Structures of ACE2–SIT1 recognized by Omicron variants of SARS-CoV-2

The continued spread of severe acute respiratory syn-drome coronavirus 2 (SARS-CoV-2) Omicron variants has caught another wave of COVID-19 pandemic and raised great public health and economic concerns 1,2 . The Omicron BA.2 subvariant has quickly outcompeted the BA.1 subvariant since Feb of 2022 3 . And now, the BA.4/ BA.5 subvariants, which share the same S protein mutations, displayed a higher transmission advantage than BA.2 and exhibit more powerful immune evasion 4 . Angiotensin-converting enzyme 2 (ACE2) is the critical cellular receptor for SARS-CoV-2, directly binding with the receptor binding domain (RBD) of spike glycoprotein (S protein) 5 – 8 . The full-length ACE2 comprises an N-terminal peptidase domain (PD) and a C-terminal collectrin-like domain (CLD) that contains a single transmembrane helix (TM) and a short intracellular seg-ment 9,10 . The PD of ACE2 is the target of SARS-CoV-2 S protein and also mediates the maturation of angiotensin (Ang) which controls vasoconstriction and blood pres-sure 11 . The CLD of ACE2 is reported as the chaperone for membrane traf ﬁ cking of amino acid transporter B 0 AT1 ( SLC6A19 ) and SIT1 ( SLC6A20 ) 12 . Recently, a

To co-express SIT1 and ACE2, about 0.75 mg plasmid for SIT1 and 0.75 mg plasmid for ACE2 were premixed with 3 mg PEIs in 50 ml of fresh medium for 15 mins before adding to cell culture. The transfected cells were cultured for 48 hours before harvesting.
For purification of the SIT1 and ACE2 complex, the cells were collected in buffer containing 25 mM HEPES, pH 7.0, 150 mM NaCl, and three protease inhibitors, aprotinin (1.3 μg/ml, AMRESCO), pepstatin (0.7 μg/ml, AMRESCO), and leupeptin (5 μg/ml, AMRESCO). The membrane fraction was solubilized at 4 °C for 2 hours with 1% (w/v) glyco diosgenin (GDN, Anatrace) and the cell debris was removed by centrifugation at 18,700 g for 45 mins. The supernatant was loaded to anti-FLAG M2 affinity resin (Sigma). After rinsing with the wash buffer 3 containing 25 mM HEPES, pH 7.0, 150 mM NaCl, and 0.01% GDN (w/v), the protein was eluted with wash buffer plus 0.2 mg/ml FLAG peptide. The eluent was further purified by Ni-NTA affinity resin (Qiagen). After elution with the wash buffer 3 supplemented with 300 mM imidazole, the eluent was then concentrated and incubated with BA.2/5 RBD at a molar ratio of about 1:2.4 for 30 mins. Then the protein mixture was subjected to size-exclusion chromatography (Superose 6 Increase 10/300 GL, GE Healthcare) in buffer containing 25 mM HEPES, pH 7.0, 150 mM NaCl and 0.01% GDN. The peak fractions were collected and concentrated for EM analysis. 3

Cryo-EM sample preparation and data acquisition
The protein of SIT1-ACE2-RBD complex was concentrated to 12 mg/mL and aliquots (3.3 μl) of the mixture were placed on glow-discharged holey carbon grids (Quantifoil Au R1.2/1.3), which were blotted for 3.0 s or 3.5 s and flash-frozen in liquid ethane cooled by liquid nitrogen with Vitrobot (Mark IV, Thermo Fisher Scientific). The cryo grids were transferred to a Titan Krios operating at 300 kV equipped with Gatan K3 Summit detector and GIF Quantum energy filter. Movie stacks were automatically collected using AutoEMation 1 , with a slit width of 20 eV on the energy filter and a defocus range from -1.4 µm to -1.8 µm in super-resolution mode at a nominal magnification of 81,000 ×. Each stack was exposed for 2.56 s with an exposure time of 0.08 s per frame, resulting in a total of 32 frames per stack. The total dose rate was approximately 50 e -/Å 2 for each stack. The stacks were motion corrected with

Data processing
Particles were automatically picked using Relion 3.0.6 ref.5-8 from manually selected micrographs. After 2D classification, good particles were selected and subjected to three cycles of heterogeneous refinement with C1 symmetry. The good particles were selected and subjected to homogeneous refinement with C2 symmetry. To further improve the map quality of ACE2-RBD interface, the particles were C2-symmetry expanded and re-extracted at the location of the interface between ACE2 and RBD. The re-extracted dataset was subject to several cycels of 3D classified and focused 4 refinement, resulting in a 3D reconstruction with better quality for ACE2-RBD interface. To further improve the map quality of the transmembrane domain of SIT1, the particles were C2-symmetry expanded and subjected to several cycels of 3D classified and focused refinement. The resolution was estimated with the gold-standard Fourier shell correlation 0.143 criterion 9 with high-resolution noise substitution 10 .

Model building and structure refinement
The model building for the part of the SIT1-ACE2-RBD complex was accomplished with Phenix 11 and Coot 12 . The atomic model of the ACE2-B 0 AT1-RBD (PDB ID: 6M17) was sequence-substituted to the SIT1, BA.2 RBD and BA.5 RBD in chainsaw and fitted into focused refined maps of transmembrane domain of SIT1 and ACE2-RBD interface using MDFF (molecular dynamics flexible fitting) 13 . Each residue was manually checked with Coot with the chemical properties taken into consideration during model building. Statistics associated with data collection, 3D reconstruction and model building are summarized in Supplementary Table S1.

The Bio-Layer Interferometry (BLI) assay
The bindings between ACE2 and Omicron subvariants RBD were performed using Octet Red96e (ForteBio), and the Wuhan-Hu-1 strain RBD was measured as well at the same time. The PD domain of ACE2 was biotinylated using biotinylation kit

Immunocytochemistry
HEK293T cells were seeded on confocal plates coated with 0.5% Matrigel. Cells were 6 washed once with DPBS and fixed with 4% PFA for 20 mins, and then permeabilized in 0.5% Triton X-100 for 10 mins at room temperature. After washing with DPBS for three times, cells were blocked in blocking buffer (5% BSA in DPBS) for 1 hour at room temperature. Cells were then stained with primary antibodies diluted in blocking buffer overnight at 4℃. After that, cells were washed three times with DPBS, and then stained with fluorophore-conjugated secondary antibodies in blocking buffer for 1 hour.
Cells were then washed with DPBS for three times and stained by DAPI. Images were acquired on a Nikon Confocal microscope (A1R+Symp64) using the confocal mode with 40X WI objective. Image analyses were preformed using Fiji