Structural definition and substrate specificity of the S28 protease family: the crystal structure of human prolylcarboxypeptidase

Abstract

Background: The unique S28 family of proteases is comprised of the carboxypeptidase PRCP and the aminopeptidase DPP7. The structural basis of the different substrate specificities of the two enzymes is not understood nor has the structure of the S28 fold been described.

Results: The experimentally phased 2.8 A crystal structure is presented for human PRCP. PRCP contains an alpha/beta hydrolase domain harboring the catalytic Asp-His-Ser triad and a novel helical structural domain that caps the active site. Structural comparisons with prolylendopeptidase and DPP4 identify the S1 proline binding site of PRCP. A structure-based alignment with the previously undescribed structure of DPP7 illuminates the mechanism of orthogonal substrate specificity of PRCP and DPP7. PRCP has an extended active-site cleft that can accommodate proline substrates with multiple N-terminal residues. In contrast, the substrate binding groove of DPP7 is occluded by a short amino-acid insertion unique to DPP7 that creates a truncated active site selective for dipeptidyl proteolysis of N-terminal substrates.

Conclusion: The results define the structure of the S28 family of proteases, provide the structural basis of PRCP and DPP7 substrate specificity and enable the rational design of selective PRCP modulators.

Figure 1

The structure of PRCP. (A) Schematic diagram of the secondary structure of PRCP. The canonical α/β hydrolase fold is shown in blue and cyan, the SKS domain in magenta, and the M and N helices of the hydrolase insert domain in yellow. (B) Ribbon diagram of tertiary structure of PRCP. The Asp-His-Ser catalytic triad is shown in red. Glycans are shown in green.

Figure 2

The PRCP Dimer. The crystallographic dimer of PRCP with monomer subunits shown in green and blue.

Figure 3

The active site of PRCP. (A) The active site of PRCP. The Asp-His-Ser catalytic triad is shown in salmon. Additional active site side chain features discussed in the text are colored according to structural element as in Figure 1. (B) Structural alignment of PRCP active site (blue), DPP4 (orange), and human pancreatic lipase (yellow) aligned on the catalytic histidine. PRCP residues are labeled.

Figure 4

The hydrophobic substrate-binding pocket of PRCP. The putative proline-binding pocket is formed by Trp 432 and Trp 359 based on structural identity with other prolyl peptidases and the spatial alignment with the Ser 179-His 455-Asp 430 catalytic triad.

Figure 5

Identification of the proline recognition site. (A) Structural alignment of PRCP (blue) with porcine PEP (yellow) in complex with the peptide substrate EFSP (PDB code: 1UOQ) in light blue. Residues discussed in the text are shown as sticks and labeled. (B) Structural alignment of PRCP (blue) with human DPP4 (orange) in complex with the peptide substrate YPSK (PDB code: 1R9N) in pink.

Figure 6

Structure-based sequence alignment of DPP7 and PRCP. Active site residues are marked in bold, underlined type. The DPP7-specific structural insertion is in red. The four disordered residues not modeled in the PRCP structure are italicized.

Figure 7

Structural comparison of DPP7 and PRCP. (A) Structural alignment of PRCP (blue) and DPP7 (yellow) and the sequence insertion of DPP7 that occludes the active-site cleft (red). (B) Enlarged view of site region with DPP7-specific insertion (red). Asp334 is shown as sticks.

Figure 8

The DPP7 substrate-binding groove is occluded by the DPP7-specific structural insertion. Views of PRCP (top) and DPP7 (bottom) showing the elongated, solvent-accessible substrate binding groove of PRCP (teal), and the binding site of DPP7 (teal) that is truncated by the DPP7-specific insert (red).