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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.


APN dynamics in catalysis and CoV recognition.(a) The active site of the closed, intermediate and open APN during peptide hydrolysis. The active site at domain II (DII, yellow) contains a modeled poly-alanine peptide coordinated to the zinc ion (cyan sphere). Side chains of APN active site residues are shown with sticks, whereas the poly-alanine is shown as a gray surface with residues as sticks (carbons in grey). N-terminal peptide residues (P1-P1′-P2′) are labeled. Nitrogens, blue; oxygens, red; hydrogen bonds are dashed lines. The helices of the ARM repeat (α25-α27) in domain IV (DIV, green) with the phenylalanine residue that contacts the peptide in the closed conformation (Phe893 in pAPN) are labeled. The crystal structure of the poly-alanine bound to the pAPN (PDB code 4HOM) was used to model it in the active site of closed, intermediate and open structures by structural superposition based on domain II. (b) Conformation of the CoV binding cavity at the domain II-IV interface in the closed and open pAPN structures. Structures were superposed based on domain IV. Ribbon diagrams of the open pAPN in complex with the porcine CoV RBD (Supplementary Fig. S1b), with residues that contact the RBD in sticks with carbons in yellow (domain II) and green (domain IV). The same residues are shown for the superposed closed structure (carbons in grey). The RBD motif that penetrates the pAPN cavity is shown with a grey surface and with residues as sticks (carbons in cyan or in magenta for Trp).
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f2: APN dynamics in catalysis and CoV recognition.(a) The active site of the closed, intermediate and open APN during peptide hydrolysis. The active site at domain II (DII, yellow) contains a modeled poly-alanine peptide coordinated to the zinc ion (cyan sphere). Side chains of APN active site residues are shown with sticks, whereas the poly-alanine is shown as a gray surface with residues as sticks (carbons in grey). N-terminal peptide residues (P1-P1′-P2′) are labeled. Nitrogens, blue; oxygens, red; hydrogen bonds are dashed lines. The helices of the ARM repeat (α25-α27) in domain IV (DIV, green) with the phenylalanine residue that contacts the peptide in the closed conformation (Phe893 in pAPN) are labeled. The crystal structure of the poly-alanine bound to the pAPN (PDB code 4HOM) was used to model it in the active site of closed, intermediate and open structures by structural superposition based on domain II. (b) Conformation of the CoV binding cavity at the domain II-IV interface in the closed and open pAPN structures. Structures were superposed based on domain IV. Ribbon diagrams of the open pAPN in complex with the porcine CoV RBD (Supplementary Fig. S1b), with residues that contact the RBD in sticks with carbons in yellow (domain II) and green (domain IV). The same residues are shown for the superposed closed structure (carbons in grey). The RBD motif that penetrates the pAPN cavity is shown with a grey surface and with residues as sticks (carbons in cyan or in magenta for Trp).

Mentions: Crystal structures are reported for mammalian APN in complex with substrates, in both intermediate (pAPN) and closed conformations (hAPN)1921. To determine how domain movement contributes to peptide processing, we modeled a pAPN-bound poly-Ala peptide in the active site of closed, intermediate and open APN (Fig. 2a). In the closed pAPN, the side chain of a phenylalanine (Phe893) at domain IV was placed at about 4.5 Å from the hydrolyzable peptide bond, whose carbonyl group is coordinated to the zinc ion. The phenylalanine was located in the loop that connects α26 and α27 in the single domain IV ARM repeat of human and pig APN (Fig. 2a); it penetrated the active site groove in the closed conformation and locked the peptide, ready for hydrolysis. Domain IV residues that precede Phe893 in the α26-α27 loop contacted domain II in the closed pAPN. A similar loop conformation is seen in a closed hAPN structure (PDB code 4FYS)19.


Allosteric inhibition of aminopeptidase N functions related to tumor growth and virus infection
APN dynamics in catalysis and CoV recognition.(a) The active site of the closed, intermediate and open APN during peptide hydrolysis. The active site at domain II (DII, yellow) contains a modeled poly-alanine peptide coordinated to the zinc ion (cyan sphere). Side chains of APN active site residues are shown with sticks, whereas the poly-alanine is shown as a gray surface with residues as sticks (carbons in grey). N-terminal peptide residues (P1-P1′-P2′) are labeled. Nitrogens, blue; oxygens, red; hydrogen bonds are dashed lines. The helices of the ARM repeat (α25-α27) in domain IV (DIV, green) with the phenylalanine residue that contacts the peptide in the closed conformation (Phe893 in pAPN) are labeled. The crystal structure of the poly-alanine bound to the pAPN (PDB code 4HOM) was used to model it in the active site of closed, intermediate and open structures by structural superposition based on domain II. (b) Conformation of the CoV binding cavity at the domain II-IV interface in the closed and open pAPN structures. Structures were superposed based on domain IV. Ribbon diagrams of the open pAPN in complex with the porcine CoV RBD (Supplementary Fig. S1b), with residues that contact the RBD in sticks with carbons in yellow (domain II) and green (domain IV). The same residues are shown for the superposed closed structure (carbons in grey). The RBD motif that penetrates the pAPN cavity is shown with a grey surface and with residues as sticks (carbons in cyan or in magenta for Trp).
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Related In: Results  -  Collection

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f2: APN dynamics in catalysis and CoV recognition.(a) The active site of the closed, intermediate and open APN during peptide hydrolysis. The active site at domain II (DII, yellow) contains a modeled poly-alanine peptide coordinated to the zinc ion (cyan sphere). Side chains of APN active site residues are shown with sticks, whereas the poly-alanine is shown as a gray surface with residues as sticks (carbons in grey). N-terminal peptide residues (P1-P1′-P2′) are labeled. Nitrogens, blue; oxygens, red; hydrogen bonds are dashed lines. The helices of the ARM repeat (α25-α27) in domain IV (DIV, green) with the phenylalanine residue that contacts the peptide in the closed conformation (Phe893 in pAPN) are labeled. The crystal structure of the poly-alanine bound to the pAPN (PDB code 4HOM) was used to model it in the active site of closed, intermediate and open structures by structural superposition based on domain II. (b) Conformation of the CoV binding cavity at the domain II-IV interface in the closed and open pAPN structures. Structures were superposed based on domain IV. Ribbon diagrams of the open pAPN in complex with the porcine CoV RBD (Supplementary Fig. S1b), with residues that contact the RBD in sticks with carbons in yellow (domain II) and green (domain IV). The same residues are shown for the superposed closed structure (carbons in grey). The RBD motif that penetrates the pAPN cavity is shown with a grey surface and with residues as sticks (carbons in cyan or in magenta for Trp).
Mentions: Crystal structures are reported for mammalian APN in complex with substrates, in both intermediate (pAPN) and closed conformations (hAPN)1921. To determine how domain movement contributes to peptide processing, we modeled a pAPN-bound poly-Ala peptide in the active site of closed, intermediate and open APN (Fig. 2a). In the closed pAPN, the side chain of a phenylalanine (Phe893) at domain IV was placed at about 4.5 Å from the hydrolyzable peptide bond, whose carbonyl group is coordinated to the zinc ion. The phenylalanine was located in the loop that connects α26 and α27 in the single domain IV ARM repeat of human and pig APN (Fig. 2a); it penetrated the active site groove in the closed conformation and locked the peptide, ready for hydrolysis. Domain IV residues that precede Phe893 in the α26-α27 loop contacted domain II in the closed pAPN. A similar loop conformation is seen in a closed hAPN structure (PDB code 4FYS)19.

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.