Short linear motif candidates in the cell entry system used by SARS-CoV-2 and their potential therapeutic implications

Abstract

The first reported receptor for SARS-CoV-2 on host cells was the angiotensin-converting enzyme 2 (ACE2). However, the viral spike protein also has an RGD motif, suggesting that cell surface integrins may be co-receptors. We examined the sequences of ACE2 and integrins with the Eukaryotic Linear Motif (ELM) resource and identified candidate short linear motifs (SLiMs) in their short, unstructured, cytosolic tails with potential roles in endocytosis, membrane dynamics, autophagy, cytoskeleton, and cell signaling. These SLiM candidates are highly conserved in vertebrates and may interact with the μ2 subunit of the endocytosis-associated AP2 adaptor complex, as well as with various protein domains (namely, I-BAR, LC3, PDZ, PTB, and SH2) found in human signaling and regulatory proteins. Several motifs overlap in the tail sequences, suggesting that they may act as molecular switches, such as in response to tyrosine phosphorylation status. Candidate LC3-interacting region (LIR) motifs are present in the tails of integrin β₃ and ACE2, suggesting that these proteins could directly recruit autophagy components. Our findings identify several molecular links and testable hypotheses that could uncover mechanisms of SARS-CoV-2 attachment, entry, and replication against which it may be possible to develop host-directed therapies that dampen viral infection and disease progression. Several of these SLiMs have now been validated to mediate the predicted peptide interactions.

Fig. 1. The RGD motif of the SARS-CoV-2 spike protein.

(A) Multiple sequence alignment of a part of the SARS-CoV-2 spike RBD region using homologous sequences from betacoronaviruses of various evolutionary distances and showing the location of potential integrin-binding motifs in black. Virus names together with the host organisms, UniProt accessions (*or GenBank accession in the case of RatG13), and sequence region numberings are shown on the left side of the alignment. The location of the region shown in the alignment is indicated in a representative diagram of the spike protein, together with the location of the RGD motif and the region responsible for ACE2 binding. (B) Neighbor-joining tree of the multiple sequence alignment, with this particular set of sequences containing the potential high affinity, low affinity, and reverse integrin-binding motifs (RGD, KGD, and NGR) shown in red, orange, and green boxes, respectively. Only the sequence regions shown in (A) were used in the calculation of the tree. (C) Structure of the SARS-CoV-2 RBD as seen in the ACE2-bound form (PDB:6m17). The RGD motif is shown in red sticks. Regions in direct contact with ACE2 are shown in blue. Residues with missing atomic coordinates (indicating flexibility) in the unbound trimeric spike protein structures (PDB:6vsb, 6vxx, and 6vyb) are shown in transparency. Alignment and tree were prepared in Jalview (226) with Clustal colors. Structure was visualized using UCSF Chimera (228).

Fig. 2. Alignment of ACE2 illustrating conservation of the MIDAS motif.

Multiple sequence alignment of a part of the ACE2 extracellular domain using 25 homologous sequences from different vertebrate lineages (mammals, birds, reptiles, and fish) and showing the conservation of the Dx[ST]xS motif as well as an NxT glycosylation site (main residues displayed above). A red box marks the conservation range of the MIDAS motif in all sequences but the hagfish. Organism names, UniProt IDs (UniParc for hagfish), and sequence numberings are listed on the left side of the alignment. The location of the region shown in the alignment is indicated in a representative diagram of the ACE2 protein. Figure was prepared with Jalview using Clustal colors. TM, transmembrane; C-ter, C-terminal.

Fig. 3. Alignment of ACE2 illustrating conserved motifs in the cytosolic C-terminal tail following the transmembrane helix.

Multiple sequence alignment of ACE2 transmembrane and C-terminal regions using 25 homologous sequences from different vertebrate lineages (mammals, birds, reptiles, and fish) and showing their motif conservation. The names (bold) and key residues of the motifs are displayed above the alignment (ɸ stands for a bulky hydrophobic residue), including a conserved tyrosine (bold) and excluded positions (red and crossed). Red boxes mark the conservation range of the PDZ-binding motif (PBM) (all sequences) and NPY motif (in mammals, birds, and some fish). Organism names, UniProt IDs (UniParc for hagfish), and sequence numberings are listed on the left side of the alignment. The location of the region shown in the alignment is indicated in a representative diagram of the ACE2 protein. Figure was prepared with Jalview using Clustal colors.

Fig. 4. The summary for the ACE2 C-terminal tail provided by PhosphoSitePlus.

No low-throughput (LTP) studies have been recorded in the database for ACE2. Thirteen high-throughput (HTP) studies have identified phosphorylation on Tyr⁷⁸¹. Phosphosites reported in the extracellular part of ACE2 have only been reported once each and therefore are likely to be misidentified peptides.

Fig. 5. Alignment of human integrins illustrating conserved motifs in the cytosolic C-terminal tail.

(A) Multiple sequence alignment of human integrin C-terminal regions, not including the two most divergent β tails (β4 and β8). The alignment shows motif conservation of the NPxY and LIR motifs (key residues displayed above). Red boxes mark the conservation range of the PTB motif in all sequences and the location of the LIR motif in integrin β3. Protein names, UniProt IDs, and sequence numberings are listed on the left side of the alignment. (B) Summary of the PTMs on the C-terminal tail of integrin β3. Details of the experimental evidence for the PTB tyrosine phosphorylations are highlighted: pTyr⁷⁷³ (pY773) and pTyr⁷⁸⁵ (pY785). Graph was obtained from PhosphoSitePlus.

Fig. 6. Model of the proposed interplay between motifs in the interface between SARS-CoV-2 and a human host cell to achieve RME.

Receptors of the SARS-CoV-2 (gray) and a human host cell (light blue) motifs involved in viral recognition and entry are shown in colored boxes. Elements shown in one of the monomers of a homotrimer (spike) or homodimer (ACE2) are also present in the other proteins forming that complex. Lines below motif boxes represent each of the overlapping motifs in that specific region. Arrows indicate the related cellular process, and the protein known to interact with their respective motif is indicated in parenthesis. Phosphorylation sites are shown as inverted triangles, with the respective sequence position indicated. For the β-integrin tail, the PTB/apoPTB phospho-switch is depicted as two separate versions of the same motif region, and the subscripts represent the motif order in the sequence. SLiMs mediating interactions are represented with boxes of different colors, protease cleavage sites with hexagons (PCs, furin-like proprotein convertases; T, TMPRSS2), phosphorylation sites with inverted triangles, and structural motifs with ovals. The color code is as follows: cleavage sites, yellow hexagon; apoPTB/PTB motif, orange; endocytic sorting signal motif, purple; I-BAR–binding motif, dark red; LIR motif, blue; MIDAS motif, gray; SH2 motif, green; PBM motif, magenta; RGD motif, bright red; and CendR motif, brown. † indicates that these motifs had been previously experimentally validated.