Cryo-EM structure of coronavirus-HKU1 haemagglutinin esterase reveals architectural changes arising from prolonged circulation in humans

Daniel L. Hurdiss; Ieva Drulyte; Yifei Lang; Tatiana M. Shamorkina; Matti F. Pronker; Frank J.M. van Kuppeveld; Joost Snijder; Raoul J. de Groot

HKU1 HE contains eight predicted N-linked glycosylation sites which are strictly conserved 128 between all HKU1 field strains studied so far. To better characterise the occupancy and semi-quantitative analysis (Table S2 ). As expected from HEK293T cell derived materials 26 , 137 glycosylation was predominantly of complex type and very heterogeneous, ranging from 8-59 138 unique glycoforms identified for each site ( Figure 3A ). For sites N145, N168 and N193, 139 situated on the LD loops ( Figure 3B ), we also detected substantial signals for the unmodified 140 asparagines, without glycosylation. Based on the combined signal intensities of all glycoforms, 141 we found that the occupancy of those sites is approximately 81% for N145, <2% for N168 and 142 44% for N193. The low occupancy of N168 agrees with the lack of density observed in the 143 cryo-EM map ( Figure 3C ). Furthermore, the high B-factors of the LD loops suggests flexibility 144 in this region ( Figure S3E ), which explains the limited density for N110, despite having 100% 145 occupancy. With the exception of N168, the first core N-acetyl glucosamine (GlcNAc) was 146 modelled for each of the LD loop glycans ( Figure 3C ). The remaining four sites are fully 147 occupied based on our MS data, in accordance with strong densities observed in the cryo-EM 148 map. Indeed, we were able to model the entire Man3,GlcNAc2 core for N314 ( Figure 3C ). Apart 149 from differences in glycan occupancy, we also observed marked differences in glycan 150 composition. Whereas the overall pattern is dominated by complex glycosylation, sites N83 151 and N328 show predominant hybrid and high-mannose glycosylation, respectively. Sites N110 152 and N145, which contain mostly complex glycans, are also heavily (core) fucosylated and 153 contain higher numbers of sialic acids. Whereas glycosylation varies substantially from site-154 to-site, and is very heterogeneous, we did identify a set of glycan compositions that are highly 155 abundant and shared at the majority of sites, as listed in Supplementary Table S3. 156 . CC-BY 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . Bottom three panels show semiquantitative analyses from extracted peak areas of sitespecific N-glycosylation by glycan type (non-glycosylated, high-mannose, hybrid, or complex), fucosylation, and sialylation. A full overview is presented in Supplementary Table S2 . B) Surface representation of the dimeric HKU1 HE atomic model, with modelled N-glycans shown as spheres and coloured according to the predominant glycan type shown in panel (A). C) EM density (blue mesh) zoned 2 Ã… around each of the modelled N-glycans and analagous region for N168. The occupancy and glycan length distribution from glycoproteomics analysis for each site is shown below.