Structural definition and substrate specificity of the S28 protease family: the crystal structure of human prolylcarboxypeptidase
© Soisson et al; licensee BioMed Central Ltd. 2010
Received: 29 April 2010
Accepted: 11 June 2010
Published: 11 June 2010
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.
The experimentally phased 2.8 Å crystal structure is presented for human PRCP. PRCP contains an α/β 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.
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.
Proteases are an important class of enzymes involved in a diverse range of physiological processes. The modulation of proteolytic activity is an established means of therapeutic intervention with currently marketed products for afflictions as diverse as type 2 diabetes, hypertension and viral infections. The human protease tree is comprised of at least 676 diverse proteins that have been systematically organized into clans and families based on similarity in sequence, structure, and function . Although the structural basis of catalytic mechanism, substrate specificity and rational drug design has been identified for numerous protease families, there has been no structural description of the S28 family of proteases that form a distinct branch of the serine carboxypeptidase clan.
The S28 family of peptidases consists of two enzymes, PRCP and DPP7. DPP7 is also called dipeptidyl peptidase 2 and quiescent cell proline dipeptidase [2–4]. PRCP is a lysosomal, serine carboxypeptidase that cleaves hydrophobic C-terminal amino acids adjacent to proline [5, 6]. In contrast, DPP7 is a serine dipeptidyl aminopeptidase that cleaves N-terminal amino acids adjacent to proline and is localized to intracellular vesicles .
Human PRCP and human DPP7 share 39.6% sequence identity and 55.4% sequence similarity. At the sequence level, the two enzymes are unrelated to other proteases; the next closest human homologues are PEP (8.4% sequence identity and 13.9% sequence similarity) and DPP4 (6.5% sequence identity and 11.2% sequence similarity). The S28 proteases PRCP and DPP7 are therefore unique within the protease superfamily.
PRCP was originally discovered as an angiotensinase  and has since been implicated in vasodilatory, proinflammatory, and metabolic pathways [6, 8, 9]. For example, angiotensin II, III and prekallikrein are all inactivated by PRCP, implicating a role for the enzyme in hypertension, tissue proliferation and smooth muscle growth. PRCP is also reported to inactivate α-melanocyte-stimulating hormone, a neuropeptide that plays a role in regulating appetite . DPP7 has been implicated in apoptosis in quiescent lymphocytes .
Here we report the crystal structure of human PRCP. The enzyme consists of an α/β hydrolase domain that contains a unique structural domain insertion that caps the active site. Comparison with the recently released coordinates of DPP7 illuminates the structural basis for the different substrate specificities of PRCP and DPP7. The results lay the foundation for understanding the structural basis of PRCP activity and for the structure-guided discovery of PRCP modulators for target validation and disease modification.
Results and Discussion
The structure of PRCP
Structure determination statistics.
Unit-cell dimensions (Å)
a = b = 181.14
c = 240.13
a = b = 179.76
c = 240.90
a = b = 181.27
c = 240.02
R merge (%)
Heavy atom sites
Isomorphous phasing power
Anomalous phasing power
Isomorphous R cullis
Anomalous R cullis
R f (%)
R free (%)
The experimental maps allowed nearly the entire structure of PRCP to be modeled and subsequently refined. The final refined model consists of residues 46-348 and 353-491, five N-linked glycans, and four disulfide bridges (residues 215-372, 233-310, 264-343 and 364-394). The final R and Rfree values are 21.8% and 24.1%, respectively. Geometry and stereochemistry are good with 95% of the residues in the most favored region of the Ramachandran plot and an overall MolProbity score of 88%. One region of unexplained tubular electron density is observed in the S1' active site area that may correspond to a structurally heterogeneous population of bound polymer (e.g., polyethylene glycol) and a second peak of unexplained electron density is observed near the putative proline S1 binding site.
The unique insertion in the PRCP hydrolase domain occurs between strand 6 and helix D and spans residues 194-398. The first part of the insertion (residues 194-334) consists of five helices packed into a novel helical bundle (the SKS domain) that caps the active site (Figure 1B). A DALI search to identify structures containing similar helical bundles to the SKS domain did not identify proteins with similar folds (Z scores < 3.3), suggesting that the SKS domain is a novel structural motif.
Four residues following the SKS domain are likely disordered as evidenced by a lack of electron density (residues 349-352). The region is followed by a pair of helices (M and N) that are linked by two long, irregular, loosely packed strands that form a concave surface at one entrance to the active site. The irregular strands and the M and N helices appear to provide additional stabilizing interactions between the SKS and hydrolase domains and form part of the substrate binding surface (Figure 1B).
Previously reported mass-spectrometry results are consistent with the CHO-expressed PRCP protein containing about 9 kDa of glycan . Sequence analysis suggests that there are six possible N-glycosylation sites at asparagines 47, 101, 317, 336, 345 and 415 that correspond to the canonical glycosylation sequence Asn-Xaa-Ser/Thr . Asn 47 is not glycosylated in the structure, in accord with mass-spectrometric mapping of glycan sites . Clear evidence of covalently attached and ordered saccharide is observed at the other five canonical glycosylation sites (Figure 1B). The presence of extensive glycosylation is likely a contributing factor to the high solvent content of the crystals.
The active site of PRCP
An unanticipated feature of the PRCP active site is an apparent charge-relay system that links the catalytic histidine (His 455) with His 456 and Arg 460 (Figure 3A). The arrangement of side chains places the imidazole nitrogen atoms of His 455 and His 456 within 3.0-3.5 Å of the catalytic serine. The guanidinium group of Arg 460 is in hydrogen bond distance (2.8 Å) of the imidazole ring of His 456. It seems likely that this unique arrangement of residues plays a role in the catalytic mechanism of PRCP. Furthermore, it is possible that the presence of the formally charged Arg 460 in close contact with the tandem histidines could alter the pKa of His 455 contributing to the acidic pH optimum (5.5) for both PRCP and DPP7 [6, 20, 21].
The tandem His-His arrangement is not seen in other serine α/β hydrolases with the exception of the lipases. For example, pancreatic lipase , contains a second histidine residue located spatially adjacent to the catalytic histidine in the active site (Figure 3B). In the lipases, the equivalent second His residue is contributed to the active site by a different structural element of the α/β hydrolase fold, and may therefore represent a convergent evolution of the S28 protease family and the lipases. The structural conservation underscores the potential importance of the histidine pair in catalysis.
Recognition of Pro-X peptide substrates by PRCP
PRCP cleaves carboxy-terminal residues of peptide substrates that contain a penultimate proline. This is exemplified by angiotensin II, the first substrate identified for PRCP (NDRVYIHP F)  and bradykinin (RPPGFSP F) . In contrast, peptides lacking the penultimate Pro, such as angiotensin I (DRVYIHP FHL), are not substrates for the enzyme .
Substrate specificity of the S28 protease family
The DPP7-specific insertion may also play an important role in substrate binding. For the aminopeptidase DPP4, substrate recognition involves coordination of the N-terminal amine of the substrate by Glu 206 of DPP4 (Figure 5B) [17, 18]. The importance of this interaction in DPP4 is illustrated by the observations that the mutation of Glu 206 in DPP4 abolishes enzymatic activity  and that N-terminal acetylation of DPP4 substrates protects against DPP4 proteolysis . The insertion loop of DPP7 also contains an acidic residue, Asp 334, which could function to coordinate with the N-terminus of the substrate in an analogous fashion to DPP4 (Figure 7B).
The structure of the human carboxypeptidase PRCP presented here provides the first structural description of the S28 family of proteases. These proteases consist of a conserved α/β hydrolase domain and a novel structural domain that caps the active site. Comparison with the previously undescribed structure of the aminopeptidase DPP7 reveals that a short insertion sequence in DPP7 sterically occludes access to the substrate binding groove to provide a simple evolutionary adaptation to change substrate specificity. These structural results provide the basis for rational design of selective PRCP regulators for the modulation of cardiovascular and metabolic diseases.
Human PRCP was expressed, purified and crystallized as described previously . Briefly, glycosylated PRCP was expressed as a secreted protein in CHO cells and purified using a combination of Ni-affinity, heparin and gel filtration chromatography. Crystals were obtained in 1.8 M ammonium sulfate, 0.1 M HEPES, pH 7.5, and 1-2% PEG 400 .
The structure of PRCP was determined using MIRAS techniques (Table 1). Two heavy-atom derivatives were prepared by soaking native PRCP crystals in stabilizing solutions containing 5 mM ethyl mercurithiosalicylate or 2.5 mM K2PtCl4 for 2 or 10 days, respectively. Data were collected at the Advanced Light Source beamline 5.0.2 by Reciprocal Space Consulting. Diffraction images were integrated using XDS  and reduced using SCALA  as implemented in autoPROC (Global Phasing Limited, Cambridge, United Kingdom). Data sets were scaled together using SCALEIT , and heavy atom sites identified with SHELXD . These heavy atom sites were used to seed runs of autoSHARP , combining native, mercury, and platinum data sets, to generate initial MIRAS phases and density-modified electron density maps. An initial model of PRCP was built into the 2.8 Å autoSHARP maps using Coot , and refined against the native data set at 2.8 Å using iterative rounds of autoBUSTER  refinement and manual rebuilding. MolProbity was used to evaluate the final refined model .
The PRCP coordinates have been deposited in the Protein Data Bank (PDB Code: 3N2Z)
dipeptidyl peptidase 4
dipeptidyl peptidase 7
figure of merit
multiple isomorphous replacement with anomalous scattering
root mean square deviation.
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