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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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Architecture of wide (top) and narrow (bottom) channels. (A) Electrostatic surface of the wide channel with yellow line indicating the 28-Å route connecting the exterior and the central chamber. (B) Details of residues lining the wide channel, showing only one set of residues from one dimer. (C) Electrostatic surface along the 33-Å length of the narrow channel. (D) Details of residues lining the narrow channel. Bound glycerol (GOL) and magnesium (Mg) molecules are shown in stick and sphere, respectively.
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Figure 3: Architecture of wide (top) and narrow (bottom) channels. (A) Electrostatic surface of the wide channel with yellow line indicating the 28-Å route connecting the exterior and the central chamber. (B) Details of residues lining the wide channel, showing only one set of residues from one dimer. (C) Electrostatic surface along the 33-Å length of the narrow channel. (D) Details of residues lining the narrow channel. Bound glycerol (GOL) and magnesium (Mg) molecules are shown in stick and sphere, respectively.

Mentions: We next performed an analysis of the wide and narrow channels in hDNPEP that represent the only access route between the twelve active sites in the central chamber and the exterior. Both channels in M18 hDNPEP are remarkably similar in topology to the M42 dodecameric tetrahedrons. The wide channels, each formed from three dimers (Additional file1, Figure S3), are 20 Å in width and 28 Å in length with a large concave surface at the entrance lined by positively-charged residues (Figures 3A and 3B). This wide channel, supported by the positive electrostatic environment that would complement the substrate acidic N-termini, likely functions as an entrance for unfolded peptide substrates. The transit function, as well as the electrostatic complementarity as a basis for substrate discrimination, has been proposed for M42 tetrahedron aminopeptidases [18,23,24]. Consistent with this theory, mutation of His363 (one of the residues lining the channel) to a non-polar residue has an adverse effect on the hDNPEP kinetic property [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)

Architecture of wide (top) and narrow (bottom) channels. (A) Electrostatic surface of the wide channel with yellow line indicating the 28-Å route connecting the exterior and the central chamber. (B) Details of residues lining the wide channel, showing only one set of residues from one dimer. (C) Electrostatic surface along the 33-Å length of the narrow channel. (D) Details of residues lining the narrow channel. Bound glycerol (GOL) and magnesium (Mg) molecules are shown in stick and sphere, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Architecture of wide (top) and narrow (bottom) channels. (A) Electrostatic surface of the wide channel with yellow line indicating the 28-Å route connecting the exterior and the central chamber. (B) Details of residues lining the wide channel, showing only one set of residues from one dimer. (C) Electrostatic surface along the 33-Å length of the narrow channel. (D) Details of residues lining the narrow channel. Bound glycerol (GOL) and magnesium (Mg) molecules are shown in stick and sphere, respectively.
Mentions: We next performed an analysis of the wide and narrow channels in hDNPEP that represent the only access route between the twelve active sites in the central chamber and the exterior. Both channels in M18 hDNPEP are remarkably similar in topology to the M42 dodecameric tetrahedrons. The wide channels, each formed from three dimers (Additional file1, Figure S3), are 20 Å in width and 28 Å in length with a large concave surface at the entrance lined by positively-charged residues (Figures 3A and 3B). This wide channel, supported by the positive electrostatic environment that would complement the substrate acidic N-termini, likely functions as an entrance for unfolded peptide substrates. The transit function, as well as the electrostatic complementarity as a basis for substrate discrimination, has been proposed for M42 tetrahedron aminopeptidases [18,23,24]. Consistent with this theory, mutation of His363 (one of the residues lining the channel) to a non-polar residue has an adverse effect on the hDNPEP kinetic property [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