The 3D structure of rat DPPIV/CD26 as obtained by cryo-TEM and single particle analysis

We present the three-dimensional structure of rat DPPIV/CD26, as determined by cryo-TEM and single particle analysis at a resolution of approximately 14A. The reconstruction confirms that the protein exists as a dimer, as predicted earlier. Since there are structural analogies to the serine peptidase POP, docking calculations of the two structures were performed. Although the docking showed a similar spatial organization (catalytic domain, beta-propeller, distal opening, central cavity), the detailed comparison revealed clear discrepancies. The most marked difference is a second (lateral) opening in DPPIV/CD26, which would enable direct access to the catalytic site. We therefore assume that substrate selectivity and binding rate are most probably driven by different mechanisms in DPPIV/CD26 and POP.     

The crystal structure of human dipeptidyl peptidase IV (DPPIV) complex with diprotin A

Dipeptidyl peptidase IV (DPPIV) is a serine protease, a member of the prolyl oligopeptidase (POP) family, and has been implicated in several diseases. Therefore, it seems important to develop selective inhibitors for human DPPIV (hDPPIV) that are able to control the biological function of hDPPIV. In order to elucidate the binding mode and substrate specificity, we determined the crystal structure complex of hDPPIV and diprotin A (IIe-Pro-IIe), a slowly hydrolyzed substrate of hDPPIV, at 2.2 A resolution. In this paper, we discuss the molecular interaction mechanism of diprotin A with hDPPIV based on the X-ray crystal structure.     

Structural and kinetic analysis of the substrate specificity of human fibroblast activation protein alpha

Fibroblast activation protein alpha (FAPalpha) is highly expressed in epithelial cancers and has been implicated in extracellular matrix remodeling, tumor growth, and metastasis. We present the first high resolution structure for the apoenzyme as well as kinetic data toward small dipeptide substrates. FAPalpha exhibits a dipeptidyl peptidase IV (DPPIV)-like fold, featuring an alpha/beta-hydrolase domain and an eight-bladed beta-propeller domain. Known DPPIV dipeptides are cleaved by FAPalpha with an approximately 100-fold decrease in catalytic efficiency compared with DPPIV. Moreover, FAPalpha, but not DPPIV, possesses endopeptidase activity toward N-terminal benzyloxycarbonyl (Z)-blocked peptides. Comparison of the crystal structures of FAPalpha and DPPIV revealed one major difference in the vicinity of the Glu motif (Glu(203)-Glu(204) for FAPalpha; Glu(205)-Glu(206) for DPPIV) within the active site of the enzyme. Ala(657) in FAPalpha, instead of Asp(663) as in DP-PIV, reduces the acidity in this pocket, and this change could explain the lower affinity for N-terminal amines by FAPalpha. This hypothesis was tested by kinetic analysis of the mutant FAPalpha/A657D, which shows on average an approximately 60-fold increase in the catalytic efficiency, as measured by k(cat)/K(m), for the cleavage of dipeptide substrates. Furthermore, the catalytic efficiency of the mutant is reduced by approximately 350-fold for cleavage of Z-Gly-Pro-7-amino-4-methylcoumarin. Our data provide a clear understanding of the molecular determinants responsible for the substrate specificity and endopeptidase activity of FAPalpha.     

Crystal structure of CD26/dipeptidyl-peptidase IV in complex with adenosine deaminase reveals a highly amphiphilic interface

Dipeptidyl-peptidase IV (DPPIV or CD26) is a homodimeric type II membrane glycoprotein in which the two monomers are subdivided into a beta-propeller domain and an alpha/beta-hydrolase domain. As dipeptidase, DPPIV modulates the activity of various biologically important peptides and, in addition, DPPIV acts as a receptor for adenosine deaminase (ADA), thereby mediating co-stimulatory signals in T-lymphocytes. The 3.0-A resolution crystal structure of the complex formed between human DPPIV and bovine ADA presented here shows that each beta-propeller domain of the DPPIV dimer binds one ADA. At the binding interface, two hydrophobic loops protruding from the beta-propeller domain of DPPIV interact with two hydrophilic and heavily charged alpha-helices of ADA, giving rise to the highest percentage of charged residues involved in a protein-protein contact reported thus far. Additionally, four glycosides linked to Asn229 of DPPIV bind to ADA. In the crystal structure of porcine DPPIV, the observed tetramer formation was suggested to mediate epithelial and lymphocyte cell-cell adhesion. ADA binding to DPPIV could regulate this adhesion, as it would abolish tetramerization.     

Crystal structure of human dipeptidyl peptidase IV in complex with a decapeptide reveals details on substrate specificity and tetrahedral intermediate formation

Dipeptidyl peptidase IV (DPPIV) is a member of the prolyl oligopeptidase family of serine proteases. DPPIV removes dipeptides from the N terminus of substrates, including many chemokines, neuropeptides, and peptide hormones. Specific inhibition of DPPIV is being investigated in human trials for the treatment of type II diabetes. To understand better the molecular determinants that underlie enzyme catalysis and substrate specificity, we report the crystal structures of DPPIV in the free form and in complex with the first 10 residues of the physiological substrate, Neuropeptide Y (residues 1-10; tNPY). The crystal structure of the free form of the enzyme reveals two potential channels through which substrates could access the active site-a so-called propeller opening, and side opening. The crystal structure of the DPPIV/tNPY complex suggests that bioactive peptides utilize the side opening unique to DPPIV to access the active site. Other structural features in the active site such as the presence of a Glu motif, a well-defined hydrophobic S1 subsite, and minimal long-range interactions explain the substrate recognition and binding properties of DPPIV. Moreover, in the DPPIV/tNPY complex structure, the peptide is not cleaved but trapped in a tetrahedral intermediate that occurs during catalysis. Conformational changes of S630 and H740 between DPPIV in its free form and in complex with tNPY were observed and contribute to the stabilization of the tetrahedral intermediate. Our results facilitate the design of potent, selective small molecule inhibitors of DPPIV that may yield compounds for the development of novel drugs to treat type II diabetes.     

N-linked glycosylation of dipeptidyl peptidase IV (CD26): effects on enzyme activity, homodimer formation, and adenosine deaminase binding

The type II transmembrane serine protease dipeptidyl peptidase IV (DPPIV), also known as CD26 or adenosine deaminase binding protein, is a major regulator of various physiological processes, including immune, inflammatory, nervous, and endocrine functions. It has been generally accepted that glycosylation of DPPIV and of other transmembrane dipeptidyl peptidases is a prerequisite for enzyme activity and correct protein folding. Crystallographic studies on DPPIV reveal clear N-linked glycosylation of nine Asn residues in DPPIV. However, the importance of each glycosylation site on physiologically relevant reactions such as dipeptide cleavage, dimer formation, and adenosine deaminase (ADA) binding remains obscure. Individual Asn-->Ala point mutants were introduced at the nine glycosylation sites in the extracellular domain of DPPIV (residues 39-766). Crystallographic and biochemical data demonstrate that N-linked glycosylation of DPPIV does not contribute significantly to its peptidase activity. The kinetic parameters of dipeptidyl peptidase cleavage of wild-type DPPIV and the N-glycosylation site mutants were determined by using Ala-Pro-AFC and Gly-Pro-pNA as substrates and varied by <50%. DPPIV is active as a homodimer. Size-exclusion chromatographic analysis showed that the glycosylation site mutants do not affect dimerization. ADA binds to the highly glycosylated beta-propeller domain of DPPIV, but the impact of glycosylation on binding had not previously been determined. Our studies indicate that glycosylation of DPPIV is not required for ADA binding. Taken together, these data indicate that in contrast to the generally accepted view, glycosylation of DPPIV is not a prerequisite for catalysis, dimerization, or ADA binding.     

Decoding the structural events in substrate‐gating mechanism of eukaryotic prolyl oligopeptidase using normal mode analysis and molecular dynamics simulations

Prolyl oligopeptidase (POP) is a serine protease, unique for its ability to cleave various small oligopeptides shorter than 30 amino acids. POP is an important drug target since it is implicated in various neurological disorders. Although there is structural evidence that bacterial POPs undergo huge interdomain movements and acquire an “open” state in the substrate‐unbound form, hitherto, no crystal structure is available in the substrate‐unbound domain‐open form of eukaryotic POPs. Indeed, there is no difference between the substrate‐unbound/bound states of eukaryotic POPs. This raises unanswered questions about whether difference in the substrate access pathway exists between bacterial and eukaryotic POPs. Here, we have used normal mode analysis and molecular dynamics to unravel the mechanism of substrate entry in mammalian POPs, which has been debated until now. Motions observed using normal modes of porcine and bacterial POPs were analyzed and compared, augmented by molecular dynamics of these proteins. Identical to bacterial POPs, interdomain opening was found to be the possible pathway for the substrate‐gating in mammals as well. On the basis of our analyses and evidences, a mechanistic model of substrate entry in POPs has been proposed. Up‐down movement of N‐terminal hydrolase domain resulted in twisting motion of two domains, followed by the conformational changes of interdomain loop regions, which facilitate interdomain opening. Similar to bacterial POPs, an open form of porcine POP is also proposed with domain‐closing motion. This work has direct implications for the development of novel inhibitors of mammalian POPs to understand the etiology of various neurological diseases. Proteins 2014; 82:1428–1443. © 2014 Wiley Periodicals, Inc.     

Active-Site Gating Regulates Substrate Selectivity in a Chymotrypsin-Like Serine Protease: The Structure of Haemophilus influenzae Immunoglobulin A1 Protease

We report here the first structure of a member of the immunoglobulin A protease (IgAP) family at 1.75-Å resolution. This protease is a founding member of the type V (autotransporter) secretion system and is considered a virulence determinant among the bacteria expressing the enzyme. The structure of the enzyme fits that of a classic autotransporter in which several unique domains necessary for protein function are appended to a central, 100-Å-long β-helical domain. The N-terminal domain of the IgAP is found to possess a chymotrypsin-like fold. However, this catalytic domain contains a unique loop D that extends over the active site acting as a lid, gating substrate access. The data presented provide a structural basis for the known ability of IgAPs to cleave only the proline/serine/threonine-rich hinge peptide unique to IgA1 (isotype 1) in the context of the intact fold of the immunoglobulin. Based upon the structural data, as well as molecular modeling, a model suggesting that the unique extended loop D in this IgAP sterically occludes the active-site binding cleft in the absence of immunoglobulin binding is presented. Only in the context of binding of the IgA1-Fc domain in a valley formed between the N-terminal protease domain and another domain appended to the β-helix spine (domain 2) is the lid stabilized in an open conformation. The stabilization of this open conformation through Fc association subsequently allows access of the hinge peptide to the active site, resulting in recognition and cleavage of the substrate.     

Crystal structure of the catalytic domain of human complement c1s: a serine protease with a handle

C1s is the highly specific modular serine protease that mediates the proteolytic activity of the C1 complex and thereby triggers activation of the complement cascade. The crystal structure of a catalytic fragment from human C1s comprising the second complement control protein (CCP2) module and the chymotrypsin-like serine protease (SP) domain has been determined and refined to 1.7 A resolution. In the areas surrounding the active site, the SP structure reveals a restricted access to subsidiary substrate binding sites that could be responsible for the narrow specificity of C1s. The ellipsoidal CCP2 module is oriented perpendicularly to the surface of the SP domain. This arrangement is maintained through a rigid module-domain interface involving intertwined proline- and tyrosine-rich polypeptide segments. The relative orientation of SP and CCP2 is consistent with the fact that the latter provides additional substrate recognition sites for the C4 substrate. This structure provides a first example of a CCP-SP assembly that is conserved in diverse extracellular proteins. Its implications in the activation mechanism of C1 are discussed.     

Crystal structure of human dipeptidyl peptidase IV/CD26 in complex with a substrate analog

Dipeptidyl peptidase IV (DPP-IV/CD26) is a multifunctional type II transmembrane serine peptidase. This enzyme contributes to the regulation of various physiological processes, including blood sugar homeostasis, by cleaving peptide hormones, chemokines and neuropeptides. We have determined the 2.5 A structure of the extracellular region of DPP-IV in complex with the inhibitor valine-pyrrolidide. The catalytic site is located in a large cavity formed between the alpha/beta-hydrolase domain and an eight-bladed beta-propeller domain. Both domains participate in inhibitor binding. The structure indicates how substrate specificity is achieved and reveals a new and unexpected opening to the active site.     

Monomeric structures of the zymogen and active catalytic domain of complement protease c1r: further insights into the c1 activation mechanism

C1r is the serine protease (SP) that mediates autoactivation of C1, the complex that triggers the classical complement pathway. We have determined the crystal structure of two fragments from the human C1r catalytic domain, each encompassing the second complement control protein (CCP2) module and the SP domain. The wild-type species has an active structure, whereas the S637A mutant is a zymogen. The structures reveal a restricted hinge flexibility of the CCP2-SP interface, and both are characterized by the unique alpha-helical conformation of loop E. The zymogen activation domain exhibits high mobility, and the active structure shows a restricted access to most substrate binding subsites. Further implications relevant to the C1r self-activation process are derived from protein-protein interactions in the crystals.     

Fluorescence resonance energy transfer (FRET) peptides and cycloretro-inverso peptides derived from bradykinin as substrates and inhibitors of prolyl oligopeptidase

Prolyl oligopeptidase (POP, EC 3.4.21.26) is a member of a family of serine peptidases with post-proline cleaving activity towards peptides. It is located in the cytosol in active form but without hydrolytic activity on proteins or peptides higher than 30 amino acids. Its function is not well defined, but it is involved in central nervous system disorders. Here, we studied the substrate specificity of wild type POP (POPwt) and its C255T variant lacking the non-catalytic Cys(255). This residue is located in the seven-bladed beta-propeller domain that regulates the activity of POP. Fluorescence resonance energy transfer (FRET) peptides were used with sequences derived from bradykinin-containing region of human kininogen and flanked by Abz (ortho-aminobenzoic acid) and EDDnp [N-ethylenediamine-(2,4-dinitrophenyl)]. The peptide Abz-GFSPFRQ-EDDnp was taken as leader substrate for the synthesis of five series of peptides modified at the P(3), P(2), P'(1), P'(2) and P'(3) residues. The optimal amino acids in each position for POPwt resulted in the sequence RRPYIR that is very similar to the C-terminal sequence of neurotensin. The cyclic peptides c(G((n))FSPFR) (n=1-4) were hydrolyzed by POP; their cycloretro and cycloretro-inverso analogues were inhibitors in the micromolar range. The differences between POPwt and its C255T mutant in the hydrolysis of the series derived from Abz-GFSPFRQ-EDDnp were restricted to the non-prime site of the substrates. The kinetic data of hydrolysis and inhibition by the cyclic peptides are consistent with the structures of POP-substrate/inhibitor complexes and with the substrate specificity data obtained with linear FRET peptides. All together, these results give information about the POP-substrate/inhibitor interactions that further complete knowledge of this important oligopeptidase.     

The oligomeric structure of human granzyme A is a determinant of its extended substrate specificity

The cell death-inducing serine protease granzyme A (GzmA) has a unique disulfide-linked quaternary structure. The structure of human GzmA bound to a tripeptide CMK inhibitor, determined at a resolution of 2.4 A, reveals that the oligomeric state contributes to substrate selection by limiting access to the active site for potential macromolecular substrates and inhibitors. Unlike other serine proteases, tetrapeptide substrate preferences do not correlate well with natural substrate cleavage sequences. This suggests that the context of the cleavage sequence within a macromolecular substrate imposes another level of selection not observed with the peptide substrates. Modeling of inhibitors bound to the GzmA active site shows that the dimer also contributes to substrate specificity in a unique manner by extending the active-site cleft. The crystal structure, along with substrate library profiling and mutagenesis, has allowed us to identify and rationally manipulate key components involved in GzmA substrate specificity.     

β-Propeller repeats and a PDZ domain in the tricorn protease: predicted self-compartmentalisation and C-terminal polypeptide-binding strategies of substrate selection

Prokaryotic proteases demonstrate a variety of substrate-selection strategies that prevent uncontrolled protein degradation. Proteasomes and ClpXP-like proteases form oligomeric structures that exclude large substrates from central solvated chambers containing their active sites. Monomeric prolyl oligopeptidases have been shown to contain beta-propeller structures that similarly reduce access to their catalytic residues. By contrast, Tsp-like enzymes contain PDZ domains that are thought to specifically target C-terminal polypeptides. We have investigated the sequence of Thermoplasma acidophilum tricorn protease using recently-developed database search methods. The tricorn protease is known to associate into a 20 hexamer capsid enclosing an extremely large cavity that is 37 nm in diameter. It is unknown, however, how this enzyme selects its small oligopeptide substrates. Our results demonstrate the presence in tricorn protease of a PDZ domain and two predicted six-bladed beta-propeller domains. We suggest that the PDZ domain is involved in targeting non-polar C-terminal peptides, similar to those generated by the T. acidophilum proteasome, whereas the beta-propeller domains serve to exclude large substrates from the tricorn protease active site in a similar manner to that previously indicated for prolyl oligopeptidase.     

Crystal structure of the proenzyme domain of plasminogen.

We have solved the X-ray crystal structure of the proenzyme form of the catalytic domain of plasminogen, with the nonessential mutations M585Q, V673M, and M788L, to 2.0 A resolution. The structure presents an inactive protease characterized by Asp740 (chymotrypsinogen 194) hydrogen bonded to His586 (chymotrypsinogen 40), preventing proper formation of the oxyanion hole and S1 specificity pocket. In addition, the catalytic triad residues are misplaced relative to the active conformation adopted by serine proteases in the chymotrypsin family. Finally, a unique form of zymogen inactivation is observed, characterized by a "foot-in-mouth" mechanism in which Trp761 (chymotrypsinogen 215) is folded into the S1 specificity pocket preventing substrate binding.     

GB virus B and hepatitis C virus NS3 serine proteases share substrate specificity.

GB virus B (GBV-B) is a recently discovered virus responsible for hepatitis in tamarins (Saguinus species). GBV-B belongs to the Flaviviridae family and is closely related to the human pathogen hepatitis C virus (HCV). Nonstructural protein 3 (NS3) of HCV has been shown to encompass a serine protease domain required for viral maturation. GBV-B and HCV share only about 30% of the amino acid sequence within the NS3 protease domain. The catalytic triad is conserved, and the residue Phe-154, presumed to be a crucial amino acid for determining the S1 specificity pocket of the HCV NS3 protease, is also conserved. We have expressed a synthetic gene encoding the GBV-B NS3 protease domain in Escherichia coli and have characterized the purified recombinant protein for its activity on HCV substrates. We have shown that the NS3 region of the GBV-B genome actually encodes a serine protease that, despite the low sequence homology, shares substrate specificity with the HCV NS3 protease.     

Evolutionary relationships of the prolyl oligopeptidase family enzymes.

The prolyl oligopeptidase (POP) family of serine proteases includes prolyl oligopeptidase, dipeptidyl peptidase IV, acylaminoacyl peptidase and oligopeptidase B. The enzymes of this family specifically hydrolyze oligopeptides with less than 30 amino acids. Many of the POP family enzymes have evoked pharmaceutical interest as they have roles in the regulation of peptide hormones and are involved in a variety of diseases such as dementia, trypanosomiasis and type 2 diabetes. In this study we have clarified the evolutionary relationships of these four POP family enzymes and analyzed POP sequences from different sources. The phylogenetic trees indicate that the four enzymes were present in the last common ancestor of all life forms and that the beta-propeller domain has been part of the family for billions of years. There are striking differences in the mutation rates between the enzymes and POP was found to be the most conserved enzyme of this family. However, the localization of this enzyme has changed throughout evolution, as three archaeal POPs seem to be membrane bound and one third of the bacterial as well as two eukaryotic POPs were found to be secreted out of the cell. There are also considerable distinctions between the mutation rates of the different substrate binding subsites of POP. This information may help in the development of species-specific POP inhibitors.     

Regulation of fibroblast migration on collagenous matrix by a cell surface peptidase complex

The invasion of migratory cells through connective tissues involves metallo- and serine types of cell surface proteases. We show that formation of a novel protease complex, consisting of the membrane-bound prolyl peptidases seprase and dipeptidyl peptidase IV (DPPIV), at invadopodia of migratory fibroblasts is a prerequisite for cell invasion and migration on a collagenous matrix. Seprase and DPPIV form a complex on the cell surface that elicits both gelatin binding and gelatinase activities localized at invadopodia of cells migrating on collagenous fibers. The protease complex participates in the binding to gelatin and localized gelatin degradation, cellular migration, and monolayer wound closure. Serine protease inhibitors can block the gelatinase activity and the localized gelatin degradation by cells. Antibodies to the gelatin-binding domain of DPPIV reduce the cellular abilities of the proteases to degrade gelatin but do not affect cellular adhesion or spreading on type I collagen. Furthermore, expression of the seprase-DPPIV complex is restricted to migratory cells involved in wound closure in vitro and in connective tissue cells during closure of gingival wounds but not in differentiated tissue cells. Thus, we have identified cell surface proteolytic activities, which are non-metalloproteases, seprase and DPPIV, that are responsible for the tissue-invasive phenotype.