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Structural characterization of the fusion active complex of severe acute respiratory syndrome SARS coronavirus Paolo Ingallinella Elisabetta Bianchi Marco Finotto Giovanna Cantoni Debra M Eckert Vinit M Supekar Chiara Bruckmann Andrea Carfi and Antonello Pessi Istituto di Ricerche di Biologia Molecolare P Angeletti Via Pontina Km 30 600 00040 Pomezia Italy and Merck Research Laboratories 126 East Lincoln Avenue Rahway NJ 07065 Communicated by Peter S Kim Merck Research Laboratories West Point PA April 19 2004 received for review September 2 2003 The causative agent of a recent outbreak of an atypical pneumonia known as severe acute respiratory syndrome SARS has been identified as a coronavirus CoV not belonging to any of the previously identified groups Fusion of coronaviruses with the host cell is mediated by the envelope spike protein Two regions within the spike protein of SARS CoV have been identified showing a high degree of sequence conservation with the other CoV which are characterized by the presence of heptad repeats HR1 and HR2 By using synthetic and recombinant peptides corresponding to the HR1 and HR2 regions we were able to characterize the fusion active complex formed by this novel CoV by CD native PAGE proteolysis protection analysis and size exclusion chromatogra phy HR1 and HR2 of SARS CoV associate into an antiparallel six helix bundle with structural features typical of the other known class I fusion proteins We have also mapped the specific boundaries of the region within the longer HR1 domain making contact with the shorter HR2 domain Notably the inner HR1 coiled coil is a stable H9251 helical domain even in the absence of interaction with the HR2 region Inhibitors binding to HR regions of fusion proteins have been shown to be efficacious against many viruses notably HIV Our results may help in the design of anti SARS therapeutics T he causative agent of the outbreak of the atypical pneumonia known as severe acute respiratory syndrome SARS 1 has been identified as a coronavirus CoV 2 9 Phylogenetic analysis of the SARS associated CoV SARS CoV shows that it is neither a mutant nor a recombinant of previously charac terized CoV 10 11 and forms a new distinct group within the genus 3 4 8 9 The fusogenic envelope glycoprotein of CoV the spike protein S protein which is displayed in H11015200 copies on the viral mem brane as a trimer 12 13 is divided into two subdomains of similar size S1 and S2 which have distinct functions 11 S1 which forms the globular portion of the spike mediates binding to host cell receptors 11 14 16 For SARS CoV the receptor binding do main is localized to amino acids 318 510 15 16 which bind angiotensin converting enzyme 2 15 17 S2 which is more conserved 9 11 forms the membrane anchored stalk region 18 and mediates viral host cell fusion 13 19 21 Cleavage between S1H20862S2 is not an absolute requirement for the fusion of CoV 4 11 17 21 24 and the available data suggest that the S protein of SARS CoV is not cleaved into subunits 4 9 17 The SARS CoV S protein shares little amino acid similarity with other CoV 9 However S2 contains two predicted amphipathic H9251 helical regions with a 4 3 heptad repeat HR characteristic of coiled coils 9 The position and sequence of these HR regions are conserved among all groups of coronaviruses 18 including SARS CoV 25 HR regions are a common feature of Type I viral fusion proteins 26 40 These usually feature an N terminal HR HR1 adjacent to the fusion peptide and a C terminal HR HR2 close to the transmembrane domain In the current paradigm of virus cell fusion mediated by these proteins the HR form a six helix bundle with an inner trimeric coiled coil formed by HR1 onto which HR2 pack in antiparallel orientation Formation of the six helix bundle brings in close proximity the fusion peptide which is N terminal to HR1 and embedded in the target cell membrane and the trans membrane domain which is immediately downstream of HR2 thereby driving the two membranes in close contact favoring the formation of a fusion pore reviewed in refs 41 43 Here we describe a systematic study of SARS CoV HR regions which allowed us to characterize the fusion active complex formed by this novel CoV Materials and Methods Peptides All of the peptides in Table 1 were prepared by standard solid phase methods whereas the HR1 peptide spanning residues 871 972 was produced as a fusion to maltose binding protein MBP by standard recombinant DNA methods For full details see Supporting Materials and Methods which is published as supporting information on the PNAS web site CD Spectroscopy CD was performed on a J 710 spectropolarimeter Jasco Easton MD and at 20 C by using a rectangular quartz cell with a 0 1 cm path length Spectra were acquired by using an 8 sec time response and a 5 nmH20862min scan speed the spectra were averaged for two acquisitions Stock solution concentration was determined by quantitative amino acid analysis The results are reported as mean residue ellipticity H9258 having units of degH18528cm 2 H18528dmol H110021 Individual peptides and mixtures of HR1H20862HR2 peptides were analyzed at 10 H9262M concentration in 20 mM sodium phosphate pH 7 3 with or without 150 mM NaCl The percentage of H9251 helix was calculated according to Chen et al 44 Thermal stability was determined at a 10 H9262M concentration by monitoring the change in the CD signal at 222 nm as a function of temperature with a 10 CH20862h increase and 16 sec integration time Native PAGE Equimolar mixtures of C1 0 6 mM in phosphate buffer pH 7 3 and one of the HR1 peptides N1 N7 were incubated at 25 C for 10 min final volume of 5 H9262l After the addition of an equal volume of 2H11003 N tris hydroxymethyl methylglycine Tricine sample buffer 0 125 M TrisH18528HCl pH6 8H2086210 glycerolH208620 004 g of bromophenol blue the mixture was analyzed by PAGE on 12 Tricine gel with a TricineH20862glycine running buffer pH 8 3 Analytical Size Exclusion Chromatography The apparent M r of the N0N3H20862C2 complex was estimated with a Superdex 75 size exclusion Abbreviations CoV coronavirus HR heptad repeat SARS severe acute respiratory syn drome S protein spike protein MBP maltose binding protein Tm melting temperature To whom correspondence may be addressed E mail elisabettaH14061bianchi or antonelloH14061pessi Present address Department of Biochemistry University of Utah Medical Research and Education Building 211 50 North Medical Drive Salt Lake City UT 84132 2004 by The National Academy of Sciences of the USA www pnas orgH20862cgiH20862doiH2086210 1073H20862pnas 0402753101 PNAS H20841 June 8 2004 H20841 vol 101 H20841 no 23 H20841 8709 8714 MEDICAL SCIENCES column Amersham Pharmacia equilibrated with 10 mM sodium phosphateH20862150 mM NaCl pH 7 5 flow rate 0 5 mlH20862min See also Supporting Materials and Methods Proteolysis Protection Experiments Stock solutions 100 H9262M of peptides C2 C3 N3 N0N3 N3N6 and HR1 871 972 MBP fusion protein were prepared in 20 mM TrisH18528HCl pH 7 5 The peptide complexes were formed by incubating equal volumes 100 H9262l of the components final concentration of 50 H9262M each for1hat4 C Proteinase K digestion was initiated by enzyme addition 1H20862150 proteinase KH20862peptide wtH20862wt and carried out at 4 C At selected time points an aliquot of the mixture was analyzed by HPLC MS on a Jupiter C 4 column 150 H11003 4 6 mm 5 H9262m 300 with linear acetonitrile gradients allowing the identification of the digested fragments Proteolysis of the individual peptides was performed in the same conditions at 50 H9262M concentration Results Location of HR1 and HR2 We used the programs LEARNCOIL VMF 45 and MULTICOIL 46 to predict the location of SARS CoV HR1 and HR2 LEARNCOIL VMF locates HR1 and HR2 at residues 900 1005 and 1151 1185 respectively whereas MULTICOIL locates HR1 and HR2 at residues 900 974 and 1148 1193 respectively see Fig 6 which is published as supporting information on the PNAS web site HR1 Peptides We initially prepared a series of partially overlapping HR1 peptides N1 N7 spanning the whole HR1 domain as predicted by MULTICOIL Table 1 The peptides were designed to include five HR a length sufficient to nucleate a stable coiled coil 47 51 shifted by one HR Our peptide set was thus designed to locate the region s within HR1 that interacts with the HR2 domain The secondary structure of the HR1 peptides was analyzed by CD Fig 1a Peptides N1 N4 are unordered peptides N5 and N7 are partially helical and remarkably peptide N6 is fully helical HR2 Peptides We prepared three peptides peptide C1 correspond ing to the entire HR2 region as predicted by LEARNCOIL VMF extended at the N terminus to residue 1148 peptide C2 corre sponding to the entire HR2 region as predicted by MULTICOIL and peptide C3 which spans residues 1116 1185 beginning 32 residues upstream of the predicted HR2 domain Table 1 CD analysis at Table 1 Synthetic SARS CoV HR1 and HR2 peptides used in the study Peptide Sequence The recombinant HR1 MBP fusion peptide encompasses residues 871 972 of the S protein see Fig 6 Fig 1 a CD spectra of peptides N1 N7 The calculated percentage of H9251 helix is indicated b CD analysis of the interaction between peptides N3 and C2 Shown is the observed spectrum solid trace and the arithmetical sum of the spectra of the individual peptides dashed trace The calculated percent age of H9251 helix of the N3H20862C2 complex is 57 8710 H20841 www pnas orgH20862cgiH20862doiH2086210 1073H20862pnas 0402753101 Ingallinella et al neutral pH revealed an equilibrium between unordered and helical structure higher in peptide C2 the calculated percentage of H9251 helix 44 was 22 42 and 26 for C1 C2 and C3 respectively Fig 7 which is published as supporting information on the PNAS web site Mapping of the HR1H20862HR2 Interaction We then mapped the interac tion between the HR1 and HR2 peptides We reasoned that by analogy with other fusion proteins 26 29 33 36 39 52 also for SARS CoV the interaction between the HR1 and HR2 regions should be mediated by interaction of helical domains Among the HR1 and HR2 peptides only N6 is fully helical whereas the others are unordered or in a conformational equilibrium between unor dered helical structure Fig 1a Therefore we expected that complex formation between one HR1H20862HR2 pair should be accom panied by a conformational transition toward H9251 helical structure To identify which region s of the longer HR1 makes direct contact with the HR2 domain each one of peptides N1 N7 was mixed with an equimolar amount of either C1 or C2 and CD spectra were recorded To highlight any conformational transition induced by complex formation each spectrum of a mixture was compared with the arithmetical sum of the spectra of the individual peptides in the absence of an interaction the two curves should be superimposable which is the case for all of the N peptides with the exception of N3 the combination of N3 with either C1 or C2 gives rise to a new spectrum with a higher content of H9251 helix Fig 1b the CD spectra of the other N peptides in complex with C1 are shown in Fig 8 which is published as supporting information on the PNAS web site Notably neither N2 nor N4 which are contiguous and mostly overlapping with N3 interact with C1 This finding is not surprising given the small size of the peptides and the highly cooperative nature of this type of interaction We then independently mapped the HR1H20862HR2 interaction by PAGE The results which were similar for C1 and C2 are shown for the former Fig 2 No interaction is observed for peptides N5 N6 and N7 whereas the combination of equimolar amounts of N3 and C1 gives rise to the disappearance of the individual bands and the formation of a new band at an intermediate position corre sponding to the complex In addition complex formation was also observed with the pairs N1H20862C1 N2H20862C1 and N4H20862C1 but without complete disappearance of the individual components which is a qualitative indication of lower affinity with respect to the fully formed N3H20862C1 complex Formation of lower stability complexes was favored by the 20 fold higher concentration used for the gel shift experiments with respect to CD Therefore the two sets of data CD and PAGE agree in locating the key region of interaction within peptide N3 HR1 residues 914 949 and peptide C1 HR2 1148 1185 Based on the finding that i the core of the HR1 region interacting with HR2 is encompassed by peptide N3 ii the interaction might extend up to peptide N1 and iii peptide N6 despite its short length is able to adopt a fully helical fold Fig 1a we prepared three longer peptides peptide N3N6 amino acids 914 970 peptide N1N3 amino acids 900 949 and peptide N0N3 amino acids 890 949 which extends beyond the predicted bound ary of HR1 and toward the predicted fusion peptide 25 53 Table 1 Peptides N1N3 and N3N6 were found to be fully helical whereas the calculated percentage of H9251 helix was slightly lower 84 for N0N3 in line with the predicted boundaries of the HR1 region Fig 3a Thermal denaturation studies were performed at the concen Although the band corresponding to the charged HR2 peptides was always visible in these conditions for most hydrophobic N peptides the band of the isolated peptide was not visible in the gel Care was taken to ensure that the combination experiments were performed with titrated equimolar amounts of the two components Observation of a residual HR2 peptide band in the presence of the complex can therefore be taken as an indication that the complex is not fully formed at equimolarity and used as a qualitative measure of the relative stability of the complexes Fig 2 Analysis of the interaction between peptides N1 N7 and peptide C1 by native PAGE The data were identical for peptide C2 Fig 3 a CD spectra of peptides N0N3 N1N3 and N3N6 Table 1 The calculated percentage of H9251 helix is indicated b Thermal denaturation curves with indicated T m values of peptides N0N3 N1N3 and N3N6 c Thermal denaturation curves with indicated T m values of the N1N3H20862C2 and N3N6H20862C2 peptide complexes Ingallinella et al PNAS H20841 June 8 2004 H20841 vol 101 H20841 no 23 H20841 8711 MEDICAL SCIENCES trationof10H9262M All three peptides showed a reversible cooper ative transition by thermal denaturation with melting points T m of 45 5 C 50 2 C and 66 8 C for N1N3 N0N3 and N3N6 respec tively Fig 3b It appears therefore that the HR1 domain of SARS CoV forms a continuous helical domain which is fully formed in the absence of any interaction with the HR2 domain This result is unlike HIV gp41 in which the N peptides are unable to form a trimeric coiled coil in the absence of the C peptide 54 and is more similar to the influenza virus hemagglutinin 26 28 55 Despite being only 84 helical N0N3 is more stable than N1N3 thus the N terminal boundary of HR1 should be positioned between residues 890 and 900 whereas the C terminal boundary should at least include residue 970 We then examined complex formation between the longer N peptides and the HR2 peptides C1 and C2 The interaction was apparent for all of the three peptides the CD of the complex between N3N6 and C1 is shown in Fig 9 which is published as supporting information on the PNAS web site Importantly the H9251 helical content was increased in all of the complexes consistent with folding of the HR2 peptides into a H9251 helical structure upon complex formation Thermal denaturation analysis shows that complex formation exerts a stabilizing effect with respect to the isolated HR1 peptides Fig 3c The increase in T m was particularly large for N0N3H20862C2 For this complex complete denaturation was not yet observed at 90 C i e H1102240 C higher than the T m measured for N0N3 Also for the N1N3H20862C2 complex we measured a T m 78 5 C that is 33 C higher than that measured for N1N3 The stabilization effect was much lower in the case of N3N6H20862C2 with a melting point of 71 0 C only 4 2 C higher than the N3N6 alone which however shows considerable stability by itself The ob served T m values are comparable with those reported for the murine hepatitis coronavirus 25 The thermal denaturation data on the complexes showing that the stability of N0N3H20862C2 complex is much higher than that of the complex N1N3H20862C2 brings further support to the hypothesis that the N terminal domain of HR1 might extend beyond residue 900 to include at least some residues that are comprised in N0N3 only M r of the HR1H20862HR2 Complex in Physiologic Conditions We performed size exclusion chromatography experiments in physiologic condi tions on the more stable N0N3H20862C2 complex Fig 4 The elution volume relative to M r standards gave an apparent M r of 39 530 Da in good agreement with the M r 35 360 Da expected for a hexameric complex formed by three molecules of N0N3 and three molecules of C2 Mapping of HR1H20862HR2 Interaction by Proteolysis Protection Experi ments To identify the boundaries and the relative orientation and position of HR2 with respect to HR1 in the complex we performed proteolysis protection experiments with proteinase K as previously done for murine hepatitis virus 25 To this aim we produced a recombinant peptide spanning residues 871 972 of the S protein i e beginning 29 residues upstream of the predicted N terminus of the HR1 domain as a fusion with MBP The proteolysis protection experiments are summarized in Fig 5 Fig 4 a Size exclusion chromatography of the N0N3H20862C2 complex on Superdex 75 Indicated are the peaks corresponding to the complex and to excess peptide C2 and the position of isolated N0N3 run in the same condi tions b M r standard calibration curve and interpolated M r of the complex arrow Fig 5 Proteolysis protection analysis of the HR1H20862HR2 complexes The indicated individual peptides and mixtures were subjected to proteinase K digestion Aliquots of the reaction mixture were analyzed by HPLC MS The results shown are all referred to the 5 h incubation time point The peptides are represented by bars with the name indicated as in Table 1 Hatched bars indicate the parts of the peptide that were readily digested by the protease black bars indicate the protease resistant parts and light gray bars indicate the regions digested at a slower rate 8712 H20841 www pnas orgH20862cgiH20862doiH2086210 1073H20862pnas 0402753101 Ingallinella et al Samples of individual peptides and the indicated HR1H20862HR2 equimolar complexes were incubated with proteinase K and the resulting fragments were analyzed by reversed phase HPLC MS to unequivocally establish the sequence of each protease resistant core peptide Digestion of isolated HR1 peptides showed the presence of a stable core in the longer peptides Although peptide N3 is com pletely digested N and C terminally extended peptides show increased stability C terminal extension up to 970 in peptide N3N6 confers stability with cleavage only occurring at the N terminus at residue 924 Also the N terminal extension of N3 to amino acid 890 in peptide N0N3 confers stability with cleavage occurring only at the N terminus at residue 896 The larger construct HR1 871 972 MBP was digested to fragments 899 972 and 908 972 The HR2 peptides C2 and C3 instead were completely digested However when the smaller peptides N3 and C2 were complexed together they were partially protected by degradation after 5 h Full degradation was instead observed after 24 h For all of the other complexes examined the pattern at 24 h was comparable to the one at 5 h and the latter common time point is shown in Fig 5 For N3H20862C2 the protease resistant core includes the entire N3 peptide amino acids 914 949 whereas C2 is trimmed at the C terminus at the two positions 1179 and 1175 We used this information to align the N terminus of N3 with respect to the C terminus of C2 The boundaries of the interaction were further probed with the longer N peptides The C terminal extension of N3 to ami