B(0)AT1 amino acid transporter complexed with SARS-CoV-2 receptor ACE2 forms a heterodimer functional unit: in situ conformation using radiation inactivation analysis

Stevens, Bruce R; Ellory, J Clive; Preston, Robert L

The SARS-CoV-2 receptor, Angiotensin Converting Enzyme-2 (ACE2), is expressed at levels of greatest magnitude in the small intestine as compared to all other human tissues. Enterocyte ACE2 is co-expressed as the apical membrane trafficking partner obligatory for expression and activity of the B(0)AT1 sodium-dependent neutral amino acid transporter. These components are assembled as an [ACE2: B(0)AT1](2) dimer-of-heterodimers quaternary complex that putatively steers SARS-CoV-2 tropism in the gastrointestinal (GI) tract. GI clinical symptomology is reported in about half of COVID-19 patients, and can be accompanied by gut shedding of virion particles. We hypothesized that within this 4-mer structural complex, each [ACE2: B(0)AT1] heterodimer pair constitutes a physiological “functional unit.” This was confirmed experimentally by employing purified lyophilized enterocyte brush border membrane vesicles that were exposed to increasing doses of high-energy electron radiation from a 16 MeV linear accelerator. Based on established target theory, the results indicated the presence of Na(+)-dependent neutral amino acid influx transport activity functional unit with target size mw = 183.7 ± 16.8 kDa in situ in intact apical membranes. Each thermodynamically stabilized [ACE2: B(0)AT1] heterodimer functional unit manifests the transport activity within the whole ∼345 kDa [ACE2: B(0)AT1](2) dimer-of-heterodimers quaternary structural complex. The results are consistent with our prior molecular docking modeling and gut-lung axis approaches to understanding COVID-19. These findings advance the understanding of the physiology of B(0)AT1 interaction with ACE2 in the gut, and thereby potentially contribute to translational developments designed to treat or mitigate COVID-19 variant outbreaks and/or GI symptom persistence in long-haul Post-Acute Sequelae of SARS-CoV-2 (PASC).