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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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Dodecameric assembly. (A) A dimer building block of hDNPEP. (B) Surface representation of the arrangement of six dimers into a tetrahedron. The dimer building block, each coloured differently, is delineated by a red dotted line. The black-dotted line indicates the monomer-monomer interface within a dimer. Each dimer sits diagonally on six faces of a cubic box that encases the tetrahedron (red line, inset). Asterisks indicate positions of the narrow (yellow) and wide (blue) channels, which are located at 3-fold axes (arrow). (C) The openings of the narrow (top) and wide (bottom) channels. (D) Electron micrograph of negatively stained hDNPEP. (E) Examples of 2D classification images with a view down the wide channel, the 3-fold symmetry imposed in the right panel. (F) Fitting of hDNPEP crystal structure onto the 2D projection.
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Figure 2: Dodecameric assembly. (A) A dimer building block of hDNPEP. (B) Surface representation of the arrangement of six dimers into a tetrahedron. The dimer building block, each coloured differently, is delineated by a red dotted line. The black-dotted line indicates the monomer-monomer interface within a dimer. Each dimer sits diagonally on six faces of a cubic box that encases the tetrahedron (red line, inset). Asterisks indicate positions of the narrow (yellow) and wide (blue) channels, which are located at 3-fold axes (arrow). (C) The openings of the narrow (top) and wide (bottom) channels. (D) Electron micrograph of negatively stained hDNPEP. (E) Examples of 2D classification images with a view down the wide channel, the 3-fold symmetry imposed in the right panel. (F) Fitting of hDNPEP crystal structure onto the 2D projection.

Mentions: Application of the crystallographic 432 symmetry to the hDNPEP monomer results in a tetrahedron-shaped dodecamer built from six homodimers, a quaternary arrangement similar to M42 enzymes [15,18,19]. Each dimer, with internal two-fold symmetry on both vertical and horizontal axes (Figure 2A), is formed by extensive contacts that involve the swapping of loop β8-β9 between the two subunits (Additional file1, Figure S1). Mediated by four-fold symmetry, the six dimers assemble into a tetrahedron (Figure 2B), with each dimer constituting one edge (~118 Å) of the tetrahedron (Figure 2B, inset). The tetrahedron has a 50 Å-diameter internal cavity harbouring all twelve active sites, which is accessible to the exterior through four wide and four narrow channels situated on the three-fold axes. The entrances of the wide channels, a triangular pore of 28 Å per side, are located at the centre of the four tetrahedron facets (Figure 2B and C, blue asterisks), while the four narrow channels have their openings (9 Å per side) on the four tetrahedron vertexes (Figure 2B and C, yellow asterisks).


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)

Dodecameric assembly. (A) A dimer building block of hDNPEP. (B) Surface representation of the arrangement of six dimers into a tetrahedron. The dimer building block, each coloured differently, is delineated by a red dotted line. The black-dotted line indicates the monomer-monomer interface within a dimer. Each dimer sits diagonally on six faces of a cubic box that encases the tetrahedron (red line, inset). Asterisks indicate positions of the narrow (yellow) and wide (blue) channels, which are located at 3-fold axes (arrow). (C) The openings of the narrow (top) and wide (bottom) channels. (D) Electron micrograph of negatively stained hDNPEP. (E) Examples of 2D classification images with a view down the wide channel, the 3-fold symmetry imposed in the right panel. (F) Fitting of hDNPEP crystal structure onto the 2D projection.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Dodecameric assembly. (A) A dimer building block of hDNPEP. (B) Surface representation of the arrangement of six dimers into a tetrahedron. The dimer building block, each coloured differently, is delineated by a red dotted line. The black-dotted line indicates the monomer-monomer interface within a dimer. Each dimer sits diagonally on six faces of a cubic box that encases the tetrahedron (red line, inset). Asterisks indicate positions of the narrow (yellow) and wide (blue) channels, which are located at 3-fold axes (arrow). (C) The openings of the narrow (top) and wide (bottom) channels. (D) Electron micrograph of negatively stained hDNPEP. (E) Examples of 2D classification images with a view down the wide channel, the 3-fold symmetry imposed in the right panel. (F) Fitting of hDNPEP crystal structure onto the 2D projection.
Mentions: Application of the crystallographic 432 symmetry to the hDNPEP monomer results in a tetrahedron-shaped dodecamer built from six homodimers, a quaternary arrangement similar to M42 enzymes [15,18,19]. Each dimer, with internal two-fold symmetry on both vertical and horizontal axes (Figure 2A), is formed by extensive contacts that involve the swapping of loop β8-β9 between the two subunits (Additional file1, Figure S1). Mediated by four-fold symmetry, the six dimers assemble into a tetrahedron (Figure 2B), with each dimer constituting one edge (~118 Å) of the tetrahedron (Figure 2B, inset). The tetrahedron has a 50 Å-diameter internal cavity harbouring all twelve active sites, which is accessible to the exterior through four wide and four narrow channels situated on the three-fold axes. The entrances of the wide channels, a triangular pore of 28 Å per side, are located at the centre of the four tetrahedron facets (Figure 2B and C, blue asterisks), while the four narrow channels have their openings (9 Å per side) on the four tetrahedron vertexes (Figure 2B and C, yellow asterisks).

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
Related in: MedlinePlus