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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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Structural comparison of the P1 pockets in hDNPEP and bacterial M18 members. (A-C) Three bacterial M18 AP structures (PDB ids in brackets) are superimposed onto hDNPEP, with particular focus on the P1 substrate pocket. This highlights variations not only in shape but also residue compositions for the P1 pocket, and may be correlated with different substrate specificities and enzyme activities among M18 enzymes. (D) A structure-based sequence alignment shows no conservation of four key residues of the hDNPEP P1 pocket among the bacterial M18 enzymes, in particular Lys374 likely to be a determinant for acidic amino acid preference.
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Figure 5: Structural comparison of the P1 pockets in hDNPEP and bacterial M18 members. (A-C) Three bacterial M18 AP structures (PDB ids in brackets) are superimposed onto hDNPEP, with particular focus on the P1 substrate pocket. This highlights variations not only in shape but also residue compositions for the P1 pocket, and may be correlated with different substrate specificities and enzyme activities among M18 enzymes. (D) A structure-based sequence alignment shows no conservation of four key residues of the hDNPEP P1 pocket among the bacterial M18 enzymes, in particular Lys374 likely to be a determinant for acidic amino acid preference.

Mentions: The modelled Asp and Glu sidechains can engage in slightly different interactions with hDNPEP (Additional file1, Figure S5). While the Asp carboxylate feasibly interacts with Lys374 and forms water-mediated hydrogen bonds with His349, the longer Glu sidechain can further penetrate this cavity and interact directly with Lys374, His349 and the nearby Tyr381. Our substrate models suggest that the strict preference for an acidic amino acid at the P1 position is conferred by positively-charged and polar residues, such as Lys374 and His349. The use of electrostatic complementarity for substrate selectivity has precedence in the M42 peptidase SpPepA [20]. Consistent with this strategy, mutation of His349 in hDNPEP has been shown to weaken substrate binding affinity [17]. Furthermore, the conservation of Lys374 only in M18 members with acidic aminopeptidase activity (e.g. yeast Ape4), but not in M18 ‘promiscuous’ peptidases (e.g. yeast Lap4, where the equivalent is Ser) (Figure 1B), provides a structure-based criteria to classify putative M18 sequences into potential aspartyl aminopeptidases (Lys374 conserved) or non-aspartyl aminopeptidases (Lys374 not conserved), facilitating subsequent enzymatic characterization. Using this criteria we propose that the structurally characterized bacterial M18 members, where the equivalent Lys374 positions are substituted (Figure 5), are unlikely to be aspartyl aminopeptidases.


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)

Structural comparison of the P1 pockets in hDNPEP and bacterial M18 members. (A-C) Three bacterial M18 AP structures (PDB ids in brackets) are superimposed onto hDNPEP, with particular focus on the P1 substrate pocket. This highlights variations not only in shape but also residue compositions for the P1 pocket, and may be correlated with different substrate specificities and enzyme activities among M18 enzymes. (D) A structure-based sequence alignment shows no conservation of four key residues of the hDNPEP P1 pocket among the bacterial M18 enzymes, in particular Lys374 likely to be a determinant for acidic amino acid preference.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 5: Structural comparison of the P1 pockets in hDNPEP and bacterial M18 members. (A-C) Three bacterial M18 AP structures (PDB ids in brackets) are superimposed onto hDNPEP, with particular focus on the P1 substrate pocket. This highlights variations not only in shape but also residue compositions for the P1 pocket, and may be correlated with different substrate specificities and enzyme activities among M18 enzymes. (D) A structure-based sequence alignment shows no conservation of four key residues of the hDNPEP P1 pocket among the bacterial M18 enzymes, in particular Lys374 likely to be a determinant for acidic amino acid preference.
Mentions: The modelled Asp and Glu sidechains can engage in slightly different interactions with hDNPEP (Additional file1, Figure S5). While the Asp carboxylate feasibly interacts with Lys374 and forms water-mediated hydrogen bonds with His349, the longer Glu sidechain can further penetrate this cavity and interact directly with Lys374, His349 and the nearby Tyr381. Our substrate models suggest that the strict preference for an acidic amino acid at the P1 position is conferred by positively-charged and polar residues, such as Lys374 and His349. The use of electrostatic complementarity for substrate selectivity has precedence in the M42 peptidase SpPepA [20]. Consistent with this strategy, mutation of His349 in hDNPEP has been shown to weaken substrate binding affinity [17]. Furthermore, the conservation of Lys374 only in M18 members with acidic aminopeptidase activity (e.g. yeast Ape4), but not in M18 ‘promiscuous’ peptidases (e.g. yeast Lap4, where the equivalent is Ser) (Figure 1B), provides a structure-based criteria to classify putative M18 sequences into potential aspartyl aminopeptidases (Lys374 conserved) or non-aspartyl aminopeptidases (Lys374 not conserved), facilitating subsequent enzymatic characterization. Using this criteria we propose that the structurally characterized bacterial M18 members, where the equivalent Lys374 positions are substituted (Figure 5), are unlikely to be aspartyl aminopeptidases.

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