Cell culture and transfection. A549 cells (ATCC® CCL-185) were grown in Ham's F-12 Nutrient Mix with 10 % fetal bovine serum, penicillin (100 U ml−1), and streptomycin (100 µg mL−1) (Gibco) at 37 °C in a humidified atmosphere with 5% CO2. pcDNA3.1(+) ACE2-eGFP was transfected using Lipofectamine LTX (Invitrogen) according to the manufacturer’s protocol.
Functionalization of AFM tips. PFQNM-LC and MSCT-D cantilevers (Bruker) were used to probe the interaction between S1-subunit (Genscript, #U5377FC120) or RBD protein (Genscript, #U5377FC120) and ACE2 protein (Sino Biological, 90211-C02H). NHS-PEG24-Ph- aldehyde linkers were used to functionalize AFM tips as previously described.25 Briefly, the cantilevers were immersed in chloroform for 10 min and further cleaned in an UV radiation and ozone (UV-O) cleaner (Jetlight), and immersed overnight in an ethanolamine solution (3.3 g of ethanolamine in 6.6 ml of DMSO). They were washed with DMSO and ethanol 3 times, respectively. Ethanolamine-coated cantilevers were immersed in NHS-PEG24-Ph-aldehyde solution (3.3 mg of it was diluted in 0.5 ml of chloroform and 30 μl of triethylamine) and finally washed 3 times with chloroform and dried with nitrogen.
For AFM tips functionalized with S1-subunit protein, 50 µl of S1-subunit protein solution (0.1 mg/ml) was put onto the cantilevers placed on Parafilm (Bemis NA) and 2 µl of fresh NaCNBH3 solution (6 % wt. vol-1 in 0.1 M NaOH(aq)) was mixed in the protein solution. The cantilevers were incubated in the solution for 1 hour on ice. Then, 5 µl of 1 M ethanolamine solution were carefully added to the protein solution and incubated 10 minutes to quench the reaction and finally washed three times with PBS.
For AFM tips derivatized with the RBD protein, 100 µl of a 100 µM tris-nitrilotriacetic amine 540 trifluoroacetate (Toronto Research Chemicals, Canada) (tris-NTA) solution was put onto the them placed on Parafilm and 2 ul of fresh NaCNBH3 solution was mixed in the protein solution. They were incubated in the solution for 1 hour on ice. Then, add 5 µl of 1 M ethanolamine solution in the protein solution and incubated 10 minutes. The mixture of 50 ul of RBD solution (0.1 mg/ml) and 2.5 µl 5 mM NiCl2 were put onto them and they were incubated for 2 hours. After incubation, they were washed in HEPES solution for 3 times.
Preparation of ACE2-coated model surfaces. ACE2 protein (Sino Biological, 90211-C02H) was immobilized using NHS-EDC chemistry. Gold-coated surfaces were first rinsed with ethanol, dried with a gentle stream of gas nitrogen, cleaned for 15 min by UV and ozone treatment (Jetlight) and incubated overnight in an alkanethiol solution (99 % 11-mercapto-1-undecanol
1 mM (Sigma Aldrich) and 1 % 16-mercaptohexadecanoic acid 1 mM (Sigma Aldrich) in ethanol). The chemically activated samples were rinsed with ethanol, dried with gas nitrogen and immersed for 30 min in the solution of 10 mg chemically activated -dimethylaminopropyl carbodiimide (Sigma Aldrich) and 40 mg of N-hydroxysuccinimide in 4 ml milliQ water. Finally, the surfaces were rinsed with milliQ water, incubated with ACE2 protein (0.1 µg/µL in PBS) on Parafilm (Bemis NA), washed in PBS.
FD-based AFM on model surfaces. FD-based AFM on model surfaces was performed in PBS at room temperature using functionalized MSCT-D probes (Bruker, nominal spring constant of
0.030 N/m and actual spring constants calculated using thermal tune).26 A Bioscope Resolve AFM (Bruker) operated in the force volume (contact) mode (Nanoscope software v9.1) was used. Areas of 5 x 5 µm were scanned, ramp size set to 500 nm and set point force of 500 pN, with a resolution of 32 x 32 pixels and a line frequency of 1 Hz.
Dynamic force spectroscopy (DFS) analysis (using a constant approach speed of 1 µm/s and variable retraction speeds of 0.1, 0.2, 1, 5, 10 and 20 µm/s) and kinetic on-rate estimation
measuring the binding probability for different hold times of 0, 50, 100, 150, 250, 500 and 1000 ms) were performed as described previously.9,27 The curves were analysed using Nanoscope analysis (v2.0, Bruker) and Origin (OriginLab).
Peptides and competition binding assays. To assess the influence of peptides on the S1- subunit-ACE2 interaction, binding probabilities were measured before and after tip incubation with 1 µM, 10 µM and 100 µM of peptide. Briefly, a first map was recorded as described above (i.e. force volume mode, 1 µm/s approach and retraction speed, ramp size of 500 nm, an applied force of 500 pN, resolution of 32 x 32 pixels and line frequency of 1 Hz, hold time of 250 ms), then the peptide at the appropriate concentration was injected and a new map was recorded.
All the peptides ([22-44], [351-357], [22-57], [22-44-g-351-357]) were synthesized by Genscript (Hong-Kong). Those peptides are designed according to the sequence of the ACE2 receptor in complex with the RBD domain of the S1-glycoprotein.
FD-based AFM and fluorescence microscopy on living cells. AFM (Bioscope Resolve, Bruker) coupled to a confocal microscope (Zeiss LSM-980) were used to acquire correlative images as described.10 The AFM was equipped with a 150 µm piezoelectric scanner. The AFM and the microscope were equipped in a cell-culture chamber allowing maintaining the temperature, the humidity and the CO2 level as described.14 Fluorescence images were recorded using an
oil immersion lens (x100, NA 1.46, Zeiss alpha Plan-Apochromat). PFQNM-LC cantilevers (Bruker) were used to record AFM images (~ 25 μm2) at imaging forces of ~ 500 pN. The cantilevers were oscillated at 0.25kHz with a 750 nm amplitude in the PeakForce Tapping mode. The sample was scanned using 256 pixels per line (256 lines) and a frequency of 0.125 Hz. AFM images and FD curves were analyzed using Nanoscope analysis software, Origin, Gwyddion and ImageJ. Optical images were analyzed using Zen software (Zeiss).
Plasma membrane staining. Plasma membrane-CFP BacMam 2.0 (Invitrogen) was used to check the co-localization of ACE2 protein and plasma membrane according to manufacturer’s protocol. Z-stack image was recorded by confocal LSM-980 (Zeiss) using a water immersion lens (x63, NA 1.20, Zeiss C-Apochromat) and 445 nm and 488 nm laser line.
Affinity measurements using biolayer interferometry. Affinity between S1-subunit or RBD and ACE2 was also investigated by biolayer interferometry (BLI), using a Blitz® device equipped with amine reactive 2nd generation (AR2G) biosensors (Pall ForteBio). After hydrating the biosensor for 10 min and performing an initial baseline (10 min), the biosensor surface was chemically activated (5 min) by a freshly prepared 20 mM EDC and 10 mM NHS (in milliQ water) solution. Then, ACE2 (0.025 µg/µL in acetate buffer pH 4) was loaded onto the biosensor during 3 min and the reaction quenched with ethanolamine 1 M (pH 8). After another baseline step (1 min in PBS), binding of S1-subunit or RBD (0.1 mg/mL) was measured for 5 min. Finally, the dissociation step (5 min) was performed in PBS. Data processing and analysis was run using a routine provided by GraphPad Prism.