Structural and Functional Basis of Plasminogen Activation by Staphylokinase

1999 ◽  
Vol 81 (04) ◽  
pp. 479-485 ◽  
Author(s):  
L. Jespers ◽  
S. Vanwetswinkel ◽  
H. R. Lijnen ◽  
N. Van Herzeele ◽  
B. Van Hoef ◽  
...  
Keyword(s):  
Active Site ◽  
Recombinant Dna ◽  
Catalytic Domain ◽  
Functional Model ◽  
Binding Pocket ◽  
Catalytic Triad ◽  
X Ray Crystallography ◽  
Serine Protease Domain ◽  
Docking Model ◽  
Binding Epitope

SummaryStaphylokinase (Sak), a 15.5-kDa bacterial protein, forms a complex with human plasmin, which in turn activates other plasminogen molecules to plasmin. Three recombinant DNA-based approaches, (i) site directed substitution with alanine, (ii) search for proximity relationships at the complex interface, and (iii) active-site accessibility to protease inhibitors have been used to deduce a coherent docking model of the crystal structure of Sak on the homology-based model of micro-plasmin (μPli), the serine protease domain of plasmin. Sak binding on μPli is primarily mediated by two surface-exposed loops, loops 174 and 215, at the rim of the active-site cleft, while the binding epitope of Sak on μPli involves several residues located in the flexible NH2-terminal arm and in the five-stranded mixed β-sheet. Several Sak residues located within the unique μ-helix and the β2 strand do not contribute to the binding epitope but are essential to induce plasminogen activating potential in the Sak:μPli complex. These residues form a topologically distinct activation epitope, which, upon binding of Sak to the catalytic domain of μPli, protrudes into a broad groove near the catalytic triad of μPli, thereby generating a competent binding pocket for micro-plasminogen (μPlg), which buries approximately 2500 Å of the Sak:μPli complex upon binding. This structural and functional model may serve as a template for the design of improved Sak-derived thrombolytic agents. Following the completion and presentation of the present study, the deduced Sak:μPli:μPlg complex was fully confirmed by X-ray crystallography, which further illustrates the power and potential of the present approach.

scholarly journals Activation and selectivity of OTUB-1 and OTUB-2 deubiquitinylases

2020 ◽  
Vol 295 (20) ◽  
pp. 6972-6982
Author(s):  
Dakshinamurthy Sivakumar ◽  
Vikash Kumar ◽  
Michael Naumann ◽  
Matthias Stein
Keyword(s):  
Active Site ◽  
Electrostatic Interactions ◽  
Active Form ◽  
Cysteine Proteases ◽  
Binding Pocket ◽  
Catalytic Triad ◽  
Catalytic Site ◽  
Dynamics Simulation ◽  
Active Site Residues ◽  
Catalytically Active

The ovarian tumor domain (OTU) deubiquitinylating cysteine proteases OTUB1 and OTUB2 (OTU ubiquitin aldehyde binding 1 and 2) are representative members of the OTU subfamily of deubiquitinylases. Deubiquitinylation critically regulates a multitude of important cellular processes, such as apoptosis, cell signaling, and growth. Moreover, elevated OTUB expression has been observed in various cancers, including glioma, endometrial cancer, ovarian cancer, and breast cancer. Here, using molecular dynamics simulation approaches, we found that both OTUB1 and OTUB2 display a catalytic triad characteristic of proteases but differ in their configuration and protonation states. The OTUB1 protein had a prearranged catalytic site, with strong electrostatic interactions between the active-site residues His265 and Asp267. In OTUB2, however, the arrangement of the catalytic triad was different. In the absence of ubiquitin, the neutral states of the catalytic-site residues in OTUB2 were more stable, resulting in larger distances between these residues. Only upon ubiquitin binding did the catalytic triad in OTUB2 rearrange and bring the active site into a catalytically feasible state. An analysis of water access channels revealed only a few diffusion trajectories for the catalytically active form of OTUB1, whereas in OTUB2 the catalytic site was solvent-accessible, and a larger number of water molecules reached and left the binding pocket. Interestingly, in OTUB2, the catalytic residues His224 and Asn226 formed a stable hydrogen bond. We propose that the observed differences in activation kinetics, protonation states, water channels, and active-site accessibility between OTUB1 and OTUB2 may be relevant for the selective design of OTU inhibitors.

Homology modelling and structural analysis of human arylamine N-acetyltransferase NAT1: evidence for the conservation of a cysteine protease catalytic domain and an active-site loop

2001 ◽  
Vol 356 (2) ◽  
pp. 327-334 ◽  
Author(s):  
Fernando RODRIGUES-LIMA ◽  
Claudine DELOMÉNIE ◽  
Geoffrey H. GOODFELLOW ◽  
Denis M. GRANT ◽  
Jean-Marie DUPRET
Keyword(s):  
Crystal Structure ◽  
Cysteine Protease ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Catalytic Domain ◽  
Homology Modelling ◽  
Metabolic Activation ◽  
Cysteine Proteases ◽  
Catalytic Triad ◽  
Quality Model

Arylamine N-acetyltransferases (EC 2.3.1.5) (NATs) catalyse the biotransformation of many primary arylamines, hydrazines and their N-hydroxylated metabolites, thereby playing an important role in both the detoxification and metabolic activation of numerous xenobiotics. The recently published crystal structure of the Salmonella typhimurium NAT (StNAT) revealed the existence of a cysteine protease-like (Cys-His-Asp) catalytic triad. In the present study, a three-dimensional homology model of human NAT1, based upon the crystal structure of StNAT [Sinclair, Sandy, Delgoda, Sim and Noble (2000) Nat. Struct. Biol. 7, 560–564], is demonstrated. Alignment of StNAT and NAT1, together with secondary structure predictions, have defined a consensus region (residues 29–131) in which 37% of the residues are conserved. Homology modelling provided a good quality model of the corresponding region in human NAT1. The location of the catalytic triad was found to be identical in StNAT and NAT1. Comparison of active-site structural elements revealed that a similar length loop is conserved in both species (residues 122–131 in NAT1 model and residues 122–133 in StNAT). This observation may explain the involvement of residues 125, 127 and 129 in human NAT substrate selectivity. Our model, and the fact that cysteine protease inhibitors do not affect the activity of NAT1, suggests that human NATs may have adapted a common catalytic mechanism from cysteine proteases to accommodate it for acetyl-transfer reactions.

scholarly journals Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2

2020 ◽  
Author(s):  
Youngchang Kim ◽  
Jacek Wower ◽  
Natalia Maltseva ◽  
Changsoo Chang ◽  
Robert Jedrzejczak ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Active Site ◽  
Catalytic Domain ◽  
Binding Pocket ◽  
Rapid Identification ◽  
Innate Immune ◽  
Manganese Ions ◽  
Antiviral Compounds ◽  
Sense Strand ◽  
Cell Assays

ABSTRACTSARS-CoV-2 Nsp15 is a uridylate-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family. It degrades the polyuridine extensions in (−) sense strand of viral RNA and some non-translated RNA on (+) sense strand. This activity seems to be responsible for the interference with the innate immune response and evasion of host pattern recognition. Nsp15 is highly conserved in coronaviruses suggesting that its activity is important for virus replication. Here we report first structures with bound nucleotides and show that SARS-CoV-2 Nsp15 specifically recognizes U in a pattern previously predicted for EndoU. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. Inhibitors of Nsp15 have been reported but not actively pursued into therapeutics. The current COVID-19 pandemic brought to attention the repurposing of existing drugs and the rapid identification of new antiviral compounds. Tipiracil is an FDA approved drug that is used with trifluridine in the treatment of colorectal cancer. Here, we combine crystallography, biochemical and whole cell assays, and show that this compound inhibits SARS-CoV-2 Nsp15 and interacts with the uridine binding pocket of the enzyme’s active site, providing basis for the uracil scaffold-based drug development.

scholarly journals Genome Mining, Phylogenetic and Structural Analysis of Bacterial Nitrilases for the Biodegradation of Nitrile Compounds

2021 ◽  
Author(s):  
Richa Salwan ◽  
Vivek Sharma ◽  
Surajit Das
Keyword(s):  
Structural Analysis ◽  
Substrate Specificity ◽  
Active Site ◽  
Structural Information ◽  
Genome Mining ◽  
Binding Pocket ◽  
Catalytic Triad ◽  
Vital Role ◽  
Active Site Residues ◽  
Motif Analysis

Abstract Microbial nitrilases play vital role in biodegradation of nitrile-containing contaminants in pollutant and effluents treatments in chemical and textile industries as well as the biosynthesis of IAA from tryptophan in plants. However, the lack of structural information hinders the correlation of its activity and substrate specificity. Here, we have identified bacterial genomes for nitrilases bearing unassigned functions including hypothetical, uncharacterized, or putative role. The genomic annotations revealed four predicted nitrilases encoding genes as uncharacterized subgroup of the nitrilase superfamily. Further, the annotation of these nitrilases revealed relatedness with nitrilase hydratases and cyanoalanine hydratases. The characterization of motif analysis of these protein sequences, predicted a single motif of 20-28 aa, and glutamate (E), lysine (K) and cysteine (C) residues as a part of catalytic triad along with several active site residues. The structural analysis of the modeled nitrilases revealed geometrical and close conformation of α-helices and β-sheets arranged in a sandwich structure. The catalytic residues constituted the substrate binding pocket and exhibited the wide nitrile substrate spectra for both aromatic and aliphatic nitriles containing compounds. The aromatic amino acid residues Y159 in active site were predicted to show importance for substrate specificity. The substitution of non-aromatic alanine residue in place of Y159 completely disrupted the catalytic activity for indole-3-acetonitrile. The present study reports several uncharacterized nitrilases which have not been reported so far for their role in the biodegradation of pollutants, xenobiotics which could find applications in industries.

scholarly journals Structural Insight into a Yeast Maltase—The BaAG2 from Blastobotrys adeninivorans with Transglycosylating Activity

2021 ◽  
Vol 7 (10) ◽  
pp. 816
Author(s):  
Karin Ernits ◽  
Christian Kjeldsen ◽  
Karina Persson ◽  
Eliis Grigor ◽  
Tiina Alamäe ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Temperature Tolerance ◽  
Catalytic Triad ◽  
Arxula Adeninivorans ◽  
Transglycosylation Activity ◽  
Potential Binding ◽  
Active Site Cleft ◽  
Prebiotic Oligosaccharides

An early-diverged yeast, Blastobotrys (Arxula) adeninivorans (Ba), has biotechnological potential due to nutritional versatility, temperature tolerance, and production of technologically applicable enzymes. We have biochemically characterized from the Ba type strain (CBS 8244) the GH13-family maltase BaAG2 with efficient transglycosylation activity on maltose. In the current study, transglycosylation of sucrose was studied in detail. The chemical entities of sucrose-derived oligosaccharides were determined using nuclear magnetic resonance. Several potentially prebiotic oligosaccharides with α-1,1, α-1,3, α-1,4, and α-1,6 linkages were disclosed among the products. Trisaccharides isomelezitose, erlose, and theanderose, and disaccharides maltulose and trehalulose were dominant transglycosylation products. To date no structure for yeast maltase has been determined. Structures of the BaAG2 with acarbose and glucose in the active center were solved at 2.12 and 2.13 Å resolution, respectively. BaAG2 exhibited a catalytic domain with a (β/α)8-barrel fold and Asp216, Glu274, and Asp348 as the catalytic triad. The fairly wide active site cleft contained water channels mediating substrate hydrolysis. Next to the substrate-binding pocket an enlarged space for potential binding of transglycosylation acceptors was identified. The involvement of a Glu (Glu309) at subsite +2 and an Arg (Arg233) at subsite +3 in substrate binding was shown for the first time for α-glucosidases.

THE CATALYTIC TRIAD RESIDUES (HIS221, ASP269, SER365) AND THE BINDING POCKET RESIDUE (ASP359) IN FACTOR IXBm ELSINORE (IXBmLE) ARE NOT ALTERED

1987 ◽  
Author(s):  
S G Spitzer ◽  
P Usharani ◽  
A D Roser ◽  
C K Kasper ◽  
S G Bajaj
Keyword(s):  
Catalytic Domain ◽  
Antithrombin Iii ◽  
Genomic Library ◽  
Indirect Evidence ◽  
Binding Pocket ◽  
Catalytic Triad ◽  
Factor Vii ◽  
Factor X ◽  
Liver Cdna Library ◽  
Synthetic Oligonucleotides

Previous studies suggested that the defect in IXBmLE (a nonfunctional variant of human IX) is either in the catalytic triad or at the site(s) of interaction with the macromolecular substrates (antithrombin III, factor VII or factor X). To distinguish between these possibilities, we isolated a complete IX cDNA clone from a human liver cDNA library. We also constructed a genomic library (in phage EMBL3) using DNA of the BmLE patient. The library was screened with normal IX cDNA and with synthetic oligonucleotides. The positive clones containing the exons coding for IX were plaque purified. Two clones which contained the coding sequence of the catalytic domain, i.e., His221 (exon VII), and Asp269, Asp359, and Ser365 (exon VIII) were selected for further studies. The phage containing exon VIII was first digested with Sail and EcoRI and a 2-Kb fragment, which hybridized with the segment of cDNA containing exon VIII, was gel purified. The 2-Kb fragment was further digested and the subfragments were cloned into M13; the length and direction of the fragments used in sequencing are shown below:The phage containing exon VII was digested with PstI and SalI, and a 1-Kb fragment that hybridized with the 19-mer His221 probe was subcloned into M13 phage for sequencing. The sequence starting with residue Vall96 through residue Arg403 was found to be normal. Thus, none of the residues in the catalytic domain of IXBmLE are different from that of normal IX. These data provide strong indirect evidence that the noncatalytic aminoterminal portion of IX plays a significant role in the structural recognition of the macromolecular substrates. The sequence of this region of IXBmLE should provide information about the putative residue(s) essential for this recognition.

scholarly journals Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Youngchang Kim ◽  
Jacek Wower ◽  
Natalia Maltseva ◽  
Changsoo Chang ◽  
Robert Jedrzejczak ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Domain ◽  
Binding Pocket ◽  
Innate Immune ◽  
Manganese Ions ◽  
Present Evidence ◽  
Transition State Analog ◽  
Cell Assays ◽  
Catalytic Mechanisms ◽  
Endoribonuclease Activity

AbstractSARS-CoV-2 Nsp15 is a uridine-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family that is highly conserved in coronaviruses. As endoribonuclease activity seems to be responsible for the interference with the innate immune response, Nsp15 emerges as an attractive target for therapeutic intervention. Here we report the first structures with bound nucleotides and show how the enzyme specifically recognizes uridine moiety. In addition to a uridine site we present evidence for a second base binding site that can accommodate any base. The structure with a transition state analog, uridine vanadate, confirms interactions key to catalytic mechanisms. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. This acquired knowledge was instrumental in identifying Tipiracil, an FDA approved drug that is used in the treatment of colorectal cancer, as a potential anti-COVID-19 drug. Using crystallography, biochemical, and whole-cell assays, we demonstrate that Tipiracil inhibits SARS-CoV-2 Nsp15 by interacting with the uridine binding pocket in the enzyme’s active site. Our findings provide new insights for the development of uracil scaffold-based drugs.

scholarly journals Structure of transmembrane prolyl 4-hydroxylase reveals unique organization of EF and dioxygenase domains

2020 ◽  
Author(s):  
Matti Myllykoski ◽  
Aleksi Sutinen ◽  
M. Kristian Koski ◽  
Juha P. Kallio ◽  
Arne Raasakka ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Active Site ◽  
Catalytic Domain ◽  
Substrate Binding ◽  
Hypoxia Inducible Factor ◽  
3D Structure ◽  
Binding Motifs ◽  
X Ray Crystallography ◽  
Binding Cavity ◽  
Negative Surface

AbstractProlyl 4-hydroxylases (P4Hs) catalyze post-translational hydroxylation of peptidyl proline residues. In addition to collagen P4Hs and hypoxia-inducible factor P4Hs, a poorly characterized endoplasmic reticulum (ER)-localized transmembrane prolyl 4-hydroxylase (P4H-TM) is found in animals. P4H-TM variants are associated with the familiar neurological HIDEA syndrome. Here, the 3D structure of the soluble human P4H-TM was solved using X-ray crystallography. The structure revealed an EF-domain with two Ca2+-binding motifs inserted to the catalytic domain. A substrate-binding cavity was formed between the EF-domain and the catalytic domain. The active site contained bound Fe2+ and N-oxalylglycine. Comparison to homologous structures complexed with peptide substrates showed that the substrate interacting residues and the lid structure that folds over the substrate are conserved in P4H-TM. Differences to homologs were found in the extensive loop structures that surround the substrate-binding cavity and generate a negative surface charge. Ca2+-binding affinity of P4H-TM was determined to be within the range of physiological Ca2+ concentration in the ER. The proximity of the EF-domain to the active site suggests that Ca2+-binding is relevant to the catalytic activity. P4H-TM was found both as a monomer and a dimer in solution, but the monomer-dimer equilibrium was not regulated by Ca2+. The solved 3D structure suggests that the HIDEA variants cause loss of P4H-TM function. In conclusion, P4H-TM shares key structural elements with the known P4Hs while possessing a unique property among the 2-oxoglutarate-dependent dioxygenases having an EF-domain and a catalytic activity potentially regulated by Ca2+.

scholarly journals The Bacillus subtilis Signaling Protein SpoIVB Defines a New Family of Serine Peptidases

2002 ◽  
Vol 184 (1) ◽  
pp. 191-199 ◽  
Author(s):  
Ngo T. Hoa ◽  
James A. Brannigan ◽  
Simon M. Cutting
Keyword(s):  
Bacillus Subtilis ◽  
Active Site ◽  
Catalytic Domain ◽  
Active Form ◽  
Catalytic Triad ◽  
Active Site Residues ◽  
Late Gene Expression ◽  
Serine Peptidase ◽  
New Family ◽  
Zinc Metalloprotease

ABSTRACT The protein SpoIVB plays a key role in signaling in the ςK checkpoint of Bacillus subtilis. This regulatory mechanism coordinates late gene expression during development in this organism and we have recently shown SpoIVB to be a serine peptidase. SpoIVB signals by transiting a membrane, undergoing self-cleavage, and then by an unknown mechanism activating a zinc metalloprotease, SpoIVFB, which cleaves pro-ςK to its active form, ςK, in the outer mother cell chamber of the developing cell. In this work we have characterized the serine peptidase domain of SpoIVB. Alignment of SpoIVB with homologues from other spore formers has allowed site-specific mutagenesis of all potential active site residues within the peptidase domain. We have defined the putative catalytic domain of the SpoIVB serine peptidase as a 160-amino-acid residue segment at the carboxyl terminus of the protein. His236 and Ser378 are the most important residues for proteolysis, with Asp363 being the most probable third member of the catalytic triad. In addition, we have shown that mutations at residues Asn290 and His394 lead to delayed signaling in the ςK checkpoint. The active site residues suggest that SpoIVB and its homologues from other spore formers are members of a new family of serine peptidases of the trypsin superfamily.

An Enzyme-Linked Immunosorbent Assay for Urokinase-Type Plasminogen Activator (u-PA) and Mutants and Chimeras Containing the Serine Protease Domain of u-PA

1992 ◽  
Vol 67 (01) ◽  
pp. 095-100 ◽  
Author(s):  
Paul J Declerck ◽  
Leen Van Keer ◽  
Maria Verstreken ◽  
Désiré Collen
Keyword(s):  
Serine Protease ◽  
Plasminogen Activator ◽  
Active Site ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Protease Domain ◽  
Serine Protease Domain ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Normal Human

SummaryAn enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 ± 0.6 ng/ml (mean ± SD, n = 27).The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising A A 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with α2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity. When purified scu-PA-32k was added to pooled normal human plasma at final concentrations ranging from 20 to 1,000 ng/ml, recoveries in the ELISA were between 84 and 110%.The assay was successfully applied for the quantitation of pharmacological levels of scu-PA and t-PA(AA87_274)/scu-PA(AA138-411) in plasma during experimental thrombolysis in baboons.Thus the present ELISA, which is specifically dependent on the presence of the serine protease part of u-PA, is useful for measurement of a wide variety of variants and chimeras of u-PA which are presently being developed for improved thrombolytic therapy.

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