Structural Changes at the Zinc Active Site of ACE2 on Binding the SARS-CoV-2 Spike Protein Receptor Binding Domain.

The causative agent of Covid-19 is the SARS-CoV-2 virus. Initiation of cell entry by SARS-CoV-2 is critically dependent upon binding of the SARS-CoV-2 spike protein to angiotensin-converting enzyme 2 (ACE2, EC 3.4.17.23). The mechanism of binding of the SARS-CoV-2 spike receptor binding domain to ACE2 is among the most intensively studied infection mechanisms of any pathogen, including a very large number of structural studies. ACE2 is a membrane-associated zinc carboxypeptidase, comprising three domains, the protease domain, a neck domain, and a membrane-spanning α-helical domain. In addition to its role as a carboxypeptidase, ACE2 is also a chaperone for a Na+-amino acid cotransporter called B0AT1, and in the presence of B0AT1, full-length ACE2 forms dimers. Most studies to date related to Covid-19 have employed just the ACE2 protease domain and have neglected any possible roles of the Zn2+-containing ACE2 active site. We show here that ACE2, including the neck domain in addition to the protease domain (and in the absence of B0AT1), is dimeric and shows distinctive allostery in its catalytic activity. In contrast, the intensively studied protease domain is monomeric and shows no allostery. Binding of the spike receptor binding domain (RBD) to dimeric ACE2 eliminates its allostery. X-ray absorption spectroscopy of Zn2+ ACE2 shows distinctive changes in the active site structure upon binding of spike RBD but only in the dimeric form. Taken together, our results indicate that the Zn2+-containing active site exhibits a notable level of flexibility and that the dimeric form of ACE2, including both protease and neck domains, likely presents a superior model for the study of ACE2-spike interactions than the monomeric ACE2.