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Structure of human aspartyl aminopeptidase complexed with substrate analogue: insight into catalytic mechanism, substrate specificity and M18 peptidase family.

Chaikuad A, Pilka ES, De Riso A, von Delft F, Kavanagh KL, Vénien-Bryan C, Oppermann U, Yue WW - BMC Struct. Biol. (2012)

Bottom Line: The DNPEP structure provides a molecular framework to understand its catalysis that is mediated by active site loop swapping, a mechanism likely adopted in other M18 and M42 metallopeptidases that form dodecameric complexes as a self-compartmentalization strategy.Small differences in the substrate binding pocket such as shape and positive charges, the latter conferred by a basic lysine residue, further provide the key to distinguishing substrate preference.Together, the structural knowledge will aid in the development of enzyme-/family-specific aminopeptidase inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Structural Genomics Consortium, Old Road Research Campus Building, Oxford OX3 7DQ, UK.

ABSTRACT

Background: Aspartyl aminopeptidase (DNPEP), with specificity towards an acidic amino acid at the N-terminus, is the only mammalian member among the poorly understood M18 peptidases. DNPEP has implicated roles in protein and peptide metabolism, as well as the renin-angiotensin system in blood pressure regulation. Despite previous enzyme and substrate characterization, structural details of DNPEP regarding ligand recognition and catalytic mechanism remain to be delineated.

Results: The crystal structure of human DNPEP complexed with zinc and a substrate analogue aspartate-β-hydroxamate reveals a dodecameric machinery built by domain-swapped dimers, in agreement with electron microscopy data. A structural comparison with bacterial homologues identifies unifying catalytic features among the poorly understood M18 enzymes. The bound ligands in the active site also reveal the coordination mode of the binuclear zinc centre and a substrate specificity pocket for acidic amino acids.

Conclusions: The DNPEP structure provides a molecular framework to understand its catalysis that is mediated by active site loop swapping, a mechanism likely adopted in other M18 and M42 metallopeptidases that form dodecameric complexes as a self-compartmentalization strategy. Small differences in the substrate binding pocket such as shape and positive charges, the latter conferred by a basic lysine residue, further provide the key to distinguishing substrate preference. Together, the structural knowledge will aid in the development of enzyme-/family-specific aminopeptidase inhibitors.

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Active site of hDNPEP. (A) /FO/- /Fc/ omit map contoured at 3σ for zinc ions and ABH molecules. (B) Insertion of the β8-β9 loop from the neighboring subunit (magenta) completes the active site construction. Bonding interactions at (C) the binuclear metal catalytic centre and (D) the P1 substrate pocket in the hDNPEP structure. (E) Proposed catalytic mechanism for hDNPEP. The substrate peptide N-terminus is shown in both amine and its protonated form, which can engage in different interactions.
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Figure 4: Active site of hDNPEP. (A) /FO/- /Fc/ omit map contoured at 3σ for zinc ions and ABH molecules. (B) Insertion of the β8-β9 loop from the neighboring subunit (magenta) completes the active site construction. Bonding interactions at (C) the binuclear metal catalytic centre and (D) the P1 substrate pocket in the hDNPEP structure. (E) Proposed catalytic mechanism for hDNPEP. The substrate peptide N-terminus is shown in both amine and its protonated form, which can engage in different interactions.

Mentions: The active site is defined by the bound substrate analogue ABH and two zinc ions (Zn1 and Zn2) (Figure 4A and B) – the latter likely carried through protein expression and purification, and confirmed by fluorescence absorption profile of the crystals (data not shown). Zn1 and Zn2, bridged by Asp264, are 3.4 Å apart, consistent with the distances observed in other binuclear metalloproteases [13]. Zn1 is further coordinated by Glu302 and His440, and Zn2 by His94 and Asp346 (Figure 4C). These five metal coordinating residues (His94, Asp264, Glu302, Asp346 and His440) form a ‘H.D.E.D.H’ signature strictly conserved among DNPEP paralogues and M18 members (Figure 1B), providing an explanation for the abolished hDNPEP activity by mutations of His94 and His440 [17].


Structure of human aspartyl aminopeptidase complexed with substrate analogue: insight into catalytic mechanism, substrate specificity and M18 peptidase family.

Chaikuad A, Pilka ES, De Riso A, von Delft F, Kavanagh KL, Vénien-Bryan C, Oppermann U, Yue WW - BMC Struct. Biol. (2012)

Active site of hDNPEP. (A) /FO/- /Fc/ omit map contoured at 3σ for zinc ions and ABH molecules. (B) Insertion of the β8-β9 loop from the neighboring subunit (magenta) completes the active site construction. Bonding interactions at (C) the binuclear metal catalytic centre and (D) the P1 substrate pocket in the hDNPEP structure. (E) Proposed catalytic mechanism for hDNPEP. The substrate peptide N-terminus is shown in both amine and its protonated form, which can engage in different interactions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472314&req=5

Figure 4: Active site of hDNPEP. (A) /FO/- /Fc/ omit map contoured at 3σ for zinc ions and ABH molecules. (B) Insertion of the β8-β9 loop from the neighboring subunit (magenta) completes the active site construction. Bonding interactions at (C) the binuclear metal catalytic centre and (D) the P1 substrate pocket in the hDNPEP structure. (E) Proposed catalytic mechanism for hDNPEP. The substrate peptide N-terminus is shown in both amine and its protonated form, which can engage in different interactions.
Mentions: The active site is defined by the bound substrate analogue ABH and two zinc ions (Zn1 and Zn2) (Figure 4A and B) – the latter likely carried through protein expression and purification, and confirmed by fluorescence absorption profile of the crystals (data not shown). Zn1 and Zn2, bridged by Asp264, are 3.4 Å apart, consistent with the distances observed in other binuclear metalloproteases [13]. Zn1 is further coordinated by Glu302 and His440, and Zn2 by His94 and Asp346 (Figure 4C). These five metal coordinating residues (His94, Asp264, Glu302, Asp346 and His440) form a ‘H.D.E.D.H’ signature strictly conserved among DNPEP paralogues and M18 members (Figure 1B), providing an explanation for the abolished hDNPEP activity by mutations of His94 and His440 [17].

Bottom Line: The DNPEP structure provides a molecular framework to understand its catalysis that is mediated by active site loop swapping, a mechanism likely adopted in other M18 and M42 metallopeptidases that form dodecameric complexes as a self-compartmentalization strategy.Small differences in the substrate binding pocket such as shape and positive charges, the latter conferred by a basic lysine residue, further provide the key to distinguishing substrate preference.Together, the structural knowledge will aid in the development of enzyme-/family-specific aminopeptidase inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Structural Genomics Consortium, Old Road Research Campus Building, Oxford OX3 7DQ, UK.

ABSTRACT

Background: Aspartyl aminopeptidase (DNPEP), with specificity towards an acidic amino acid at the N-terminus, is the only mammalian member among the poorly understood M18 peptidases. DNPEP has implicated roles in protein and peptide metabolism, as well as the renin-angiotensin system in blood pressure regulation. Despite previous enzyme and substrate characterization, structural details of DNPEP regarding ligand recognition and catalytic mechanism remain to be delineated.

Results: The crystal structure of human DNPEP complexed with zinc and a substrate analogue aspartate-β-hydroxamate reveals a dodecameric machinery built by domain-swapped dimers, in agreement with electron microscopy data. A structural comparison with bacterial homologues identifies unifying catalytic features among the poorly understood M18 enzymes. The bound ligands in the active site also reveal the coordination mode of the binuclear zinc centre and a substrate specificity pocket for acidic amino acids.

Conclusions: The DNPEP structure provides a molecular framework to understand its catalysis that is mediated by active site loop swapping, a mechanism likely adopted in other M18 and M42 metallopeptidases that form dodecameric complexes as a self-compartmentalization strategy. Small differences in the substrate binding pocket such as shape and positive charges, the latter conferred by a basic lysine residue, further provide the key to distinguishing substrate preference. Together, the structural knowledge will aid in the development of enzyme-/family-specific aminopeptidase inhibitors.

Show MeSH