Coronavirus receptor switch explained from the stereochemistry of protein–carbohydrate interactions and a single mutation Mark J. G. Bakkersa, Qinghong Zengb,1, Louris J. Feitsmab,1, Ruben J. G. Hulswita, Zeshi Lic,d, Aniek Westerbekea, Frank J. M. van Kuppevelda, Geert-Jan Boonsc,d,e, Martijn A. Langereisa, Eric G. Huizingab,2, and Raoul J. de Groota,2 aVirology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CH Utrecht, The Netherlands; bCrystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Faculty of Sciences, Utrecht University, 3584 CH Utrecht, The Netherlands; cDepartment of Chemistry, University of Georgia, Athens, GA 30602; dDepartment of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CG Utrecht, The Netherlands; and eComplex Carbohydrate Research Center, University of Georgia, Athens, GA 30602 Edited by John J. Skehel, National Institute for Medical Research, London, United Kingdom, and approved April 19, 2016 (received for review October 9, 2015) Hemagglutinin-esterases (HEs) are bimodular envelope proteins of orthomyxoviruses, toroviruses, and coronaviruses with a carbohydrate-binding “lectin” domain appended to a receptor- destroying sialate-O-acetylesterase (“esterase”). In concert, these do- mains facilitate dynamic virion attachment to cell-surface sialoglycans. Most HEs (type I) target 9-O-acetylated sialic acids (9-O-Ac-Sias), but one group of coronaviruses switched to using 4-O-Ac-Sias instead (type II). This specificity shift required quasisynchronous adaptations in the Sia-binding sites of both lectin and esterase domains. Previ- ously, a partially disordered crystal structure of a type II HE revealed how the shift in lectin ligand specificity was achieved. How the switch in esterase substrate specificity was realized remained unresolved, however. Here, we present a complete structure of a type II HE with a receptor analog in the catalytic site and identify the mutations underlying the 9-O- to 4-O-Ac-Sia substrate switch. We show that (i) common principles pertaining to the stereochemistry of protein– carbohydrate interactions were at the core of the transition in lectin ligand and esterase substrate specificity; (ii) in consequence, the switch in O-Ac-Sia specificity could be readily accomplished via con- vergent intramolecular coevolution with only modest architectural changes in lectin and esterase domains; and (iii) a single, inconspicuous Ala-to-Ser substitution in the catalytic site was key to the emergence of the type II HEs. Our findings provide fundamental insights into how proteins “see” sugars and how this affects protein and virus evolution. coronavirus | hemagglutinin-esterase | sialic acid | crystal structure | sialate-O-acetyl esterase A mong host cell surface determinants for pathogen adherence, sialic acids (Sias) rank prominently (1, 2). Representatives of at least 11 families of vertebrate viruses use Sia as primary entry receptor and/or attachment factor (3, 4). Viral adherence to sia- loglycans, however, comes with inherent complexities related to (i) the sheer ubiquity of receptor determinants that may act as “decoys” when present on off-target cells and non–cell-associated glycoconjugates, and (ii) the dense clustering that is characteristic to glycotopes and that may augment the apparent affinity of ligand– lectin interactions by orders of magnitude (5, 6). Viruses may avoid inadvertent virion binding to nonproductive sites by being selective for particular sialoglycan subtypes so that attachment is dependent on Sia linkage type, the underlying glycan chain, and/or the absence or presence of specific postsynthetic Sia modifications (2, 7, 8). Moreover, as an apparent strategy to evade irremediable binding to decoy receptors, viral sialolectins typically are of low affinity, with dissociation constants in the millimolar range (reviewed in ref. 3). In consequence, virion–Sia interactions are intrinsically dynamic and the affinity of the virolectins would appear to be fine-tuned such as to ensure reversibility of virion attachment. In most viruses, reversibility is exclusively subject to the lectin–ligand binding equilibrium. Some, however, take this principle one step further by encoding virion-associated enzymes to promote catalytic virion elution through progressive local receptor depletion (3, 4). In lineage A betacoronaviruses (A-βCoVs), a group of envel- oped positive-strand RNA viruses of human clinical and veterinary relevance (9), catalysis-driven reversible binding to O-acetylated Sias (O-Ac-Sias) is mediated by the hemagglutinin-esterase (HE), a homodimeric type I envelope glycoprotein (10–15). HE mono- mers resemble cellular carbohydrate-modifying proteins (16, 17), in that they have a bimodular structure with a lectin appended to the enzyme domain. The lectin domain mediates virion attach- ment to specific O-Ac-Sia subtypes with binding hinging on the all- important sialate-O-acetyl moiety, whereas removal of this O-acetyl by the catalytic sialate-O-acetylesterase (“esterase”) do- main results in receptor destruction (18–21). Intriguingly, HE homologs also occur in toroviruses (22–25) as well as in three genera of orthomyxoviruses (Influenza virus C, Influenza virus D, and Isavirus) (26–32), but, among coronavi- ruses, exclusively in A-βCoVs (9). HE was added to the proteome of an A-βCoV common progenitor through horizontal gene Significance A wide variety of vertebrate viruses, representative of at least 11 families, use sialic acid (Sia) for host cell attachment. In betacoronaviruses, the hemagglutinin-esterase envelope pro- tein (HE) mediates dynamic attachment to O-acetylated Sias. HE function relies on the concerted action of carbohydrate-binding lectin and receptor-destroying esterase domains. Although most betacoronaviruses target 9-O-acetylated Sias, some switched to using 4-O-acetylated Sias instead. The crystal structure of a “type II” HE now reveals how this was achieved. Common principles pertaining to the stereochemistry of protein–carbohydrate interac- tions facilitated the ligand/substrate switch such that only modest architectural changes were required in lectin and esterase domains. Our findings provide fundamental insights into how proteins “see” sugars and how this affects protein and virus evolution. Author contributions: M.J.G.B., Q.Z., L.J.F., M.A.L., E.G.H., and R.J.d.G. designed research; M.J.G.B., Q.Z., L.J.F., R.J.G.H., A.W., and M.A.L. performed research; Z.L. and G.-J.B. contributed new reagents/analytic tools; M.J.G.B., Q.Z., L.J.F., R.J.G.H., F.J.M.v.K., M.A.L., E.G.H., and R.J.d.G. analyzed data; and M.J.G.B., Q.Z., L.J.F., E.G.H., and R.J.d.G. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: The three crystal structures have been deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 4ZXN (RCoV-NJ HE0), 5JIF (MHV-DVIM HE), and 5JIL (RCoV-NJ HE0 in complex with 4-N-acetylated sialic acid)]. 1Q.Z. and L.J.F. contributed equally to this work. 2To whom correspondence may be addressed. Email: