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. 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 sequence Name Modification KD ± SEM (μM) ACE2796-805*
FITC-QNTDDVQTSF-COO−MAGI1 PDZ1 Unphos. N.B. NHERF3 PDZ1 13.9 ± 0.3 NHERF3 PDZ3 N.B. SCRIB PDZ1 N.B. SHANK1 PDZ 8.3 ± 0.2 SNTA1 PDZ N.B. SNX27 PDZ 4.7 ± 0.3 TAX1BP3 PDZ N.B. ACE2787–798
FITC-SKGENNPGFQNT-NH2DOK1 PTB Unphos. N.B. FRS2 PTB N.B. IRS1 PTB N.B. TLN1 PTB N.B. TLN2 PTB N.B. SNX17 FERM (100)† ACE2775–790
FITC-RSGENPYASIDISKGE-NH2AP2 μ2 Unphos. (60)† pTyr781 N.B. pSer783 (30)† FYN SH2 Unphos. N.B. pTyr781 7.0 ± 0.5 pSer783 N.B. NCK1 SH2 Unphos. N.B. pTyr781 N.B. pSer783 N.B. LYN SH2 Unphos. N.B. pTyr781 (60) pSer783 N.B. MAP1LC3A ATG8 Unphos. N.B. pTyr781 N.B. pSer783 N.B. MAP1LC3B ATG8 Unphos. N.B. pTyr781 N.B. pSer783 N.B. GABARAP ATG8 Unphos. N.B. pTyr781 N.B. pSer783 N.B. GABARAPL2 ATG8 Unphos. N.B. pTyr781 N.B. pSer783 N.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 sequence Protein domains Protein domain KD ± SEM (μM) ATG9A4–14
FITC-FDTEYQRLEAS-NH2AP2 μ2 1.64 ± 0.07 SQSTM1335–344
FITC-DDDWTHLSSK-NH2GABARAP ATG8 0.9 ± 0.1 GABARAPL2 ATG8 11 ± 1 MAP1LC3A ATG8 0.81 ± 0.04 MAP1LC3B ATG8 1.9 ± 0.1 MAP1LC3C ATG8 0.33 ± 0.01 Tir10371–280
FITC-EHIpYDEVAAD-NH2NCK1 SH2 0.055 ± 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 sequences KI values ± SEM (μM) MAP1LC3A
ATG8MAP1LC3B
ATG8MAP1LC3C
ATG8GABARAPL2
ATG8GABARAP
ATG8Unphosphorylated
CH3CO-
KEATSTFTNITYRG-NH2N.B. N.B. N.B. N.B. N.B. pThr777
CH3CO-
KEApTSTFTNITYRG-NH2N.B. N.B. 73 ± 4 N.M. 47 ± 3 pSer778
CH3CO-
KEATpSTFTNITYRG-NH229.4 ± 0.5 N.B. 16.1 ± 0.5 80 ± 5 N.M. pThr779
CH3CO-
KEATSpTFTNITYRG-NH2N.B. N.B. 82 ± 3 N.B. N.M. pTyr785
CH3CO-
KEATSTFTNITpYRG-NH238 ± 2 81 ± 2 83 ± 4 89 ± 12 N.M. pThr779/pTyr785
CH3CO-
KEATSpTFTNITpYRG-NH28.5 ± 0.5 15 ± 1 8 ± 2 N.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.
Name UniProt Plasmid AP2 μ2 (160–435) Q96CW1 pGEX-4 T1 DOK1 PTB (151–256) Q99704 pH1003 FRS2 PTB (8–136) Q8WU20 pH1003 FYN SH2 (142–251) P06241 pGEX GABARAP ATG8 (2-115) O95166 pGEX-4 T1 GABARAPL2 ATG8 (2–115) P60520 pGEX-4 T1 IRS1 PTB (157–267) P35568 pH1003 LYN SH2 (106–213) P07948 pGEX MAGI1 PDZ1 (1–106) Q96QZ7 pETM41 MAP1LC3A ATG8 (2–119) Q9H492 pGEX-4 T1 MAP1LC3B ATG8 (2–119) Q9GZQ8 pGEX-4 T1 MAP1LC3C ATG8 (2–125) Q9BXW4 pGEX-4 T1 Mouse SNX17 FERM (109–388) Q15036 pMCSG10 NCK1 SH2 (275–377) P16333 pGEX-2 T NHERF3 PDZ1 (2–107) Q5T2W1 pETM41 NHERF3 PDZ2 (132–224) Q5T2W1 pETM41 SCRIB PDZ1 (718–814) Q14160 pETM41 SHANK1 PDZ (655–761) Q9Y566 pETM41 SNX27 PDZ (40–141) Q96L92 pETM41 SNTA1 PDZ (84–171) Q13424 pETM41 TAX1B3 PDZ (7–124) O14907 pETM41 TLN1 PTB (442–770) Q9Y490 pH1003 TLN2 PTB (309–401) Q9Y4G6 pH1003
