Putative Receptor Binding Domain of Bat-Derived Coronavirus HKU9 Spike Protein: Evolution of Betacoronavirus Receptor Binding Motifs

Abstract

The suggested bat origin for Middle East respiratory syndrome coronavirus (MERS-CoV) has revitalized the studies of other bat-derived coronaviruses with respect to interspecies transmission potential. Bat coronavirus (BatCoV) HKU9 is an important betacoronavirus (betaCoV) that is phylogenetically affiliated with the same genus as MERS-CoV. The bat surveillance data indicated that BatCoV HKU9 has been widely spreading and circulating in bats. This highlights the necessity of characterizing the virus for its potential to cross species barriers. The receptor binding domain (RBD) of the coronavirus spike (S) protein recognizes host receptors to mediate virus entry and is therefore a key factor determining the viral tropism and transmission capacity. In this study, the putative S RBD of BatCoV HKU9 (HKU9-RBD), which is homologous to other betaCoV RBDs that have been structurally and functionally defined, was characterized via a series of biophysical and crystallographic methods. By using surface plasmon resonance, we demonstrated that HKU9-RBD binds to neither SARS-CoV receptor ACE2 nor MERS-CoV receptor CD26. We further determined the atomic structure of HKU9-RBD, which as expected is composed of a core and an external subdomain. The core subdomain fold resembles those of other betaCoV RBDs, whereas the external subdomain is structurally unique with a single helix, explaining the inability of HKU9-RBD to react with either ACE2 or CD26. Via comparison of the available RBD structures, we further proposed a homologous intersubdomain binding mode in betaCoV RBDs that anchors the external subdomain to the core subdomain. The revealed RBD features would shed light on the evolution route of betaCoV.

Figure 1

Sequence features of HKU9-RBD. (A) Schematic representation of BatCoV HKU9 S. The indicated domain elements were defined on the basis of either the pairwise sequence alignment results or the bioinformatics predictions. The signal peptide (SP), transmembrane domain (TM), and heptad repeats 1 and 2 (HR1 and HR2, respectively) were predicted with the SignalP 4.0 server, TMHMM server, and Learncoil-VMF program, respectively, while the N-terminal domain (NTD) and RBD were deduced by alignment with the N-terminal galectin-like domain of murine hepatitis virus S and MERS-RBD, respectively. The S1/S2 site potentially cleaved by furin-like proteases could not be ascertained and is therefore labeled with a question mark. (B and C) Structure-based alignment of the HKU9-, SARS-, MERS-, and HKU4-RBD sequences. The arrows and spiral lines indicate strands and helices, respectively. These secondary structure elements were labeled as illustrated in Figure 3. The conserved cysteine residues that form three disulfide bonds in the structures are marked with Arabic numerals 1–3. The core subdomain is conserved among the four RBD structures, but the external subdomain is structurally irrelevant. We therefore present the sequences separately. The two elements that anchor the external subdomain to the core subdomain are highlighted with black boxes. (B) Core subdomain sequence. (C) External subdomain sequence.

Figure 2

Characterization of HKU9-RBD by SPR assays. The indicated RBD proteins expressed by insect cells were immobilized on CM5 chips and tested for the binding with gradient concentrations of human ACE2 or CD26 using a BIAcore 3000 machine. The recorded kinetic profiles are shown: (A) human ACE2 and SARS-RBD, (B) human CD26 and MERS-RBD, (C) human ACE2 and HKU9-RBD, and (D) human CD26 and HKU9-RBD. Clearly shown is the fact that HKU9-RBD does not bind either ACE2 or CD26, in the context of which SARS-RBD and MERS-RBD bind their respective receptors. Then we purified the mFc-fused HKU9-RBD proteins in mammalian (293T) cells and assembled the abilities to bind CD26 or ACE2 proteins using a captured SPR method by a BIAcore T100 system. The anti-mouse antibodies were immobilized on CM5 chips. The mFc-fused RBD proteins were then captured (3 μg/mL for 60 s) by the antibodies and tested for binding to human ACE2 or CD26. (E) The mFc-fused SARS-RBD (SARS-RBD-mFc) did not bind to CD26 but bound to ACE2 well. (F) The mFc-fused MERS-RBD (MERS-RBD-mFc) did not bind to ACE2 but bound to CD26 well. (G) The mFc-fused HKU9-RBD (HKU9-RBD-mFc) does not bind either ACE2 or CD26.

Figure 3

Crystal structure of HKU9-RBD. The core and external subdomains are colored magenta and green, respectively. The core subdomain is further divided into a center region (core-center) and a peripheral region (core-peripheral), which are encircled. The core-center strands and helices are labeled βc1−βc5 and H1–H6, respectively, while the core-peripheral strands are marked βp1−βp3. The disulfide bonds and the RBD termini are labeled. The core subdomain is further presented in a surface representation in the right panel to highlight the top positioning of the external subdomain like a hat.

Figure 4

Structural and topological comparison of available betaCoV RBD structures. Four structures, including those of HKU9-, SARS-, MERS-, and HKU4-RBD, were oriented similarly and are presented as cartoons in parallel. The core-center, core-peripheral, and the external subdomain are encircled and highlighted in yellow. For each structure, the topological arrangement of the core-center and core-peripheral strands as well as of the external components is depicted. The core strands that flank the external subdomain are colored red and blue, respectively. Yellow lines indicate disulfide bonds. The N- and C-termini are highlighted: (A) HKU9-RBD, (B) SARS-RBD, (C) MERS-RBD, and (D) HKU4-RBD. The similarity in the topological arrangement of the external subdomain as an insertion between two core strands is illustrated.

Figure 5

Homologous intersubdomain amino acid interactions anchoring the external subdomain to the core subdomain. (A) Superimposition of the betaCoV RBD (HKU9-RBD in green, SARS-RBD in yellow, MERS-RBD in blue, and HKU4-RBD in cyan) structures highlighting the external elements that can be well-aligned. These two elements, with seven (element 1) and eight (element 2) amino acids, respectively, engage mainly core subdomain helices H2 and H6 for the intersubdomain interactions. To facilitate comparison, the element residues were successively assigned a position marker (a–g for element 1 and a–h for element 2), which is highlighted. (B) Characterization of the element residues for their contributions to the intersubdomain binding. The two external elements are presented as cartoons, while the core subdomain is shown at the surface. At each position, the residue is marked sequentially with the position marker, the amino acid identity and numbering, the interacting mode/type, and the side-chain orientation. For the interaction mode, the hydrophobic or van der Waals interactions are indicated with encircled Ps, the side-chain H-bonds with encircled Ss, and main-chain H-bonds with encircled Ms. The side-chain orientations are indicated with arrows. (C) Summary of the intersubdomain interactions specified in panel B. The element sequences of the four RBDs are aligned and listed. A + indicates that a certain type of interaction is commonly observed at the position, while a +/– indicates that the interaction type is specific to some but not all of the four RBDs. The arrows mark the side-chain orientations.