Research ArticleBiochemistry

Cytoplasmic short linear motifs in ACE2 and integrin β3 link SARS-CoV-2 host cell receptors to mediators of endocytosis and autophagy

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Science Signaling  12 Jan 2021:
Vol. 14, Issue 665, eabf1117
DOI: 10.1126/scisignal.abf1117

Figures

  • Fig. 1 Overview of domains and peptides tested for binding.

    (A to C) Representative structures of peptide-binding domain assessed for their potential interactions with peptide sequences from the cytoplasmic tails of ACE2 (B) and integrin β3 (C). Green: Region containing predicted overlapping binding sites for NCK SH2 domain, the ATG8 domains of MAP1LC3As and GABARAPs, and AP2 μ2. Magenta: Predicted PTB binding site. Orange: Predicted class I PDZ-binding site. Blue: Predicted ATG8 binding site in integrin β3. PDB, Protein Data Bank.

  • Fig. 2 Assessment of PDZ and PTB domain binding to the ACE2 cytoplasmic tail.

    (A and B) Saturation curves obtained by fluorescence polarization (FP) experiments for selected class I PDZ domains (A) and a selection of PTB domains and SNX17 FERM (B). Curves were obtained by titrating the protein domains against the respective FITC-labeled ACE2 peptide, containing either a class I PDZ or a predicted PTB binding motif. Data are means ± SEM; n = 3 experiments.

  • Fig. 3 Testing the predicted overlapping SH2, ATG8, and AP2 μ2 domain binding motifs in the ACE2 tail (ACE2775–790) by displacement experiments.

    (A) Saturation curves of AP2 μ2, the ATG8 domains, and NCK1 SH2 for the respective FITC-labeled peptides (ATG9A, SQSTM1, and Tir10). (B) Displacement curves of FP experiments using a peptide from the cytoplasmic ACE2 tail predicted to contain AP2 μ2, ATG8, and NCK SH2 domain binding motifs. Preferential binding of the different domains for unphosphorylated and pTyr781 ACE2 peptide was tested. Data are means ± SEM; n = 3 experiments.

  • Fig. 4 Testing predicted overlapping SH2, ATG8, and AP2 μ2 domain binding motifs in the ACE2 tail by direct binding.

    Saturation curves of AP2 μ2, SH2, and ATG8 domains of the indicated proteins for the respective FITC-labeled ACE2 peptide (key above). Data are means ± SEM; n = 3 experiments.

  • Fig. 5 Investigating the phospho-regulation of the LIR motif in the integrin β3 tail.

    Fluorescence polarization displacement curves obtained using unphosphorylated and phosphorylated peptides representing the cytoplasmic integrin β3 tail. Peptide sequence and sampled phosphosites are indicated in the figure. Data are means ± SEM; n = 3 experiments.

Tables

  • Table 1 Affinities of PDZ, PTB, FERM, SH2, and ATG8 domains as well as AP2 μ2 for the ACE2 C-terminal peptide as determined by FP.

    Equilibrium dissociation constant (KD) values of the tested domains for FITC-labeled ACE2 C-terminal peptides. Indicated errors are the errors of the mean (SEM); n = 3 replicates. N.B., no or low affinity (KD >100 μM).

    Peptide sequenceNameModificationKD ± SEM (μM)
    ACE2796-805*
    FITC-QNTDDVQTSF-COO
    MAGI1 PDZ1Unphos.N.B.
    NHERF3 PDZ113.9 ± 0.3
    NHERF3 PDZ3N.B.
    SCRIB PDZ1N.B.
    SHANK1 PDZ8.3 ± 0.2
    SNTA1 PDZN.B.
    SNX27 PDZ4.7 ± 0.3
    TAX1BP3 PDZN.B.
    ACE2787–798
    FITC-SKGENNPGFQNT-NH2
    DOK1 PTBUnphos.N.B.
    FRS2 PTBN.B.
    IRS1 PTBN.B.
    TLN1 PTBN.B.
    TLN2 PTBN.B.
    SNX17 FERM(100)†
    ACE2775–790
    FITC-RSGENPYASIDISKGE-NH2
    AP2 μ2Unphos.(60)†
    pTyr781N.B.
    pSer783(30)†
    FYN SH2Unphos.N.B.
    pTyr7817.0 ± 0.5
    pSer783N.B.
    NCK1 SH2Unphos.N.B.
    pTyr781N.B.
    pSer783N.B.
    LYN SH2Unphos.N.B.
    pTyr781(60)
    pSer783N.B.
    MAP1LC3A ATG8Unphos.N.B.
    pTyr781N.B.
    pSer783N.B.
    MAP1LC3B ATG8Unphos.N.B.
    pTyr781N.B.
    pSer783N.B.
    GABARAP ATG8Unphos.N.B.
    pTyr781N.B.
    pSer783N.B.
    GABARAPL2 ATG8Unphos.N.B.
    pTyr781N.B.
    pSer783N.B.

    *MBP was tested as a control for binding (N.B.) due to the PDZ domains being MBP-tagged. †Values in brackets indicate estimated affinities due to incomplete saturation curves.

    • Table 2 Affinities of AP2 μ2, ATG8 domains of GABARAPs and MAP1LC3s, and NCK1 SH2 domains for probe peptides as determined by FP.

      Equilibrium dissociation constant (KD) values of AP2 μ2, the ATG8 domains, and NCK1 SH2 for the respective FITC-labeled peptides (ATG9A, SQSTM1, and Tir10). Indicated errors are the errors of the mean (SEM); n = 3 replicates.

      Peptide sequenceProtein domains
      Protein domainKD ± SEM (μM)
      ATG9A4–14
      FITC-FDTEYQRLEAS-NH2
      AP2 μ21.64 ± 0.07
      SQSTM1335–344
      FITC-DDDWTHLSSK-NH2
      GABARAP ATG80.9 ± 0.1
      GABARAPL2 ATG811 ± 1
      MAP1LC3A ATG80.81 ± 0.04
      MAP1LC3B ATG81.9 ± 0.1
      MAP1LC3C ATG80.33 ± 0.01
      Tir10371–280
      FITC-EHIpYDEVAAD-NH2
      NCK1 SH20.055 ± 0.006
    • Table 3 Affinities of ATG8 domains for the integrin β3 peptide.

      KI values for ATG8 domains calculated from displacement experiments using unphosphorylated integrin β3 peptide and phosphorylated peptides (pThr777, pSer778, pThr779, pTyr785, or pThr779 pTyr785). Indicated error is the error of the mean (SEM); n = 3 replicates. N.B., no or low affinity (KI > 100 μM); N.M., not measured.

      Peptide sequencesKI values ± SEM (μM)
      MAP1LC3A
      ATG8
      MAP1LC3B
      ATG8
      MAP1LC3C
      ATG8
      GABARAPL2
      ATG8
      GABARAP
      ATG8
      Unphosphorylated
      CH3CO-
      KEATSTFTNITYRG-NH2
      N.B.N.B.N.B.N.B.N.B.
      pThr777
      CH3CO-
      KEApTSTFTNITYRG-NH2
      N.B.N.B.73 ± 4N.M.47 ± 3
      pSer778
      CH3CO-
      KEATpSTFTNITYRG-NH2
      29.4 ± 0.5N.B.16.1 ± 0.580 ± 5N.M.
      pThr779
      CH3CO-
      KEATSpTFTNITYRG-NH2
      N.B.N.B.82 ± 3N.B.N.M.
      pTyr785
      CH3CO-
      KEATSTFTNITpYRG-NH2
      38 ± 281 ± 283 ± 489 ± 12N.M.
      pThr779/pTyr785
      CH3CO-
      KEATSpTFTNITpYRG-NH2
      8.5 ± 0.515 ± 18 ± 2N.M.17.8 ± 0.8
    • Table 4 Constructs used for expression of protein domains.

      Protein domains and the encoding plasmids. Species is human unless otherwise stated.

      NameUniProtPlasmid
      AP2 μ2 (160–435)Q96CW1pGEX-4 T1
      DOK1 PTB (151–256)Q99704pH1003
      FRS2 PTB (8–136)Q8WU20pH1003
      FYN SH2 (142–251)P06241pGEX
      GABARAP ATG8 (2-115)O95166pGEX-4 T1
      GABARAPL2 ATG8 (2–115)P60520pGEX-4 T1
      IRS1 PTB (157–267)P35568pH1003
      LYN SH2 (106–213)P07948pGEX
      MAGI1 PDZ1 (1–106)Q96QZ7pETM41
      MAP1LC3A ATG8 (2–119)Q9H492pGEX-4 T1
      MAP1LC3B ATG8 (2–119)Q9GZQ8pGEX-4 T1
      MAP1LC3C ATG8 (2–125)Q9BXW4pGEX-4 T1
      Mouse SNX17 FERM (109–388)Q15036pMCSG10
      NCK1 SH2 (275–377)P16333pGEX-2 T
      NHERF3 PDZ1 (2–107)Q5T2W1pETM41
      NHERF3 PDZ2 (132–224)Q5T2W1pETM41
      SCRIB PDZ1 (718–814)Q14160pETM41
      SHANK1 PDZ (655–761)Q9Y566pETM41
      SNX27 PDZ (40–141)Q96L92pETM41
      SNTA1 PDZ (84–171)Q13424pETM41
      TAX1B3 PDZ (7–124)O14907pETM41
      TLN1 PTB (442–770)Q9Y490pH1003
      TLN2 PTB (309–401)Q9Y4G6pH1003

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