Abstract
X-prolyl aminopeptidases catalyze the removal of a penultimate prolyl residue from the N termini of peptides. Mammalian X-prolyl aminopeptidases are shown to be responsible for the degradation of bradykinin, a blood pressure regulator peptide, and have been linked to myocardial infarction. The x-ray crystal structure of human cytosolic X-prolyl aminopeptidase (XPN-PEP1) was solved at a resolution of 1.6 angstroms. The structure reveals a dimer with a unique three-domain organization in each subunit, rather than the two domains common to all other known structures of X-prolyl aminopeptidase and prolidases. The C-terminal catalytic domain of XPNPEP1 coordinates two metal ions and shares a similar fold with other prolyl aminopeptidases. Metal content analysis and activity assays confirm that the enzyme is double Mn(II) dependent for its activity, which contrasts with the previous notion that each XPNPEP1 subunit contains only one Mn(II) ion. Activity assays on an E41A mutant demonstrate that the acidic residue, which was considered as a stabilizing factor in the protonation of catalytic residue His498, plays only a marginal role in catalysis. Further mutagenesis reveals the significance of the N-terminal domain and dimerization for the activity of XPNPEP1, and we provide putative structural explanations for their functional roles. Structural comparisons further suggest mechanisms for substrate selectivity in different X-prolyl peptidases.
Highlights
X-prolyl aminopeptidases catalyze the removal of a penultimate prolyl residue from the N termini of peptides
Whereas other X-prolyl peptidases with known structures all contain two domains, the XPNPEP1 structure possesses a novel three-domain organization with a conserved C-terminal catalytic domain
In contrast to previous reports, we identified the presence of a double Mn2ϩ binding site in the catalytic domain, both in the crystal structure and in solution
Summary
Cloning and Expression—The cDNA of wild type (WT) XPNPEP1 and that of a domain I-truncated mutant (residues 162– 623) were cloned into the pET28a vector (Novagen) between SalI and HindIII sites. The released His6-tagged protein was purified following standard protocols of nickel-nitrilotriacetic acid resin (Qiagen) It was eluted from the resin with buffer B (20 mM Tris-HCl (pH 7.9), 500 mM NaCl, 10% (v/v) glycerol, and 300 mM imidazole) and dialyzed against a salt-free buffer (20 mM Tris-HCl (pH 8.0)). Purified protein samples without crystallization trial were extensively dialyzed against 100 mM Tris-HCl (pH 8.0) and 100 mM NaCl before ICP-MS analysis. The protein samples for AUC were prepared at a concentration of about 0.5 mg/ml in a buffer of 100 mM Tris-HCl (pH 8.0) and 100 mM NaCl. The AUC data were processed as a c(M) distribution model [17]
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