Putative conformations of the receptor-binding domain in S protein of hCoV-EMC in complex with its receptor dipeptidyl peptidase-4
Authors: Shibo Jiang, Lu Lu, Lanying Du, Asim K. Debnath
Journal: Journal of Infection (2013)

Based on sequence alignment and homology modeling analysis, we previously predicted that the receptor-binding domain (RBD) of the novel human betacoronavirus 2c EMC/2012 (hCoV-EMC) is located in a region spanning residues 377–662 in hCoV-EMC spike (S) protein.1Jiang S. Lu L. Du L. Debnath A.K. A predicted receptor-binding and critical neutralizing domain in S protein of the novel human coronavirus HCoV-EMC.J Infect. 2013; 66: 464-466Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar Similar to the RBD in the S protein of SARS coronavirus (SARS–CoV), the predicted hCoV-EMC S-RBD also contains a core domain consisting of 5 β-sheets (β1-β4, β7) and 3 α-helices (αA-αC).2Li F. Li W.H. Farzan M. Harrison S.C. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor.Science. 2005; 309: 1864-1868Crossref PubMed Scopus (1522) Google Scholar, 3Prabakaran P. Gan J. Feng Y. Zhu Z. Choudhry V. Xiao X. et al.Structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody.J Biol Chem. 2006; 281: 15829-15836Crossref PubMed Scopus (192) Google Scholar However, they have different extended loops between β4 and β7. SARS–CoV S-RBD has a 71-amino-acid (aa)(residues 424–494) extended loop, including two anti-parallel β-sheets (β5–β6)2. Its receptor-binding motif (RBM) is located in this extended loop, which is responsible for directly contacting the residues in the virus-binding site in the SARS–CoV's receptor, angiotensin-converting enzyme 2 (ACE2) on the target cell.2Li F. Li W.H. Farzan M. Harrison S.C. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor.Science. 2005; 309: 1864-1868Crossref PubMed Scopus (1522) Google Scholar, 3Prabakaran P. Gan J. Feng Y. Zhu Z. Choudhry V. Xiao X. et al.Structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody.J Biol Chem. 2006; 281: 15829-15836Crossref PubMed Scopus (192) Google Scholar The hCoV-EMC S-RBD contains a much longer (143-aa: residues 440–582) extended loop, consisting of two anti-parallel β-sheets (β5-β6) as well as three α-helices. The first two α-helices form a V-shaped structure.1Jiang S. Lu L. Du L. Debnath A.K. A predicted receptor-binding and critical neutralizing domain in S protein of the novel human coronavirus HCoV-EMC.J Infect. 2013; 66: 464-466Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar Since the sequence length and conformation of the extended loop in the predicted hCoV-EMC S-RBD significantly differ from those in SARS–CoV S-RBD, we have predicted that hCoV-EMC must have a receptor different from that of SARS–CoV. As we expected, hCoV-EMC does not use SARS–CoV's coreceptor ACE2 for entry.4Muller M.A. Raj V.S. Muth D. Meyer B. Kallies S. Smits S.L. et al.Human coronavirus EMC does not require the SARS-coronavirus receptor and maintains broad replicative capability in mammalian cell lines.MBio. 2012; 3Crossref Scopus (168) Google Scholar Most recently, Raj et al.5Raj V.S. Mou H. Smits S.L. Dekkers D.H. Muller M.A. Dijkman R. et al.Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC.Nature. 2013; 495: 251-254Crossref PubMed Scopus (1586) Google Scholar have demonstrated that hCoV-EMC's receptor is the dipeptidyl peptidase-4 (DPP4, also known as CD26). To identify the RBM in the hCoV-EMC S-RBD, we performed a protein–protein docking simulation analysis using our predicted hCoV-ECM S-RBD1Jiang S. Lu L. Du L. Debnath A.K. A predicted receptor-binding and critical neutralizing domain in S protein of the novel human coronavirus HCoV-EMC.J Infect. 2013; 66: 464-466Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 5Raj V.S. Mou H. Smits S.L. Dekkers D.H. Muller M.A. Dijkman R. et al.Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC.Nature. 2013; 495: 251-254Crossref PubMed Scopus (1586) Google Scholar as the ligand and DPP4 as the receptor. Since the native DPP4 is presented in a dimeric form on the cell surface, we used the X-ray structure of the DPP4 dimer (pdb: 1PFQ)6Oefner C. D'Arcy A. Mac S.A. Pierau S. Gardiner R. Dale G.E. High-resolution structure of human apo dipeptidyl peptidase IV/CD26 and its complex with 1-[([2-[(5-iodopyridin-2-yl)amino]-ethyl]amino)-acetyl]-2-cyano-(S)-pyrrolidine.Acta Crystallogr D Biol Crystallogr. 2003; 59: 1206-1212Crossref PubMed Scopus (103) Google Scholar, 7Comeau S.R. Kozakov D. Brenke R. Shen Y. Beglov D. Vajda S. ClusPro: performance in CAPRI rounds 6-11 and the new server.Proteins. 2007; 69: 781-785Crossref PubMed Scopus (76) Google Scholar and the fully automated web server ClusPro 2.0 (http://cluspro.bu.edu) for rigid body docking. The ClusPro 2.0 server uses the newly developed docking program PIPER,8Kozakov D. Brenke R. Comeau S.R. Vajda S. PIPER: an FFT-based protein docking program with pairwise potentials.Proteins. 2006; 65: 392-406Crossref PubMed Scopus (653) Google Scholar which performs a rigid body docking using the Fast Fourier Transform (FFT) approach with pairwise interaction potential. About 1000 conformations with lowest scores are retained and clustered. We have selected one of the lowest scored docking poses from the 10 best clusters where the RBD of hCoV-EMC docked on the dimer interface of DPP4. As shown in Fig. 1A, the V-shaped structure formed by the first two α-helices in the extended loop of the hCoV-EMC S-RBD deeply inserts into the interface of DPP4 dimer. The tip of the V-shaped structure could even touch the last α-helix (residues 746–762) in DPP4. This finding suggests that the extended loop region in hCoV-EMC S-RBD, especially the first two α-helices, contains the RBM that may mediate the binding of hCoV-EMC S-RBD with the virus-binding site of the receptor DPP4 dimer on the target cell. We have also used the Hex server (http://hexserver.loria.fr/),9Ritchie D.W. Venkatraman V. Ultra-fast FFT protein docking on graphics processors.Bioinformatics. 2010; 26: 2398-2405Crossref PubMed Scopus (294) Google Scholar which utilizes an ultra-fast FFT protein docking technique, to dock the hCoV-ECM S-RBD on DPP4 dimer. We found that the best docking pose from this server had very similar orientation to that selected from the ClusPro 2.0 server and docked at the similar dimer interface location (Fig. 1B). By superimposing the two images obtained from ClusPro 2.0 servers and Hex server, we found, surprisingly, that the V-shaped structures in the extended loop of the hCoV-ECM S-RBD docked almost at the same location in the interface of DPP4 dimer (Fig. 1C). This provided additional confidence on the selection of the docking pose of RBD of hCoV-ECM on DPP4. We determined the possible contact residues of the ClusPro 2.0-based docking complex of the RBD of hCoV-ECM and DPP4 dimer using the PDBePISA web server (http://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver).10Krissinel E. Henrick K. Inference of macromolecular assemblies from crystalline state.J Mol Biol. 2007; 372: 774-797Crossref PubMed Scopus (7508) Google Scholar The total buried surface area in combined dimer binding interface was ∼3000 Å representing about 10% of the total surface area of the dimer. The major binding interface is located in one of the monomers of DPP4 within residues Tyr120 – Gln123, Ile149 – Gln153, Ile185 – Asp192, Ser239 – Arg253 and Asn281 – Ser284. Major salt bridges were formed between Lys190 and Asp163, Glu191 and Lys117, Asp192 and Arg166, Glu738 and Lys211 and Asp739 and Lys211 of the RBD. In the other monomer of DPP4, major binding interface were located within Thr186 – Ile194, Gln227 – Glu237 and Ala259 and Lys267. In this monomer of DPP4 only one salt bridge was located between Glu232 and Arg238 of the RBD. On March 26, 2013, WHO was informed of a new confirmed case of hCoV-ECM infection, resulting in a global total of 17 cases, including 11 deaths.11WHO. Novel coronavirus infection – update. http://www.who.int/csr/don/2013_03_26/en/index.html. 3-26-2013.Google Scholar A recent report of a family cluster of hCoV-EMC infections in the UK suggests its person-to-person transmissibility,12The Health Protection Agency (HPA) UK Novel Coronavirus Investigation teamEvidence of person-to-person transmission within a family cluster of novel coronavirus infections, United Kingdom, February 2013.Euro Surveill. 2013; 18 (pii=20427)Google Scholar raising great concerns about its potential pandemic like SARS in 2003.7Comeau S.R. Kozakov D. Brenke R. Shen Y. Beglov D. Vajda S. ClusPro: performance in CAPRI rounds 6-11 and the new server.Proteins. 2007; 69: 781-785Crossref PubMed Scopus (76) Google Scholar Therefore, development of effective and save vaccine and therapeutics is urgently needed. The predicted RBM in the hCoV-EMC S-RBD provides a critical target for developing subunit vaccines and virus entry inhibitors to prevent and treat hCoV-EMC infection as well as combat future pandemic of this lethal SARS-like disease. No reported conflicts. SJ was supported by grants from the National Institutes of Health (R01AI098775) and the 973 Programme of China (#2012CB519001). LL was supported by the "Chen Guang" Project of SMEC and SEDF (11CG03).