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In order to elucidate the effect of modification of the hinge region on structural polymorphism associated with conformational transitions, structural studies of hinge-modified oligopeptidase B from Serratia proteamaculans (SpOpBmod) in the crystalline state and solution were carried out. A new crystal structure of SpOpBmod in the intermediate conformation was obtained, and a molecular model of SpOpBmod in the open conformation was created using a combination of small-angle X-ray scattering with MD simulations. The improved electron density of the mobile H-loop carrying the catalytic H652 distinguished the obtained crystal structure from that which was previously reported. Good electron density in this region was previously found only in the inhibitor-bound SpOpBmod structure, in which one of the inhibitor molecules was covalently bound to H652. Comparison of the above structures of free and inhibitor-bound enzymes showed that both tertiary folds are the result of the internal conformational dynamics of SpOpBmod, which were captured by inhibitor binding. Comparison of the SpOpBmod structures with the structures of the same enzyme with a native hinge peptide made it possible to establish the influence of hinge modification on the rearrangement of the interdomain interface during conformational transitions. The above analysis also used models of native and hinge-modified enzymes in open conformations. We found that the interdomain interface observed in the crystal structures of hinge-modified enzymes could be considered an extreme version of the H-loop arrangement, in which closure of the domains does not lead to the assembly of the catalytic triad, whereas the intermediate conformation observed in the structure of the enzyme with the native hinge sequence illustrates a productive transition to the catalytically active closed conformation. [ABSTRACT FROM AUTHOR]

In order to elucidate the effect of modification of the hinge region on structural polymorphism associated with conformational transitions, structural studies of hinge-modified oligopeptidase B from Serratia proteamaculans (SpOpBmod) in the crystalline state and solution were carried out. A new crystal structure of SpOpBmod in the intermediate conformation was obtained, and a molecular model of SpOpBmod in the open conformation was created using a combination of small-angle X-ray scattering with MD simulations. The improved electron density of the mobile H-loop carrying the catalytic H652 distinguished the obtained crystal structure from that which was previously reported. Good electron density in this region was previously found only in the inhibitor-bound SpOpBmod structure, in which one of the inhibitor molecules was covalently bound to H652. Comparison of the above structures of free and inhibitor-bound enzymes showed that both tertiary folds are the result of the internal conformational dynamics of SpOpBmod, which were captured by inhibitor binding. Comparison of the SpOpBmod structures with the structures of the same enzyme with a native hinge peptide made it possible to establish the influence of hinge modification on the rearrangement of the interdomain interface during conformational transitions. The above analysis also used models of native and hinge-modified enzymes in open conformations. We found that the interdomain interface observed in the crystal structures of hinge-modified enzymes could be considered an extreme version of the H-loop arrangement, in which closure of the domains does not lead to the assembly of the catalytic triad, whereas the intermediate conformation observed in the structure of the enzyme with the native hinge sequence illustrates a productive transition to the catalytically active closed conformation.

A covalent serine protease inhibitor—Na-p-Tosyl-Lysyl Chloromethylketone (TCK) is a modified lysine residue tosylated at the N-terminus and chloromethylated at the C-terminus, one molecule of which is capable of forming two covalent bonds with both Ser and His catalytic residues, was co-crystallized with modified oligopeptidase B (OpB) from Serratia proteomaculans (PSPmod). The kinetics study, which preceded crystallization, shows that the stoichiometry of TCK-dependent inhibition of PSPmod was 1:2 (protein:inhibitor). The crystal structure of the PSPmod-TCK complex, solved at a resolution of 2.3 Å, confirmed a new type of inhibitor binding. Two TCK molecules were bound to one enzyme molecule: one with the catalytic Ser, the other with the catalytic His. Due to this mode of binding, the intermediate state of PSPmod and the disturbed conformation of the catalytic triad were preserved in the PSPmod-TCK complex. Nevertheless, the analysis of the amino acid surroundings of the inhibitor molecule bound to the catalytic Ser and its comparison with that of antipain-bound OpB from Trypanosoma brucei provided an insight in the structure of the PSPmod substrate-binding pocket. Supposedly, the new type of binding is typical for the interaction of chloromethylketone derivatives with two-domain OpBs. In the open conformational state that these enzymes are assumed in solution, the disordered configuration of the catalytic triad prevents simultaneous interaction of one inhibitor molecule with two catalytic residues.

Oligopeptidase B (OpB) is a two-domain, trypsin-like serine peptidase belonging to the S9 prolyloligopeptidase (POP) family. Two domains are linked by a hinge region that participates in the transition of the enzyme between two major states—closed and open—in which domains and residues of the catalytic triad are located close to each other and separated, respectively. In this study, we described, for the first time, a structure of OpB from bacteria obtained for an enzyme from Serratia proteomaculans with a modified hinge region (PSPmod). PSPmod was crystallized in a conformation characterized by a disruption of the catalytic triad together with a domain arrangement intermediate between open and closed states found in crystals of ligand-free and inhibitor-bound POP, respectively. Two additional derivatives of PSPmod were crystallized in the same conformation. Neither wild-type PSP nor its corresponding mutated variants were susceptible to crystallization, indicating that the hinge region modification was key in the crystallization process. The second key factor was suggested to be polyamine spermine since all crystals were grown in its presence. The influences of the hinge region modification and spermine on the conformational state of PSP in solution were evaluated by small-angle X-ray scattering. SAXS showed that, in solution, wild-type PSP adopted the open state, spermine caused the conformational transition to the intermediate state, and spermine-free PSPmod contained molecules in the open and intermediate conformations in dynamic equilibrium.

Non-commercial cooked Whiteleg shrimp ( Penaeus vannamei ) was valorized by application of protein hydrolysis treatments with different proteases (Giant catfish ( Pangasianodon gigas ) viscera proteases, commercial trypsin, and Alcalase®) for the obtainment of functional components (protein hydrolysates and carotenoids). Functional properties and in vitro inhibitory effect of the resulting protein hydrolysates on Dipeptidyl Peptidase-IV (DPP-IV) and Prolyloligopeptidase (PO) were evaluated. After hydrolysis, more than 73% of protein was recovered in soluble form, whereas 11–15% was insoluble. Carotenoids, determined as astaxanthin, were mainly present (>97%) in the insoluble protein fraction, presumably in form of complexes of high molecular weight. Protein hydrolysates showed excellent solubility (>97%) in a wide pH range (3–10), good oil holding capacity (0.86–1.83 g oil/g hydrolysates) and discrete inter-facial properties. Besides, all shrimp hydrolysates at concentration of 1 mg/mL provided DPP-IV inhibition activity (22.7–61.7%) and those prepared with trypsin and Alcalase® also inhibited PO (35–40% inhibition). The enzymatic processing of non-commercial boiled shrimp could be a useful way to valorize it, obtaining soluble proteins/peptides, potential hypoglycemic and antidepressant compounds (DPP-IV and PO inhibition peptides) and astaxanthin with interesting functional properties for food applications. [ABSTRACT FROM AUTHOR]

Aim/Purpose of the Study:Activation of the renin-angiotensin system leading to increased angiotensin-(1–7) (Ang-(1–7)) and decreased angiotensin 2 (Ang 2) levels may be a new therapeutic approach to reduce acute lung injury. Prolylcarboxypeptidase (PRCP) and prolyloligopeptidase (PREP) are capable of hydrolyzing Ang 2 into Ang-(1–7). However, their relation with circulating Ang 2 levels after lung ischemia–reperfusion injury (LIRI) has never been explored. This study determines whether the activity and expression of PRCP and PREP in plasma and lung tissue is related to circulating Ang 2 levels in a murine model of LIRI.Materials and Methods:LIRI in Swiss mice (6 animals per group) was induced by temporary left lung hilar clamping (1 h) followed by 0, 1 or 24 h of reperfusion. Animals in the sham group received thoracotomy only. PRCP activity was measured via RP-HPLC, PREP activity using a fluorogenic substrate and plasma Ang 2 levels via ELISA. Western blotting was used to determine the PRCP and PREP protein expression profiles in left lung tissue.Results:Plasma Ang 2 levels significantly rise after lung ischemia and remain increased after 1 h and 24 h of reperfusion compared to the sham group. While a significant decrease in plasma PREP activity was found after 24 h of reperfusion, a transient increase in plasma PRCP activity was observed after ischemia. However, no correlation with plasma Ang 2 levels could be demonstrated. The activity profiles of PRCP and PREP and the protein expression of PRCP in the lung tissues remained unchanged after LIRI.Conclusions:LIRI causes a dysregulation of circulating Ang 2 levels and plasma PREP activity, although no direct link between both phenomena could be shown. The activity profile of pulmonary PRCP and PREP was not significantly changed after LIRI, which implies a minor role for local PRCP and PREP in the ischemic lung itself. [ABSTRACT FROM PUBLISHER]

Dipeptidyl-peptidase IV/CD26 (DPP IV) is a cell-surface protease belonging to the prolyloligopeptidase family. It selectively removes the N-terminal dipeptide from peptides with proline or alanine in the second position. Apart from its catalytic activity, it interacts with several proteins, for instance, adenosine deaminase, the HIV gp120 protein, fibronectin, collagen, the chemokine receptor CXCR4, and the tyrosine phosphatase CD45. DPP IV is expressed on a specific set of T lymphocytes, where it is up-regulated after activation. It is also expressed in a variety of tissues, primarily on endothelial and epithelial cells. A soluble form is present in plasma and other body fluids. DPP IV has been proposed as a diagnostic or prognostic marker for various tumors, hematological malignancies, immunological, inflammatory, psychoneuroendocrine disorders, and viral infections. DPP IV truncates many bioactive peptides of medical importance. It plays a role in glucose homeostasis through proteolytic inactivation of the incretins. DPP IV inhibitors improve glucose tolerance and pancreatic islet cell function in animal models of type 2 diabetes and in diabetic patients. The role of DPP IV/ CD26 within the immune system is a combination of its exopeptidase activity and its interactions with different molecules. This enables DPP IV/CD26 to serve as a co-stimulatory molecule to influence T cell activity and to modulate chemotaxis. DPP IV is also implicated in HIV-1 entry, malignant transformation, and tumor invasion. [ABSTRACT FROM AUTHOR]

A covalent serine protease inhibitor—Na-p-Tosyl-Lysyl Chloromethylketone (TCK) is a modified lysine residue tosylated at the N-terminus and chloromethylated at the C-terminus, one molecule of which is capable of forming two covalent bonds with both Ser and His catalytic residues, was co-crystallized with modified oligopeptidase B (OpB) from Serratia proteomaculans (PSPmod). The kinetics study, which preceded crystallization, shows that the stoichiometry of TCK-dependent inhibition of PSPmod was 1:2 (protein:inhibitor). The crystal structure of the PSPmod-TCK complex, solved at a resolution of 2.3 Å, confirmed a new type of inhibitor binding. Two TCK molecules were bound to one enzyme molecule: one with the catalytic Ser, the other with the catalytic His. Due to this mode of binding, the intermediate state of PSPmod and the disturbed conformation of the catalytic triad were preserved in the PSPmod-TCK complex. Nevertheless, the analysis of the amino acid surroundings of the inhibitor molecule bound to the catalytic Ser and its comparison with that of antipain-bound OpB from Trypanosoma brucei provided an insight in the structure of the PSPmod substrate-binding pocket. Supposedly, the new type of binding is typical for the interaction of chloromethylketone derivatives with two-domain OpBs. In the open conformational state that these enzymes are assumed in solution, the disordered configuration of the catalytic triad prevents simultaneous interaction of one inhibitor molecule with two catalytic residues. [ABSTRACT FROM AUTHOR]

Simple Summary: Oligopeptidase B is a two-domain, trypsin-like peptidase from parasitic protozoa and bacteria which belongs to the least studied group of prolyloligopeptidases. In this study, we describe for the first time a crystal structure of bacterial oligopeptidase B and compare it with those of protozoan oligopeptidases B and related prolyloligopeptidases. The enzyme was crystallized in the presence of spermine and contained a modified sequence of the interdomain linker. Both factors were key for crystallization. The structure showed an uncommon intermediate conformation with a domain arrangement intermediate between open and closed conformations found in the crystals of ligand-free and inhibitor-bound prolyloligopeptidases, respectively. To evaluate the impact of the modification and spermine in the obtained conformation, small-angle X-ray scattering was applied, which showed that in solution wild-type enzymes adopt the open conformation and spermine causes a transition to the intermediate state, while the modification is associated with a partial transition. We suggest that spermine-dependent conformational transition replicates the behavior of the enzyme in bacterial cells and the intermediate state, which is rarely detected in vitro, and might be widely distributed in vivo, and so should be considered during computational studies, including those aimed wanting to develop the small molecule inhibitors targeting prolyloligopeptidases. Oligopeptidase B (OpB) is a two-domain, trypsin-like serine peptidase belonging to the S9 prolyloligopeptidase (POP) family. Two domains are linked by a hinge region that participates in the transition of the enzyme between two major states—closed and open—in which domains and residues of the catalytic triad are located close to each other and separated, respectively. In this study, we described, for the first time, a structure of OpB from bacteria obtained for an enzyme from Serratia proteomaculans with a modified hinge region (PSPmod). PSPmod was crystallized in a conformation characterized by a disruption of the catalytic triad together with a domain arrangement intermediate between open and closed states found in crystals of ligand-free and inhibitor-bound POP, respectively. Two additional derivatives of PSPmod were crystallized in the same conformation. Neither wild-type PSP nor its corresponding mutated variants were susceptible to crystallization, indicating that the hinge region modification was key in the crystallization process. The second key factor was suggested to be polyamine spermine since all crystals were grown in its presence. The influences of the hinge region modification and spermine on the conformational state of PSP in solution were evaluated by small-angle X-ray scattering. SAXS showed that, in solution, wild-type PSP adopted the open state, spermine caused the conformational transition to the intermediate state, and spermine-free PSPmod contained molecules in the open and intermediate conformations in dynamic equilibrium. [ABSTRACT FROM AUTHOR]

Backbone N-methylation as a posttranslational modification was recently discovered in a class of ribosomally encoded peptides referred to as borosins. The founding members of the borosins are the omphalotins (A-I), backbone N-methylated, macrocyclic dodecapeptides produced by the mushroom Omphalotus olearius. Omphalotins display a strong and selective toxicity toward the plant parasitic nematode Meloidogyne incognita. The primary product omphalotin A is synthesized via a concerted action of the omphalotin precursor protein (OphMA) and the dual function prolyloligopeptidase/macrocyclase (OphP). OphMA consists of α-N-methyltransferase domain that autocatalytically methylates the core peptide fused to its C-terminus via a clasp domain. Genome mining uncovered over 50 OphMA homologs from the fungal phyla Ascomycota and Basidiomycota. However, the derived peptide natural products have not been described yet, except for lentinulins, dendrothelins and gymnopeptides produced by the basidiomycetes Lentinula edodes, Dendrothele bispora and Gymnopus fusipes, respectively. In this chapter, we describe methods used to isolate and characterize these backbone N-methylated peptides and their precursor proteins both in their original hosts and in the heterologous hosts Escherichia coli and Pichia pastoris. These methods may pave the path for both the discovery of novel borosins with interesting bioactivities. In addition, understanding of borosin biosynthetic pathways may allow setting up a biotechnological platform for the production of pharmaceutical leads for orally available peptide drugs., (Copyright © 2021 Elsevier Inc. All rights reserved.)