{"doi":"10.1101/2024.03.07.583884","title":"Visualizing the Rapid Degradation and Differential Binding of Membrane-Bound Human ACE2 Protein Upon Binding and Internalization of the SARS-CoV-2 Spike RBD Protein","abstract":"Abstract The SARS-CoV-2 betacoronavirus infects humans through binding the Angiotensin Converting Enzyme 2 (ACE2) protein that lines the nasal cavity and lungs, followed by import into a cell utilizing the Transmembrane Protease, Serine 2 (TMPRSS2) cofactor. ACE2 binding is mediated by an approximately 200-residue portion of the SARS-CoV-2 extracellular spike protein, the receptor binding domain (RBD). Robust interactions are shown using a novel cell-based assay between an RBD membrane tethered-GFP fusion protein and a membrane-bound ACE2-Cherry fusion protein. Several observations were not predicted, including rapid and sustained interactions leading to internalization of the RBD fusion protein into ACE2-expressing cells and rapid downregulation of ACE2-Cherry fluorescence, suggesting that a membrane-associated form of RBD found on the viral coat may have long-term system-wide consequences on ACE2-expressing cells. Targeted mutation in the RBD disulfide Loop 4 led to a loss of internalization for several variants tested. However, a secreted RBD did not cause ACE2 downregulation of ACE2-Cherry fluorescence. Omicron BA.1 and BA.2 variants have altered their dependency on the amino terminus (Nt) of the ACE2 protein. In contrast, the H-CoV-NL63 RBD is only dependent on the ACE2 internal region for binding, leading to the conclusion that the RBD binding surface of ACE2 appears relatively fluid and amenable to internalizing a range of novel variants.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2024,"id":495076,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9641,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":716588,"name":"Paul Feinstein","orcid":"0000-0002-4924-1732","position":0,"is_corresponding":true}],"reference_count":54,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:09:15.528853Z","pmid":"38496410","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}