JOURNAL OF VIROLOGY Sept 2008 p 8647 8655 Vol 82 No 17 0022 538X 08 08 00H110010 doi 10 1128 JVI 00298 08 Copyright 2008 American Society for Microbiology All Rights Reserved Structure of the Main Protease from a Global Infectious Human Coronavirus HCoV HKU1 H17188 Qi Zhao 1 Shuang Li 1 Fei Xue 1 Yilong Zou 1 Cheng Chen 1 Mark Bartlam 2 and Zihe Rao 1 2 3 Tsinghua Nankai IBP Joint Research Group for Structural Biology Tsinghua University Beijing 100084 China 1 College of Life Sciences and Tianjin State Laboratory of Protein Sciences Nankai University Tianjin 300071 China 2 and National Laboratory of Biomacromolecules Institute of Biophysics IBP Chinese Academy of Sciences Beijing 100101 China 3 Received 11 February 2008 Accepted 10 June 2008 The newly emergent human coronavirus HKU1 HCoV HKU1 was first identified in Hong Kong in 2005 Infection by HCoV HKU1 occurs worldwide and causes syndromes such as the common cold bronchitis and pneumonia The CoV main protease M pro which is a key enzyme in viral replication via the proteolytic processing of the replicase polyproteins has been recognized as an attractive target for rational drug design In this study we report the structure of HCoV HKU1 M pro in complex with a Michael acceptor inhibitor N3 The structure of HCoV HKU1 provides a high quality model for group 2A CoVs which are distinct from group 2B CoVs such as severe acute respiratory syndrome CoV The structure together with activity assays supports the relative conservation at the P1 position that was discovered by sequencing the HCoV HKU1 genome Combined with structural data from other CoV M pro s the HCoV HKU1 M pro structure reported here provides insights into both substrate preference and the design of antivirals targeting CoVs Coronaviruses CoVs are positive strand RNA viruses that have been identified as the main etiologic agents responsible for a vast number of enteric gastric and respiratory syndromes of both humans and animals 14 17 19 21 23 26 30 31 34 45 CoVs can be divided into three groups group 1 including human CoV 229E HCoV 229E and transmissible gastric en teritis virus TGEV group 2 including HCoV OC43 murine hepatitis virus MHV and bovine CoV BCoV and group 3 including avian infectious bronchitis virus IBV Shortly af ter the emergence of severe acute respiratory syndrome CoV SARS CoV in 2003 group 2 CoVs were further divided into two subgroups termed 2A and 2B 46 The classical group 2 viruses constitute subgroup 2A while the newly emergent SARS CoV and its animal counterparts 37 form subgroup 2B Group 1 and group 2 CoVs have more impact on human health than group 3 since group 3 CoVs such as avian IBV can only infect avian species Following the outbreak of SARS group 2 CoVs have continued to attract greater attention for two reasons First they consist of human viruses SARS CoV and HCoV OC43 as well as several important animal viruses MHV and BCoV that serve as useful models for CoV host interactions Second group 2 CoVs are reported to have crossed the animal to human species barrier in two instances one bat to human transmission in group 2B 27 37 and one transmission event in group 2A CoVs in which BCoV led to the emergence of HCoV OC43 36 Group 2A HCoVs were less widely studied prior to the global SARS epidemic in 2003 However they are closely as sociated with a wide range of acute or chronic respiratory syndromes 3 4 7 9 11 12 15 20 22 35 39 40 47 In the wake of the SARS outbreak several novel HCoVs have been discovered one of which is HCoV HKU1 9 39 HCoV HKU1 has achieved global distribution since it was first iden tified in 2005 infections were first characterized in Hong Kong 26 followed by the identification of several strains of the virus in Korea 9 Europe 5 17 Australia 31 and North America 14 In contrast to the lethal SARS CoV infection by HCoV HKU1 usually leads to self limiting syndromes affecting the lower respiratory tract Nevertheless the consequences could be more severe in patients with a compromised or im mature immune system such as asthma sufferers or newborn infants 24 Genome sequencing has confirmed that the HCoV HKU1 virus belongs to CoV group 2A and shares high sequence homology with MHV and BCoV 39 The functional components of the CoV replication machin ery are released via posttranslational cleavage by two or three proteases These proteases were first designated the papain like protease PLP and 3C like protease 3CL for their re spective sequence homology to the papain and rhinovirus 3C proteases The 3CL protease also is commonly known as the main protease M pro because of the major role it plays in the proteolytic pathway which makes it the most attractive phar macological target for anti CoV drug design CoV M pro s have been intensively studied and crystal structures have been de termined for the M pro s from the following CoVs HCoV strain 229E HCoV 229E 2 porcine TGEV 1 avian IBV 41 and SARS CoV 44 These structures are representative of group 1 HCoV 229E and TGEV group 2B SARS CoV and group 3 IBV CoVs However no structure of the M pro from a group 2A CoV MHV HCoV HKU1 and HCoV OC43 has been determined to date The absence of structural data presents a major obstacle for structure aided drug opti mization targeting group 2A CoVs Corresponding author Mailing address Laboratory of Structural Biology Life Sciences Building Tsinghua University Beijing 100084 China Phone 86 10 62771493 Fax 86 10 62773145 E mail raozh Supplemental material for this article may be found at http jvi asm org H17188 Published ahead of print on 18 June 2008 8647 on April 1 2015 by guest http jvi asm org Downloaded from The M pro s from different CoV groups are homologous in both sequence and main chain architecture They share a sim ilar substrate binding sequence with a requirement for glu tamine at the P1 position and a strong preference for leucine methionine at P2 Based on this information broad spectrum lead compounds 43 with micromolar K i values have been designed that target CoV M pro s However structural data for the M pro s from classical group 2A CoVs still are not available posing a problem for further optimization Although CoV M pro s exhibit absolute specificity for glu tamine in the P1 position recent research 38 has shown that the M pro from HCoV HKU1 may possess an unusual substrate preference at P1 site quite different from that of other CoV M pro s Here we report the structure of HCoV HKU1 M pro which serves as a model for group 2A CoVs in complex with a synthetic peptidomimetic inhibitor N3 The structure and sub sequent enzyme activity assays help to resolve the issue of the relative conservation at the P1 position based on genome se quencing Moreover this complex structure provides further structural data for rational drug design against HCoVs MATERIALS AND METHODS cDNA and plasmid The cDNA encoding HCoV HKU1 M pro was kindly provided by K Y Yuen from the Department of Microbiology Hong Kong University Hong Kong Special Administrative Region China BamHI and XhoI restriction sites were attached to the 5H11032 and 3H11032 ends separately by PCR and the PCR product first was inserted into the pMD 18T vector Takara The DNA of interest then was cleaved from the T vector and subcloned into a glutathione S transferase tagged expression vector pGEX 4T 1 The validity of the whole procedure was confirmed by DNA sequencing Protein expression and purification The plasmid was first transformed into the commercial Escherichia coli strain BL21 DE3 Rosetta Invitrogen After incubation at 37 C overnight on an Amp H11001 algae Luria Bertani LB plate fresh transformants were inoculated into 5 ml LB medium in the presence of 100 H9262g ml ampicillin After growth for 12 h the incubation system was scaled up to 1 liter LB medium with the same concentration of antibiotics in a 2 liter flask and the solution was shaken vigorously at 37 C until the optical density at 600 nm reached 0 6 Cells were induced by 0 5 mM isopropyl H9252 D thiogalactopyranoside Sigma at 16 C overnight Cell pellets were harvested by centrifugation resuspended in 40 ml phosphate buffered saline buffer with 2 mM dithiothreitol and 7 mM H9252 mercaptoethanol and sonicated on ice for 25 min The supernatant was collected after the cen trifugation of the sonicant at 15 000 rpm for 40 min Affinity purification was achieved by letting the supernatant flow through 2 ml glutathione S transferase affinity medium twice On column digestion lasted for 16 h at 4 C with thrombin New England BioLabs and the protein of interest was harvested and concentrated to 30 mg ml The N3 inhibitor then was added to a final molar ratio of 1 1 and incubated at 4 C overnight Finally the HCoV HKU1 M pro inhibitor complex was purified by gel filtration using a Superdex 200 10 30 column GE Healthcare The protein concentration was adjusted to 20 mg ml for crystallization trials Crystallization and structure determination Crystals of HCoV HKU1 M pro were grown in 0 1 M imidazole pH 6 0 and 0 6 M sodium acetate by the hanging drop vapor diffusion method Synchrotron X ray diffraction data were collected on beamline BL 5A of the Photon Factory Tsukuba Japan and processed to 2 5 resolution using HKL2000 29 for data indexing and scaling Molecular replacement using the SARS M pro structure Protein Data Bank entry 2AMQ 48 identity as a template was performed with PHASER 32 The manual rebuilding of the structure was performed using Coot 13 and the structure was refined using REFMAC in the CCP4 suite 10 Final modification was carried out using CNS 6 The volume of the S1 cavity was calculated using VOIDOO 25 Enzyme activity assays Substrates and analogs were designed through three rounds of affinity optimization 42 by substrate mimicry and from a library of substrate analogs The substrate and analogs were synthesized by Dawei Ma from the Institute of Organic Chemistry Chinese Academy of Sciences Shanghai China The strategy employed for enzyme activity assays of HCoV HKU1 M pro has been described previously 43 Activity assays for HCoV HKU1 M pro against the CoV consensus substrate and the HCoV HKU1 specific substrate followed a similar protocol which is described briefly below The consensus substrate and HCoV HKU1 specific substrates were fluorescent compounds with the se quences MCA AVLQSGFR Lys Dnp Lys NH 2 and MCA PRLHCTTN Lys Dnp Lys NH 2 respectively greater than 95 purity GL Biotech Shanghai Ltd Shanghai China A P1 single mutant substrate also was synthesized with sequence MCA AVLHSGFR Lys Dnp Lys NH 2 The excitation and emission wavelengths of the fluorescent substrates were 320 and 405 nm respectively A buffer consisting of 50 mM Tris HCl pH 7 3 and 1 mM EDTA was used for enzyme activity assays at 30 C The reaction was initiated by adding protease final concentration 2 H9262M to a solution containing different final concentrations of the substrate 3 2 to 40 H9262M Strict kinetic parameters for the inhibition assay were determined according to the previously reported protocol 43 All results from enzyme activity assays were calculated using data based on at least three independent parallel experiments Coordinate accession number Coordinates and structure factors for the HKU1 M pro in complex with inhibitor N3 have been deposited in the Protein Data Bank under entry ID 3D23 RESULTS Structural overview Four protein molecules denoted A B C and D occupy one asymmetric unit with one N3 molecule per protomer Two of the protomers form a typical ho modimer while the remaining two protomers dimerize with their adjacent symmetry related counterparts Fig 1a Each protomer exhibits a three domain I to III architecture that is common to other CoV M pro structures 1 2 42 44 domains I and II have chymotrypsin like folds and domain III displays a globular H9251 helical cluster that is unique to CoV M pro The catalytic site including the Cys His dyad and the relatively shallow substrate binding pocket of HCoV HKU1 M pro are located in the cleft between domains I and II The substrate binding pocket features two deeply buried sites P1 and P2 and several sites with different levels of solvent exposure P3 P4 and P5 Fig 1b X ray data collection and refinement statistics are summarized in Table 1 Michael acceptor and catalytic dyad Clear and continuous electron density was observed between the reactive backbone carbon atom of the N3 substrate and the SH9253 atom of Cys145 in the inhibitor bound HCoV HKU1 M pro structure We con clude that this reaction can be categorized as an electrophilic addition mediated by a Michael acceptor obeying the K i H11002 k 3 kinetics where K i is the dissociation constant and k 3 is the turnover number according to the following scheme EH11001S O K m ESO k cat EH11002S 1 As the covalently bound inhibitor is a mimic of the real peptide substrate it is possible to model the transition state by treating the enzyme inhibitor complex structure as a snapshot of the catalytic dyad and hence to predict parameters of the K m H11002 k cat kinetics according to the following scheme EH11001I O K i EIO k 3 EH11002I 2 This catalytic dyad involves residues His41 and Cys145 and the intermediate state might be stabilized by the oxyanion hole 28 formed by the backbone amides of the oxyanion loop from Phe140 to Cys145 Fig 2a The oxyanion hole is crucial to the 8648 ZHAO ET AL J VIROL on April 1 2015 by guest http jvi asm org Downloaded from stabilization of the intermediate state so the formation of the oxyanion hole has a significant influence on k cat As discussed for the inhibitor design targeting human rhinovirus 3C pro teases 28 the correct organization of this oxyanion loop also is essential to the k 3 step for mechanism based suicide inhibi tors Surrounding the SH9253 atom of Cys145 we observe well defined amides from the loop from residues 142 to 145 Similarly to FIG 1 a Structural overview of four protomers A green B cyan C magenta and D yellow in one asymmetric unit represented as cartoons N3 inhibitors are shown as blue sticks b Structural overview of the enzyme inhibitor complex of one monomer unit The main chain of the enzyme is represented as blue cartoons and the synthetic inhibitor is shown as yellow sticks The three domains are labeled VOL 82 2008 HUMAN CORONAVIRUS HKU1 M pro STRUCTURE 8649 on April 1 2015 by guest http jvi asm org Downloaded from human rhinovirus 3C proteases 28 these amide dipoles con struct a tetrahedral oxyanion hole From native and complex structures of SARS CoV M pro the correct orientation of these backbone amides is triggered and maintained by substrate binding in particular by the binding of the P1 residue and interaction between the N finger and the substrate 2 44 in which the backbone carbonyl of Leu141 is hydrogen bonded to the side chain oxygen of Ser144 The correct position of Leu141 is maintained by a hydrogen bond between the car bonyl group of Phe140 and the amide group of the P1 side chain and by hydrophobic stacking between His163 and Phe140 Although the analysis of the HCoV HKU1 M pro struc ture in complex with N3 Fig 2a shows that the P1 side chain exerts no direct influence on the residues forming the oxyanion hole its side chain oxygen atom forms a strong hydrogen bond 2 6 with His163 and helps to strengthen the stacking in teraction with Phe140 Furthermore the nitrogen atom of the P1 side chain also forms a hydrogen bond 3 1 with the backbone of Phe140 thus helping to maintain the oxyanion loop Phe140 Cys145 in its proper conformation For the above reasons we conclude that the P1 side chain is important for the network of interactions stabilizing the oxyanion hole The S1 pocket has a smaller size to accommodate P1 histi dine Given its crucial role in the catalytic process glutamine outperforms other residues as the signature of the M pro sub strate at the P1 position In addition to this advantage the side chain of glutamine in the P1 position suitably fits with residues forming the S1 subsite via Van der Waals interactions Fig 2b From the HCoV HKU1 genome sequence 11 out of 12 M pro recognition sites have Gln at the P1 position In our structure the N3 molecule has a lactam ring as an analog to the glutamine residue the cross linking between the CH9253 and N atoms helps to select the stretching conformation from the ensemble of rotamers and better occupy the binding cleft In HCoV HKU1 M pro structures the lactam ring protrudes into the S1 pocket via a hydrogen bond to the imidazole ring NH of His162 at a distance of 2 6 However unlike the SARS CoV M pro structure in complex with N3 the NH of the HKU1 N3 lactam ring fails to recruit a water molecule to satisfy a second S1 hydrogen bond Instead the N terminal OH9253 atom might FIG 2 a Details of the interaction between the P1 side chain and the defined oxyanion loop shown in stereo representation Side chains are shown as sticks and the crucial hydrogen bond between His163 and the substrate side chain is shown by a cyan dashed line b Details of the substrate binding pocket The inhibitor is shown in the following color scheme C white O red and N blue The crucial residues of the enzyme are shown in the following color scheme C cyan O red N blue and S yellow Hydrogen bonds are shown as red dashed lines TABLE 1 X ray data processing and refinement statistics Statistic Value for the HCoV HKU1 M pro N3 complex e Data collection Wavelength A 1 0 Resolution limit A 50 0 2 48 2 62 2 48 Space group P4 1 Cell parameters a A 91 770 b A 91 770 c A 187 914 H9252 90 Total no of reflections 510 408 No of unique reflections 108 501 Completeness 99 1 98 64 Redundancy 5 0 4 9 R merge a 0 11 Sigma cutoff 0 I H9268 I 16 5 Refinement Resolution range A 50 0 2 5 R work b 22 9 R free 28 5 rmsd c from ideal geometry Bonds A 0 012 Angles 1 59 Avg B factor A 2 Protein 38 7 Small molecule 43 7 Ramachandran plot d Favored 86 8 Allowed 12 2 Generously allowed 0 9 Disallowed 0 1 a R merge H11005H9018I i H11002H11021IH11022 H9018I where I i is the intensity of an individual reflection and H11021IH11022 is the average intensity of that reflection b R work H11005H9018F p H11002F c H9018F p where F c is the calculated and F p is the observed structure factor amplitude c rmsd root mean squares deviation d Ramachandran plots were generated by using the program PROCHECK e Numbers in parentheses correspond to the highest resolution shell 8650 ZHAO ET AL J VIROL on April 1 2015 by guest http jvi asm org Downloaded from provide a weak electronegative interaction to stabilize the NH atom the interaction likely is stronger due to the presence of redundant residues as a cloning artifact hindering the N ter minal Ser from coming any closer to the NH of P1 side chain Nevertheless compared with the M pro s from other CoV groups the structure of the HCoV HKU1 M pro has an S1 pocket with a relatively smaller volume of H1101118 1 3 Incon trast the volume of the S1 pocket of TGEV M pro is H1101119 1 3 that of of IBV M pro is H1101121 7 3 and that of SARS CoV M pro is H1101119 5 3 The reduced size of the S1 pocket might be caused by the position of the loop Leu167 Cys171 which is bent up ward by about 90 As a result the smaller S1 pocket might tolerate mutation to short chain residues at the P1 position in which case a weakened oxyanion hole is to be expected Novel substrate specificity already has been found in the HCoV HKU1 genome in which the M pro recognition site between the helicase and exonuclease utilizes histidine instead of glutamine at the P1 position Mimicking proteolysis in the cell enzyme activity assays using a synthetic fluorogenic substrate confirm the existence of such a cleavage event in vitro and exhibit novel enzymatic properties not seen with the consensus substrate Table 2 Enzyme activity assays indicate that the affinity for a sub strate containing a single mutation at the P1 position decreases to 30 of the affinity for the native consensus substrate which can be attributed to the loss of a hydrogen bond