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SARS-CoV-2 utilizes angiotensin-converting enzyme 2 (ACE2) as its primary receptor to initiate viral entry into host cells. While ACE2 is highly conserved across different species, genetic variability in the interacting surfaces between ACE2 orthologs and SARS-CoV-2 spike (S) protein can modulate viral binding affinity and entry efficiency. This study investigates the impact of amino acid substitutions in human ACE2 (hACE2) interacting with the receptor-binding domain of SARS-CoV-2 S protein. Site-directed mutagenesis, combined with molecular dynamics simulations and pseudovirus assays, revealed that D30V and H34R substitutions reduce hACE2 binding affinity and fusogenic activity, impairing SARS-CoV-2 entry. However, the double mutant D30V-H34R did not reduce viral entry efficiency further, suggesting compensatory molecular interactions at the ACE2-S binding interface. These insights contribute to a deeper understanding of SARS-CoV-2-host interactions and may guide future therapeutic development targeting viral entry mechanisms.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      Given the pivotal role of angiotensin-converting enzyme 2 (ACE2) in mediating viral entry and the genetic divergence observed in ACE2 orthologs across different species, we aimed to elucidate further the molecular intricacies underlying the interactions between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike (S) protein and ACE2. In this study, we examined the amino acid residues in ACE2 orthologs interacting with SARS-CoV-2 spike receptor-binding domain to identify those with discernible effects on viral binding and entry. Through\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      mutagenesis and\n                      <jats:italic toggle=\"yes\">in silico<\/jats:italic>\n                      modeling studies of ACE2 variants, we have pinpointed the amino acid substitutions in human ACE2 that affect SARS-CoV-2 binding and entry. This work can significantly advance our understanding of the molecular mechanisms of SARS-CoV-2-host interactions, receptor recognition, viral entry process, and potential therapeutic options targeting coronavirus entry.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.03244-24","type":"journal-article","created":{"date-parts":[[2025,6,20]],"date-time":"2025-06-20T09:00:57Z","timestamp":1750410057000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":5,"title":["Critical amino acid residues in human ACE2 for SARS-CoV-2 spike protein binding and virus entry"],"prefix":"10.1128","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3175-2718","authenticated-orcid":true,"given":"Weiyi","family":"Chen","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03q8dnn23","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Infectious Diseases and 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