Sialoglycan binding triggers spike opening in a human coronavirus

Abstract

Coronavirus spike proteins mediate receptor binding and membrane fusion, making them prime targets for neutralizing antibodies. In the cases of severe acute respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus 2 and Middle East respiratory syndrome coronavirus, spike proteins transition freely between open and closed conformations to balance host cell attachment and immune evasion^1,2,3,4,5. Spike opening exposes domain S1^B, allowing it to bind to proteinaceous receptors^6,7, and is also thought to enable protein refolding during membrane fusion^4,5. However, with a single exception, the pre-fusion spike proteins of all other coronaviruses studied so far have been observed exclusively in the closed state. This raises the possibility of regulation, with spike proteins more commonly transitioning to open states in response to specific cues, rather than spontaneously. Here, using cryogenic electron microscopy and molecular dynamics simulations, we show that the spike protein of the common cold human coronavirus HKU1 undergoes local and long-range conformational changes after binding a sialoglycan-based primary receptor to domain S1^A. This binding triggers the transition of S1^B domains to the open state through allosteric interdomain crosstalk. Our findings provide detailed insight into coronavirus attachment, with possibilities of dual receptor usage and priming of entry as a means of immune escape.

Main

Long before the advent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), four coronaviruses (CoVs) colonized the human population. Two of these, human CoVs HKU1 and OC43 in the betacoronavirus subgenus Embecovirus, independently arose from rodent reservoirs—either directly or through intermediate hosts^8,9,10. Unlike other human CoVs, HKU1 and OC43 rely on cell surface glycans as indispensable primary receptors^11,12. Their attachment and fusion spike proteins specifically bind to 9-O-acetylated sialosides^{11,13,14,15,16,17}. Underlining the importance of glycan attachment, embecoviruses uniquely code for an additional envelope protein, haemagglutinin esterase, a sialate-O-acetylesterase serving as a receptor-destroying enzyme^13,18,19. Recent observations suggest that HKU1 spike particularly targets α2,8-linked 9-O-acetylated disialosides (9-O-Ac-Sia(α2,8)Sia; that is, glycan motifs typical of oligosialogangliosides such as GD3). Accordingly, following overexpression of GD3 synthase ST8SIA1, HEK293T cells become susceptible to HKU1 S-pseudotyped viruses¹⁷.

CoV spike proteins are homotrimeric class I fusion proteins²⁰. The spike protomer can be divided into an amino- and carboxy-terminal region designated S1 and S2, respectively. Distinct S1 domains mediate receptor binding²¹, whereas S2 comprises the fusion machinery (Fig. 1a). In HKU1 and OC43, attachment to 9-O-Ac-sialosides occurs through a well-conserved receptor-binding site located in spike protein domain S1^A (Fig. 1a)^15,16. There are indications, however, for the existence of a secondary receptor engaged through domain S1^B, as epitopes of virus-neutralizing antibodies map to subdomain S1^B2 (refs. ^22,23,24). Moreover, in the case of HKU1, recombinantly expressed S1^B blocks infection²³, with single-site substitutions in S1^B2 abolishing this activity²⁴.

The spike proteins of SARS-CoV, SARS-CoV-2 and Middle East respiratory syndrome coronavirus (MERS-CoV) occur in different conformations with their receptor-binding S1^B domains either partially buried between neighbouring protomers (‘closed’ or ‘down’) or with one or more S1^B domains exposed (1-, 2- and 3-up, ‘open’)^2,5,7,25. The conformational dynamics of S1^B, and modulation thereof, would provide CoVs with a means to balance host cell attachment and immune escape¹. Recently, spontaneous conversion of S1^B into the up conformation was also described for porcine epidemic diarrhoea virus²⁶. However, available structures of all other CoV spike proteins, including those of HKU1 and OC43 (refs. ^16,27), have been observed only in a closed conformation (Supplementary Table 1), shielding S1^B from neutralizing antibodies but preventing S1^B-mediated receptor engagement^1,22. Adding to the conundrum, the transition from a closed to an open spike conformation has been linked to the elaborate conformational changes in S2 that drive fusion^4,28,29. The question thus arises whether specific mechanisms might exist that trigger S1^B conversion to the open state. Here we describe cryogenic electron microscopy (cryo-EM) structures of a serotype A HKU1 (HKU1-A) spike protein in four conformations, one in a closed apo state, the others in complex with the HKU1 disialoside receptor 9-O-Ac-Sia(α2,8)Sia. We show that glycan receptor binding by S1^A specifically prompts a conformational transition of S1^B domains into 1- and eventually 3-up positions, apparently through an allosteric mechanism.

Structure of the apo HKU1-A spike protein

HKU1 field strains are divided into three genotypes with evidence of intertypic recombination, but essentially occur in two distinct serotypes, with either A- or B-type spike proteins³⁰. Single-particle cryo-EM analysis of spike protein ectodomains of HKU1-A strain Caen1 yielded a reconstruction for the unbound state at a global resolution of 3.4 Å (Fig. 1b, Extended Data Fig. 1, Supplementary Figs. 1 and 2 and Supplementary Table 2). Notably, the HKU1-A spike protein trimers were found exclusively in a closed, pre-fusion conformation as reported for a serotype B HKU1 (HKU1-B) spike protein²⁷. The HKU1-A and HKU1-B spike proteins, at 84% sequence identity (Supplementary Fig. 3), are highly similar in global structure with an average Cα root mean square deviation of 1.1 Å for pruned atom pairs (Extended Data Fig. 2a). Compared to the HKU1-B model, our data allowed building an additional 231 residues per protomer. Among newly built segments are the membrane-proximal connecting domain (residues 776–796 and 1152–1225) and the linker between the S1/S2 and S2′ protease cleavage sites (residues 878–907; Fig. 1c). We could also model a major portion of S1^B2 (residues 480–575) such that this subdomain—purportedly crucial for protein receptor binding—is now fully resolved in the context of an intact HKU1 spike trimer, our findings essentially confirming the crystal structure of a HKU1-A S1^B-C fragment (residues 310–677)²⁴ (Extended Data Fig. 2b). In addition, 20 N-linked glycans per protomer were built, all well supported by the density map (Fig. 1b). Several glycans are engaged in interprotomer contacts (for example, N1215; Supplementary Fig. 4), among which the S1^B N355-glycan may help stabilize the HKU1-A spike trimer in the closed conformation by contacting the clockwise neighbouring protomer via Y528 (Supplementary Fig. 5). Using site-specific glycosylation patterns of HKU1-B (ref. ³¹), we carried out molecular dynamics simulations of the fully glycosylated spike ectodomain trimer. HKU1-A spike is largely shielded by glycans leaving only a few regions exposed, most notably the sialic acid-binding site in domain S1^A (Extended Data Fig. 2c).

Predictably similar in overall arrangement, the apo structures of A- and B-type spike trimers differ in the orientation of their S1^A domains, with those of HKU1-A tilted outwards (Extended Data Fig. 2a). The S1^A 9-O-Ac-Sia-binding site is conserved in HKU1-A S1^A, as expected, with key ligand contact residues K80, T/S82 and W89 (ref. ¹⁵) aligning with those in HKU1-B spike (Extended Data Fig. 2d,e). There are, however, notable differences in binding site topology. In HKU1-B, the 9-O-Ac-Sia-binding site is located within a narrow crevice between loop elements e1 (residues 29–37) and e2 (residues 246–252)^15,16. In the HKU1-A spike apo structure, the p1 and p2 pockets that accommodate the sialoside 9-O-Ac and 5-N-Ac moieties, respectively, are much less prominent owing to a consequential outward displacement of the e1 loop (see below).

Glycan binding triggers opening of S1^B

Incubation of the HKU1-A spike protein with the receptor analogue 9-O-Ac-Neu5Ac-α2,8-Neu5Ac-Lc-biotin (Supplementary Fig. 6) led to marked conformational changes yielding a surprising heterogeneity in structures. We identified and modelled three distinct conformations: a fully closed state (3.8 Å resolution), a partially opened state with a single S1^B domain rotated upwards by 101° (1-up, 5 Å resolution) and a fully opened state (3-up, 3.7 Å resolution; Fig. 2, Extended Data Fig. 3, Supplementary Figs. 2, 7 and 8 and Supplementary Table 2). A 2-up state was not detected. In all holo structures, clear densities for the disialoside were observed within S1^A receptor-binding sites (Fig. 2 and Supplementary Figs. 8 and 9). Apparently, binding of a specific 9-O-Ac-Sia-based primary receptor analogue by the S1^A domain triggers an allosteric mechanism, causing the exposure of S1^B domains located 40 Å from the S1^A binding pocket (Fig. 2 and Supplementary Fig. 10)….