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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


The dynamic conformation of mammalian APN ectodomains.(a) Ribbon representation of the closed (pAPN), intermediate (hAPN) and open (pAPN-RBD, PDB code 4F5C) dimeric APN structures (Table 1). The APN domains colored in orange (domain I, DI), yellow (domain II, DII), red (domain III, DIII) and green (domain IV, DIV). N-linked glycans omitted. (b) Interdomain movement between the open and closed APN conformations. The arrow indicates the swing movement of the domain I-II-III module toward domain IV after ectodomain closure in each monomer of the APN dimer, with domain IV fixed by dimerization. The hinge residues at the N terminus of α13 and α15 in domain IV are marked with black dots. See also Supplementary Video S1. (c) Surface representation of the open and closed APN. Front view of the active site, with the zinc ion in domain II as a cyan sphere.
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f1: The dynamic conformation of mammalian APN ectodomains.(a) Ribbon representation of the closed (pAPN), intermediate (hAPN) and open (pAPN-RBD, PDB code 4F5C) dimeric APN structures (Table 1). The APN domains colored in orange (domain I, DI), yellow (domain II, DII), red (domain III, DIII) and green (domain IV, DIV). N-linked glycans omitted. (b) Interdomain movement between the open and closed APN conformations. The arrow indicates the swing movement of the domain I-II-III module toward domain IV after ectodomain closure in each monomer of the APN dimer, with domain IV fixed by dimerization. The hinge residues at the N terminus of α13 and α15 in domain IV are marked with black dots. See also Supplementary Video S1. (c) Surface representation of the open and closed APN. Front view of the active site, with the zinc ion in domain II as a cyan sphere.

Mentions: In the past we reported a pAPN ectodomain crystal structure in complex with a CoV spike (S) fragment (PDB code 4F5C)16, here we show three new structures for APN (Table 1). In the four structures, the N-terminal HA tag and ~30 ectodomain residues were very disordered, indicating a large degree of flexibility in the membrane proximal polypeptide. The ectodomains have a hook-like conformation formed by domain I to IV and contained a zinc ion at the active site in domain II (Fig. 1). The exposed convex side of domain IV mediates similar protein dimerization in the distinct crystals. Approximately 950 Å2 of each monomer is buried at the dimer interface (Table 2), indicative of a stable protein-protein interaction. Domain IV is the largest APN domain and the most divergent in the M1 aminopeptidase family. In APN, domain IV has seven helix-turn-helix HEAT repeats and a single ARM repeat formed by three alpha helices (α25-α27). The ARM repeat is the most variable domain IV region in the hAPN and pAPN structures, and can contact the peptide substrate bound to the active site (see below).


Allosteric inhibition of aminopeptidase N functions related to tumor growth and virus infection
The dynamic conformation of mammalian APN ectodomains.(a) Ribbon representation of the closed (pAPN), intermediate (hAPN) and open (pAPN-RBD, PDB code 4F5C) dimeric APN structures (Table 1). The APN domains colored in orange (domain I, DI), yellow (domain II, DII), red (domain III, DIII) and green (domain IV, DIV). N-linked glycans omitted. (b) Interdomain movement between the open and closed APN conformations. The arrow indicates the swing movement of the domain I-II-III module toward domain IV after ectodomain closure in each monomer of the APN dimer, with domain IV fixed by dimerization. The hinge residues at the N terminus of α13 and α15 in domain IV are marked with black dots. See also Supplementary Video S1. (c) Surface representation of the open and closed APN. Front view of the active site, with the zinc ion in domain II as a cyan sphere.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5385526&req=5

f1: The dynamic conformation of mammalian APN ectodomains.(a) Ribbon representation of the closed (pAPN), intermediate (hAPN) and open (pAPN-RBD, PDB code 4F5C) dimeric APN structures (Table 1). The APN domains colored in orange (domain I, DI), yellow (domain II, DII), red (domain III, DIII) and green (domain IV, DIV). N-linked glycans omitted. (b) Interdomain movement between the open and closed APN conformations. The arrow indicates the swing movement of the domain I-II-III module toward domain IV after ectodomain closure in each monomer of the APN dimer, with domain IV fixed by dimerization. The hinge residues at the N terminus of α13 and α15 in domain IV are marked with black dots. See also Supplementary Video S1. (c) Surface representation of the open and closed APN. Front view of the active site, with the zinc ion in domain II as a cyan sphere.
Mentions: In the past we reported a pAPN ectodomain crystal structure in complex with a CoV spike (S) fragment (PDB code 4F5C)16, here we show three new structures for APN (Table 1). In the four structures, the N-terminal HA tag and ~30 ectodomain residues were very disordered, indicating a large degree of flexibility in the membrane proximal polypeptide. The ectodomains have a hook-like conformation formed by domain I to IV and contained a zinc ion at the active site in domain II (Fig. 1). The exposed convex side of domain IV mediates similar protein dimerization in the distinct crystals. Approximately 950 Å2 of each monomer is buried at the dimer interface (Table 2), indicative of a stable protein-protein interaction. Domain IV is the largest APN domain and the most divergent in the M1 aminopeptidase family. In APN, domain IV has seven helix-turn-helix HEAT repeats and a single ARM repeat formed by three alpha helices (α25-α27). The ARM repeat is the most variable domain IV region in the hAPN and pAPN structures, and can contact the peptide substrate bound to the active site (see below).

View Article: PubMed Central - PubMed

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.

No MeSH data available.