Fig 2: Structural determinants for ACE2 binding to the PDZ-scaffold protein NHERF1(A) Domain organization of ACE2 and NHERF1. ACE2 is shown as a dimer (PDB code 6M18) prepared by PyMol (Schrödinger). Monomeric ACE2 colored in yellow or cyan is a single-pass transmembrane protein containing an N-terminal peptidase domain (PD) and a C-terminal collectrin-like domain (CLD). The missing intracellular C-terminal tail containing a typical PDZ-binding motif 802QTSF805 from the solved structure is represented as yellow or cyan dotted lines assuming a random coil conformation. NHERF1 possesses a carboxy-terminal Ezrin/Radixin/Moesin/Merlin-binding domain (EBD) and two N-terminal PDZ domains, each of which has a same conserved GYGF core-binding motif. The schematic diagram of NHERF1 was created by Domain Graph (DOG) (Ren et al., 2009).(B) Co-immunoprecipitation of ACE2 with wild-type (WT), S1 where GYGF was mutated to GAGA in PDZ1, or S2 with GAGA substitution for GYGF. Proteins from HEK293 GnTI- cell lysates were immunoprecipitated with anti-FLAG M2 agarose beads followed by immunoblot analysis using FLAG, myc, or actin antibodies. The molecular markers (kDa) are shown on the right.(C) Quantification analysis of Figure 1B by ImageJ (Schneider et al., 2012). Intensity of ACE2 binding to wild-type NHERF1 was set to 100%. Relative binding of various NHERF1 to ACE2 was calculated and plotted using GraphPad Prism 9. Data are means ± S.D. (error bars) of n = 3 independent experiments (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).(D) Reverse co-immunoprecipitation analysis to that shown in Figure 1B. Samples were immunoprecipitated with anti-c-Myc agarose beads followed by immunoblot analysis using FLAG, myc, or actin antibodies. The illustrated immunoblots are representative of three replicates. Quantification analysis is shown in Figure S1.(E) Co-immunoprecipitation of NHERF1 with wild-type ACE2 or a mutant with quadruple Ala replacement for the PDZ -QTSF recognition motif. Purified proteins from HEK293 GnTI- cell lysates were immunoprecipitated with anti-FLAG M2 agarose beads followed by immunoblot analysis using FLAG, myc, or actin antibodies. Results represent three independent experiments.(F) Computational modeling of NHERF1 PDZ1 (red) or PDZ2 (green) structure complexed with the C-terminal eight-residue tail of ACE2 by molecular dynamics simulation as detailed previously (Mamonova et al., 2015). For the sake of simplicity, only the last four resides 802QTSF805 were shown in the structures. The hydrophobic pocket is formed by Phe26 (yellow), Leu28 (cyan), Val76 (green), and Ile79 (orange) in PDZ1 and Phe166 (yellow), Val216 (cyan), and Ile219 (purple) in PDZ2. The hydrogen bonds are shown as dashed lines.

Fig 3: Direct interaction between endogenous ACE2 and NHERF1 in lung and intestine cells susceptible to SARS-CoV-2 infection(A) Constitutive expression of ACE2 and NHERF1 in HEK293T, Caco-2, Calu-3, and HK2 cells detected by western blotting (left panel). Molecular markers are indicated in kDa on the right. Relative protein expression of ACE2 and NHERF1 from the whole-cell lysate was normalized to actin and plotted in the right panel, where the highest level of ACE2 in Calu-3 (red) and NHERF1 in Caco-2 (blue) was set to 1, respectively. Shown are representative immunoblots of three experiments with similar results. Data are means ± S.D. (error bars).(B) Visualization of ACE2/NHERF1 interactions in Calu-3 and Caco-2 cells labeled by PLA probes (red) and DAPI (blue). Representative images of confocal microscopy were shown on the left panel. Scale bar (white), 10 µm. Quantification of PLA signals per cell was graphed in the right panel (error bars are SD).

Fig 4: Proposed SARS-CoV-2 cell entry mechanism regulated by PDZ-mediated interaction(A) ß-Arrestin 1 or ß-arrestin 2 binds to ACE2 with nearly equivalent affinity. FLAG-ß-arrestin 1 or 2 was transiently coexpressed in HEK293 GnTI cells with Myc-ACE2. The cleared cell lysates were incubated with anti-c-Myc agarose beads. Proteins are eluted by 2x Laemmli sample buffer (Bio-Rad) supplemented with ß-mercaptoethanol and detected by western blotting using the indicated specific antibodies (left panel). ß-Actin was used as a loading control. The molecular markers are indicated in kDa on the right. Given the equivalent expression of myc-ACE2 observed in the cell lysate and immunoprecipitates, pull-down of ß-arrestin 1 or ß-arrestin 2 by myc-ACE2 was quantified by normalizing the signal intensities to their individual lysate expression, respectively (right panel). Error bars indicate SD.(B) NHERF1 is not required to promote ACE2 dimerization. FLAG-NHERF1 and Myc-ACE2 were transiently transfected into HK2 cells. Forty-eight hours post transfection, the cell lysates were cleared by centrifugation and incubated with anti-c-Myc agarose beads (only the sample in the lower left panel was prepared using anti-FLAG agarose beads and therefore eluted by 3x FLAG peptide). Purified proteins by c-Myc peptide elution were detected by the indicated antibodies. The boxed blots in red are from SDS-PAGE (top panel) and the blots in the lower panel are from blue native PAGE performed as described before (Dewson, 2015). Molecular weights are indicated at the right side of the blots. In the lower right panel, the same samples were loaded in the first and third lane to better show the slight difference of protein size from different samples.(C) Sequence alignment of ACE2 and NRP1 from SARS-CoV-2 hosts human (Protein ID: NP_001358344.1 for ACE2 and ADN93470.1 for NRP1) and bat (Protein ID: NP_001231902.2 for ACE2 and KAF6372190.1 for NRP1). Human and bat ACE2/Ace2 and NRP1/Nrp1 are used because they are primary hosts of SARS-CoV-2 (Burki, 2020). Both identified SARS-CoV-2 receptors are single-pass transmembrane proteins and have a typical type I PDZ-binding motif (QTSF and YSEA) implying a possible common mechanism by which NHERF1 regulates SARS-CoV-2 receptor internalization. The single-pass transmembrane regions are colored in red, and the intracellular C-terminal tail is colored in blue. The conserved serine and threonine residues are indicated by asterisks and are likely to be potential phosphorylation sites denoted by an encircled P. These sites may mediate SARS-CoV-2 receptor binding to ß-arrestin as in the case of ß2AR, where the phosphorylated receptor C terminus is required for binding to ß-arrestin and receptor internalization (Nobles et al., 2011).(D) NHERF1 functions in ACE2-mediated SARS-CoV-2 cell entry in lung, kidney, and intestine and possibly NRP1-mediated virus entry in brain. NHERF1 enhances plasma membrane localization of B0AT1-stabilized ACE2 receptor and regulates its internalization and hence, ACE2-mediated SARS-CoV-2 cell entry. Except NHERF1, the virion-receptor complex also requires ß-arrestin to facilitate its internalization process in which clathrin is involved. Once internalized, SAS-CoV-2 is either degraded in lysosomes (pink arrow) or released into the cytosol where the viral genomic RNA is released and immediately translated into viral RNA polymerase. The ACE2 receptor may be recycled back to the plasma membrane. Viral RNA genome and its structural nucleocapsid protein is replicated and synthesized in the cytoplasm. Other viral structural proteins are translated in the endoplasmic reticulum and further glycosylated in the Golgi. Mature progeny virions are then formed and bud out from plasma by exocytosis.