Limits...
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.

Show MeSH

Related in: MedlinePlus

Overview of hDNPEP structure. (A) hDNPEP protomer organizes into the dimerization (blue) and proteolytic domains, the latter is further comprised of subdomain A (orange) and subdomain B (magenta). Zinc ions are shown as blue spheres and the ABH ligand as yellow sticks. (B) Sequence alignment of DNPEP (human, h; bovine, b) and M18 aminopeptidases from yeast Saccharomyces cerevisiae Lap4 (ScLAP4), yeast Ape4 (ScApe4) and two bacterial enzymes (Pseudomonas aeruginosa PaApeB and Thermotoga maritima TmApeA). Secondary structure elements, catalytic residues (yellow) and residues in the P1 substrate pocket (cyan) of hDNPEP are highlighted. Structural superimposition of hDNPEP with bacterial M18 APs (C) and with M20, M28 and M42 representatives from the MH clan (D) reveals highly conserved topology of the proteolytic domain. The superimposed structures include M18 APs: Thermotoga maritima ApeA (TmApeA), Clostridium acetobutylicum ApeA (CaApeA), Borrelia burgdorferi ApeA (BbApeA), Pseudomonas aeruginosa ApeB (PaApeB); M20 APs: Pseudomonas CPG2 (PsCPG2), Lactobacillus delbrueckii PepV (LdPepV), Legionella pneumophila DapE (LpDapE); M28; Aeromonas proteolytica LAP (ApLAP), Streptomyces griseus Ap (SgApS); and M42: Pyrococcus horikoshii TET1 and TET2 (PhTET1, PhTET2), Streptococcus pneumonia PepA (SpPepA). PDB IDs of all structures are given.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3472314&req=5

Figure 1: Overview of hDNPEP structure. (A) hDNPEP protomer organizes into the dimerization (blue) and proteolytic domains, the latter is further comprised of subdomain A (orange) and subdomain B (magenta). Zinc ions are shown as blue spheres and the ABH ligand as yellow sticks. (B) Sequence alignment of DNPEP (human, h; bovine, b) and M18 aminopeptidases from yeast Saccharomyces cerevisiae Lap4 (ScLAP4), yeast Ape4 (ScApe4) and two bacterial enzymes (Pseudomonas aeruginosa PaApeB and Thermotoga maritima TmApeA). Secondary structure elements, catalytic residues (yellow) and residues in the P1 substrate pocket (cyan) of hDNPEP are highlighted. Structural superimposition of hDNPEP with bacterial M18 APs (C) and with M20, M28 and M42 representatives from the MH clan (D) reveals highly conserved topology of the proteolytic domain. The superimposed structures include M18 APs: Thermotoga maritima ApeA (TmApeA), Clostridium acetobutylicum ApeA (CaApeA), Borrelia burgdorferi ApeA (BbApeA), Pseudomonas aeruginosa ApeB (PaApeB); M20 APs: Pseudomonas CPG2 (PsCPG2), Lactobacillus delbrueckii PepV (LdPepV), Legionella pneumophila DapE (LpDapE); M28; Aeromonas proteolytica LAP (ApLAP), Streptomyces griseus Ap (SgApS); and M42: Pyrococcus horikoshii TET1 and TET2 (PhTET1, PhTET2), Streptococcus pneumonia PepA (SpPepA). PDB IDs of all structures are given.

Mentions: The structure of the hDNPEP·Zn2+·ABH complex (Figure 1A), determined at 2.2 Å resolution, is homologous to four unpublished bacterial M18 homologues with undefined enzyme and substrate properties (DALI Z-scores ~40, rmsd 1.9-2.7 Å and sequence identity 23-35%). Superposition of the structures reveals a common two-domain architecture consisting of the proteolytic and dimerization domains (Figure 1A and C), with the active site located in a concave groove at the domain interface. The globular proteolytic domain (aa 7–98 and 249–468 in hDNPEP) features a core nine-stranded β-sheet sandwiched between several α-helices and has a small five-stranded β-subdomain resting on top (Figure 1A). This proteolytic domain is highly similar among all M18 structures (rmsd ~1.5 Å). The dimerization domain, contributed from the central polypeptide stretch (aa 99–248 in hDNPEP), sits on top of the proteolytic domain (Figure 1A). This butterfly-shaped domain is built of two orthogonal β-sheets (five- and three-stranded respectively) that share in common two tilted strands β5 and β6, and also includes an extended β8-β9 loop that is important for active site formation (see next sections). Variations in the dimerization domain are observed among M18 enzymes, particularly with the location and spatial orientation of helices α3 and α4 and the connecting loop α3-α4. In hDNPEP loop α3-α4 is longer than the bacterial equivalents (Figure 1B), although it is partially disordered in our structure.


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)

Overview of hDNPEP structure. (A) hDNPEP protomer organizes into the dimerization (blue) and proteolytic domains, the latter is further comprised of subdomain A (orange) and subdomain B (magenta). Zinc ions are shown as blue spheres and the ABH ligand as yellow sticks. (B) Sequence alignment of DNPEP (human, h; bovine, b) and M18 aminopeptidases from yeast Saccharomyces cerevisiae Lap4 (ScLAP4), yeast Ape4 (ScApe4) and two bacterial enzymes (Pseudomonas aeruginosa PaApeB and Thermotoga maritima TmApeA). Secondary structure elements, catalytic residues (yellow) and residues in the P1 substrate pocket (cyan) of hDNPEP are highlighted. Structural superimposition of hDNPEP with bacterial M18 APs (C) and with M20, M28 and M42 representatives from the MH clan (D) reveals highly conserved topology of the proteolytic domain. The superimposed structures include M18 APs: Thermotoga maritima ApeA (TmApeA), Clostridium acetobutylicum ApeA (CaApeA), Borrelia burgdorferi ApeA (BbApeA), Pseudomonas aeruginosa ApeB (PaApeB); M20 APs: Pseudomonas CPG2 (PsCPG2), Lactobacillus delbrueckii PepV (LdPepV), Legionella pneumophila DapE (LpDapE); M28; Aeromonas proteolytica LAP (ApLAP), Streptomyces griseus Ap (SgApS); and M42: Pyrococcus horikoshii TET1 and TET2 (PhTET1, PhTET2), Streptococcus pneumonia PepA (SpPepA). PDB IDs of all structures are given.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Overview of hDNPEP structure. (A) hDNPEP protomer organizes into the dimerization (blue) and proteolytic domains, the latter is further comprised of subdomain A (orange) and subdomain B (magenta). Zinc ions are shown as blue spheres and the ABH ligand as yellow sticks. (B) Sequence alignment of DNPEP (human, h; bovine, b) and M18 aminopeptidases from yeast Saccharomyces cerevisiae Lap4 (ScLAP4), yeast Ape4 (ScApe4) and two bacterial enzymes (Pseudomonas aeruginosa PaApeB and Thermotoga maritima TmApeA). Secondary structure elements, catalytic residues (yellow) and residues in the P1 substrate pocket (cyan) of hDNPEP are highlighted. Structural superimposition of hDNPEP with bacterial M18 APs (C) and with M20, M28 and M42 representatives from the MH clan (D) reveals highly conserved topology of the proteolytic domain. The superimposed structures include M18 APs: Thermotoga maritima ApeA (TmApeA), Clostridium acetobutylicum ApeA (CaApeA), Borrelia burgdorferi ApeA (BbApeA), Pseudomonas aeruginosa ApeB (PaApeB); M20 APs: Pseudomonas CPG2 (PsCPG2), Lactobacillus delbrueckii PepV (LdPepV), Legionella pneumophila DapE (LpDapE); M28; Aeromonas proteolytica LAP (ApLAP), Streptomyces griseus Ap (SgApS); and M42: Pyrococcus horikoshii TET1 and TET2 (PhTET1, PhTET2), Streptococcus pneumonia PepA (SpPepA). PDB IDs of all structures are given.
Mentions: The structure of the hDNPEP·Zn2+·ABH complex (Figure 1A), determined at 2.2 Å resolution, is homologous to four unpublished bacterial M18 homologues with undefined enzyme and substrate properties (DALI Z-scores ~40, rmsd 1.9-2.7 Å and sequence identity 23-35%). Superposition of the structures reveals a common two-domain architecture consisting of the proteolytic and dimerization domains (Figure 1A and C), with the active site located in a concave groove at the domain interface. The globular proteolytic domain (aa 7–98 and 249–468 in hDNPEP) features a core nine-stranded β-sheet sandwiched between several α-helices and has a small five-stranded β-subdomain resting on top (Figure 1A). This proteolytic domain is highly similar among all M18 structures (rmsd ~1.5 Å). The dimerization domain, contributed from the central polypeptide stretch (aa 99–248 in hDNPEP), sits on top of the proteolytic domain (Figure 1A). This butterfly-shaped domain is built of two orthogonal β-sheets (five- and three-stranded respectively) that share in common two tilted strands β5 and β6, and also includes an extended β8-β9 loop that is important for active site formation (see next sections). Variations in the dimerization domain are observed among M18 enzymes, particularly with the location and spatial orientation of helices α3 and α4 and the connecting loop α3-α4. In hDNPEP loop α3-α4 is longer than the bacterial equivalents (Figure 1B), although it is partially disordered in our structure.

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