Figure 1.
Stereochemical differences between 9-O- and 4-O-acetylated sialic acid.
Stick representation of (left) αNeu5,9Ac22Me and (right) αNeu4,5Ac22Me. Backbone αNeu5Ac2Me is colored in gray (carbon), red (oxygen) and blue (nitrogen). The 9-O-Ac group of αNeu5,9Ac22Me and 4-O-Ac group of αNeu4,5Ac22Me are highlighted in cyan (carbon).
Figure 2.
HE-Fc fusion protein displays proper receptor-binding and enzymatic activities.
(A) Binding of two-fold serial dilutions (starting at 100 µg/ml) of esterase-deficient Fc-fusion proteins (HE0-Fc) of BCoV-Mebus and MHV-S in a solid-phase lectin-binding assay towards horse serum glycoproteins (HSG) and bovine submaxillary mucins (BSM). Relative binding in percentages is calculated with the binding of the highest concentration lectin set at a 100%. Wells incubated without lectin (“mock”) were included as negative control. (B) Receptor destroying enzyme activity towards HSG. Coated HSG was treated with two-fold serial dilutions (starting at 100 ng/ml) of enzymatically-active BCoV-Mebus and MHV-S HE Fc-fusion proteins and 4-O-Ac-Sia content was detected by solid phase lectin binding assay with MHV-S HE0-Fc. Decrease in signal as compared to untreated HSG is plotted in percentages. (C) MHV-S HE ectodomain displays sialate-4-O-acetylesterase activity towards the synthetic di-O-acetylated sialic acid analogue αNeu4,5,9Ac32Me. Graphs show total ion current gas-chromatograms and Sia subtypes were identified by mass spectrometry: Sia (αNeu5Ac2Me [peak 1]), 4-O-Ac-Sia (αNeu4,5Ac22Me [peak 2]), 9-O-Ac-Sia (αNeu5,9Ac22Me [peak 4], 4,9-di-O-Ac-Sia (αNeu4,5,9Ac32Me [peak 5]). Peak 3 represents a non-sialic acid compound. (D) Receptor binding activity of MHV-S HE ectodomain was assessed by hemagglutination assay with rat erythrocytes and twofold serial dilutions of the HE proteins (10,000 to 5 ng per well, arrow).
Table 1.
Data collection and refinement statistics.
Figure 3.
Overall structure and comparison to BCoV-Mebus HE.
(A) Ribbon representation of the dimeric MHV-S (residues 25–395) and BCoV-Mebus HE (residues 19–376) structures. One monomer is colored grey, the other by domain: lectin domain (R, blue) with bound αNeu4,5Ac22Me (MHV-S HE) or αNeu4,5,9Ac32Me (BCoV-Mebus HE; cyan sticks) and potassium ion (magenta sphere); esterase domain (E, green); membrane-proximal domain (MP, red). (B) Linear representation of MHV HE with domains color-coded as in panel A. Grey segments indicate the signal-peptide (SP) and transmembrane (TM) domain. The bracket indicates the part of the protein for which the structure has been solved. (C) Structure- (MHV-S and BCoV-Mebus) and sequence-based (MHV-DVIM) alignment of HE sequences. Colored boxes above the sequences indicate domain organization as in panel A and B and black lines indicate loops involved in receptor binding. Note that in MHV-S HE two insertions increase the length of loops R3 and R4. Asterisks indicate the highly conserved residues of the potassium binding site and boxes indicate the critical serine, histidine and aspartic acid residues of the catalytic site. Residues that interact with the ligand are indicated in bold; those conserved among all three HEs are highlighted by grey shading. Other residues also conserved in all three HEs are highlighted in yellow. The residues in disordered loops of the esterase domain are indicated in light gray lettering.
Figure 4.
Comparison of the MHV-S and BCoV-Mebus HE receptor binding sites.
(A) Ribbon superposition of the MHV-S and BCoV-Mebus HE receptor binding sites. BCoV-Mebus HE is colored gray, coloring of MHV-S HE as in panel A. Bound receptor analogues are shown as cyan sticks and potassium ions as magenta spheres. The five surface exposed loops and the RBS-hairpin that interact with the receptor are indicated. Note that only the R3- and R4-loops differ in conformation. (B) Close-up of the HE-potassium binding-site of MHV-S HE and BCoV-Mebus HE. Shown in ribbon representation are the R3-loop (salmon) and RBS-hairpin (purple) that interacts with the potassium ion (magenta sphere).
Figure 5.
MHV-S HE has a unique receptor-binding site that binds specifically 4-O-acetylated sialic acid.
(A) Surface and (B) stick representation of the MHV-S HE receptor-binding site in complex with a receptor analogue. The ligand bound to the HE receptor-binding site is shown in stick representation and the potassium ion as a magenta sphere, indicated by a black arrow in panel A. Hydrogen bonds between HE and the receptor are shown as black dashed lines. Surface representation of the MHV-S HE receptor-binding site reveals two pockets accommodating the 4-O- and 5-N-acetyl groups of the receptor, respectively. Note that crystals were soaked with αNeu4,5,9Ac32Me, but most likely as a result of the low pH crystallization conditions, the 9-O-Ac group was lost [42]. (C) Surface and (D) stick representation of the BCoV-Mebus HE receptor-binding site. Note that the topology of the two hydrophobic pockets is conserved, except they bind different substituents of the receptor analogue. (E) The effect of Ala substitutions on receptor binding. Relative binding affinity of wild-type HE0 (wt) and its derivatives was assessed by hemagglutination assay with rat erythrocytes and twofold serial dilutions of each of the HE0-Fc chimeras (5,000 to 10 ng per well, arrow). (F) Binding of twofold serial dilutions of wild-type (wt) HE0-Fc chimera and its derivatives in a solid-phase lectin-binding assay towards horse serum glycoproteins (HSG) as described in Figure 2A.