Allosteric inhibition of aminopeptidase N functions related to tumor growth and virus infection

Abstract

Cell surface aminopeptidase N (APN) is a membrane-bound ectoenzyme that hydrolyzes proteins and peptides and regulates numerous cell functions. APN participates in tumor cell expansion and motility, and is a target for cancer therapies. Small drugs that bind to the APN active site inhibit catalysis and suppress tumor growth. APN is also a major cell entry receptor for coronavirus, which binds to a region distant from the active site. Three crystal structures that we determined of human and pig APN ectodomains defined the dynamic conformation of the protein. These structures offered snapshots of closed, intermediate and open APN, which represent distinct functional states. Coronavirus envelope proteins specifically recognized the open APN form, prevented ectodomain progression to the closed form and substrate hydrolysis. In addition, drugs that bind the active site inhibited both coronavirus binding to cell surface APN and infection; the drugs probably hindered APN transition to the virus-specific open form. We conclude that allosteric inhibition of APN functions occurs by ligand suppression of ectodomain motions necessary for catalysis and virus cell entry, as validated by locking APN with disulfides. Blocking APN dynamics can thus be a valuable approach to development of drugs that target this ectoenzyme.

Characterization of the interaction between recombinant porcine aminopeptidase N and spike glycoprotein of porcine epidemic diarrhea virus

“…Purified pAPN ectodomain was crystallized with one molecule in each asymmetric unit and its crystal structure was determined at 2.65 Å in a C121 space group (Table 3). As described previously [22,24,43,63,64], pAPN ectodomain adopts a hook-like conformation or so-called seahorse shape ( Fig. 5B and C).…”

Receptor-binding loops in alphacoronavirus adaptation and evolution

“…The observation that HCoV-229E and PRCoV bind to different sites on APN has important consequences. Among species, APN is found in open/intermediate and closed conformations and conversion between them is thought to be important for the catalysis of its substrates 39,42 . The HCoV-229E RBD binds to hAPN in its closed conformation and structural comparison shows that the H-site does not differ between the open and closed conformations.…”

“…This is to be contrasted with the P-site of pAPN that differs in the open and closed conformations. Indeed, the PRCoV RBD has recently been shown to bind to pAPN in the open conformation as a result of P-site interactions made possible in the open form 42 . These differences in binding and receptor conformation are reflected in the fact that enzyme inhibitors that promote the closed conformation of APN block TGEV infection 42 , but not HCoV-229E infection 8 , and the fact that the PRCoV S-protein 42 , but not HCoV-229E 43 , inhibits APN catalytic activity.…”

The Role of Conformational Dynamics in Antigen Trimming by Intracellular Aminopeptidases

“…This conformation was apparently induced by binding of a selective potent inhibitor, exhibited structural changes in the active site loop containing the conserved exo-peptidase G-A-M-E-N motif and was overall very similar to the “closed” conformations of ERAP1 and ERAP2, suggesting that it may constitute the active conformation of the enzyme. It should be noted that this conformational plasticity is not unique to ERAP1 or IRAP, but seems to extend to other members of the M1 family of aminopeptidases since structures of the tricorn-interacting factor F3 ( 30 ) and aminopeptidase N (APN) ( 31 ) have also revealed closed, intermediate and open conformations.…”