scholarly journals Structural Analyses on the Deamidation of N-Terminal Asn in the Human N-Degron Pathway

Biomolecules ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 163 ◽  
Author(s):  
Joon Sung Park ◽  
Jae-Young Lee ◽  
Yen Thi Kim Nguyen ◽  
Nae-Won Kang ◽  
Eun Kyung Oh ◽  
...  
Keyword(s):  
High Specificity ◽  
Catalytic Triad ◽  
Structural Basis ◽  
Side Chain ◽  
Peptide Substrates ◽  
Hydrophobic Residues ◽  
Structural Principles ◽  
Peptide Complexes ◽  
Biochemical Analyses

The N-degron pathway is a proteolytic system in which a single N-terminal amino acid acts as a determinant of protein degradation. Especially, degradation signaling of N-terminal asparagine (Nt-Asn) in eukaryotes is initiated from its deamidation by N-terminal asparagine amidohydrolase 1 (NTAN1) into aspartate. Here, we have elucidated structural principles of deamidation by human NTAN1. NTAN1 adopts the characteristic scaffold of CNF1/YfiH-like cysteine hydrolases that features an α-β-β sandwich structure and a catalytic triad comprising Cys, His, and Ser. In vitro deamidation assays using model peptide substrates with varying lengths and sequences showed that NTAN1 prefers hydrophobic residues at the second-position. The structures of NTAN1-peptide complexes further revealed that the recognition of Nt-Asn is sufficiently organized to produce high specificity, and the side chain of the second-position residue is accommodated in a hydrophobic pocket adjacent to the active site of NTAN1. Collectively, our structural and biochemical analyses of the substrate specificity of NTAN1 contribute to understanding the structural basis of all three amidases in the eukaryotic N-degron pathway.

scholarly journals On the substrate specificity of bacterial DD-peptidases: evidence from two series of peptidoglycan-mimetic peptides

2003 ◽  
Vol 373 (3) ◽  
pp. 949-955 ◽  
Author(s):  
John W. ANDERSON ◽  
Suara A. ADEDIRAN ◽  
Paulette CHARLIER ◽  
Martine NGUYEN-DISTÈCHE ◽  
Jean-Marie FRÈRE ◽  
...  
Keyword(s):  
Molecular Mass ◽  
High Activity ◽  
High Specificity ◽  
Structural Variable ◽  
Structural Element ◽  
Peptide Substrates ◽  
Current State ◽  
The One ◽  
Species Specific

The reactions between bacterial DD-peptidases and β-lactam antibiotics have been studied for many years. Less well understood are the interactions between these enzymes and their natural substrates, presumably the peptide moieties of peptidoglycan. In general, remarkably little activity has previously been demonstrated in vitro against potential peptide substrates, although in many cases the peptides employed were non-specific and not homologous with the relevant peptidoglycan. In this paper, the specificity of a panel of DD-peptidases against elements of species-specific d-alanyl-d-alanine peptides has been assessed. In two cases, those of soluble, low-molecular-mass DD-peptidases, high activity against the relevant peptides has been demonstrated. In these cases, the high specificity is towards the free N-terminus of the peptidoglycan fragment. With a number of other enzymes, particularly high-molecular-mass DD-peptidases, little or no activity against these peptides was observed. In separate experiments, the reactivity of the enzymes against the central, largely invariant, peptide stem was examined. None of the enzymes surveyed showed high activity against this structural element although weak specificity in the expected direction towards the one structural variable (d-γGln versus d-γGlu) was observed. The current state of understanding of the activity of these enzymes in vitro is discussed.

New Chromogenic Substrates for Thrombin with Increased Specificity

1986 ◽  
Vol 56 (02) ◽  
pp. 155-159 ◽  
Author(s):  
H-J Kolde ◽  
R Eberle ◽  
H Hebert ◽  
N Heimburger
Keyword(s):  
Factor Xa ◽  
High Specificity ◽  
Side Chain ◽  
Colorimetric Determination ◽  
Chromogenic Substrates ◽  
Peptide Substrates ◽  
New Approach ◽  
Order Of Magnitude ◽  
Derivatives Of

SummaryChromogenic substrates for thrombin with high specificity are necessary for several functional assays, especially for the performance of photometric PT and APTT. A new approach to improve the specificity of chromogenic peptide substrates is made coupling tripeptide sequences selective for thrombin to derivatives of 5-amino-2-nitro benzoic acid (ANBA). Especially when the chromophore’s side chain is substituted by amines or amino acids hydrolysis rates by other enzymes like kallikrein, plasmin or factor Xa are decreased significantly compared to corresponding para-nitroanilides of the same amino acid sequence. On the other hand, most of these compounds are still sensitive thrombin substrates. KM-values for thrombin and other enzymes are in the same order of magnitude as corresponding pNA-peptides. ANBA peptide substrates may be useful to measure thrombin selectively in a mixture of other proteases like plasmin, factor Xa or kallikrein and for the colorimetric determination of PT and APTT.

scholarly journals Structural and biochemical analyses of aClostridium perfringenssortase D transpeptidase

2015 ◽  
Vol 71 (7) ◽  
pp. 1505-1513 ◽  
Author(s):  
Randy Suryadinata ◽  
Shane A. Seabrook ◽  
Timothy E. Adams ◽  
Stewart D. Nuttall ◽  
Thomas S. Peat
Keyword(s):  
Catalytic Activity ◽  
Antimicrobial Agents ◽  
Catalytic Triad ◽  
Covalent Attachment ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
C Terminus ◽  
Arginine Residues ◽  
Biochemical Analyses

The assembly and anchorage of various pathogenic proteins on the surface of Gram-positive bacteria is mediated by the sortase family of enzymes. These cysteine transpeptidases catalyze a unique sorting signal motif located at the C-terminus of their target substrate and promote the covalent attachment of these proteins onto an amino nucleophile located on another protein or on the bacterial cell wall. Each of the six distinct classes of sortases displays a unique biological role, with sequential activation of multiple sortases often observed in many Gram-positive bacteria to decorate their peptidoglycans. Less is known about the members of the class D family of sortases (SrtD), but they have a suggested role in spore formation in an oxygen-limiting environment. Here, the crystal structure of the SrtD enzyme fromClostridium perfringenswas determined at 1.99 Å resolution. Comparative analysis of theC. perfringensSrtD structure reveals the typical eight-stranded β-barrel fold observed in all other known sortases, along with the conserved catalytic triad consisting of cysteine, histidine and arginine residues. Biochemical approaches further reveal the specifics of the SrtD catalytic activityin vitro, with a significant preference for the LPQTGS sorting motif. Additionally, the catalytic activity of SrtD is most efficient at 316 K and can be further improved in the presence of magnesium cations. SinceC. perfringensspores are heat-resistant and lead to foodborne illnesses, characterization of the spore-promoting sortase SrtD may lead to the development of new antimicrobial agents.

scholarly journals Necator americanus Ancylostoma Secreted Protein-2 (Na-ASP-2) Binds an Ascaroside (ascr#3) in Its Fatty Acid Binding Site

2020 ◽  
Vol 8 ◽  
Author(s):  
Ola El Atab ◽  
Rabih Darwiche ◽  
Nathanyal J. Truax ◽  
Roger Schneiter ◽  
Kenneth G. Hull ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Small Molecules ◽  
Binding Sites ◽  
Structural Basis ◽  
Side Chain ◽  
Secreted Protein ◽  
Competition Assay ◽  
Fatty Acid Binding ◽  
Binding Cavity

During their infective stages, hookworms release excretory-secretory (E-S) products, small molecules, and proteins to help evade and suppress the host's immune system. Small molecules found in E-S products of mammalian hookworms include nematode derived metabolites like ascarosides, which are composed of the sugar ascarylose linked to a fatty acid side chain. The most abundant proteins found in hookworm E-S products are members of the protein family known as Ancylostoma secreted protein (ASP). In this study, two ascarosides and their fatty acid moieties were synthesized and tested for in vitro binding to Na-ASP-2 using both a ligand competition assay and microscale thermophoresis. The fatty acid moieties of both ascarosides tested and ascr#3, an ascaroside found in rat hookworm E-S products, bind to Na-ASP-2's palmitate binding cavity. These molecules were confirmed to bind to the palmitate but not the sterol binding sites. An ascaroside, oscr#10, which is not found in hookworm E-S products, does not bind to Na-ASP-2. More studies are required to determine the structural basis of ascarosides binding by Na-ASP-2 and to understand the physiological significance of these observations.

scholarly journals Escherichia coli DegP Protease Cleaves between Paired Hydrophobic Residues in a Natural Substrate: the PapA Pilin

2002 ◽  
Vol 184 (20) ◽  
pp. 5762-5771 ◽  
Author(s):  
C. Hal Jones ◽  
Paul Dexter ◽  
Amy K. Evans ◽  
Christopher Liu ◽  
Scott J. Hultgren ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein A ◽  
Pdz Domains ◽  
Peptide Arrays ◽  
Peptide Substrates ◽  
Hydrophobic Residues ◽  
E Coli ◽  
Hydrophobic Amino Acids ◽  
Carboxyl Terminal

ABSTRACT The DegP protein, a multifunctional chaperone and protease, is essential for clearance of denatured or aggregated proteins from the inner-membrane and periplasmic space in Escherichia coli. To date, four natural targets for DegP have been described: colicin A lysis protein, pilin subunits and MalS from E. coli, and high-molecular-weight adherence proteins from Haemophilus influenzae. In vitro, DegP has shown weak protease activity with casein and several other nonnative substrates. We report here the identification of the major pilin subunit of the Pap pilus, PapA, as a natural DegP substrate and demonstrate binding and proteolysis of this substrate in vitro. Using overlapping peptide arrays, we identified three regions in PapA that are preferentially cleaved by DegP. A 7-mer peptide was found to be a suitable substrate for cleavage by DegP in vitro. In vitro proteolysis of model peptide substrates revealed that cleavage is dependent upon the presence of paired hydrophobic amino acids; moreover, cleavage was found to occur between the hydrophobic residues. Finally, we demonstrate that the conserved carboxyl-terminal sequence in pilin subunits, although not a cleavage substrate for DegP, activates the protease and we propose that the activating peptide is recognized by DegP's PDZ domains.

scholarly journals Structural basis for catalysis and ubiquitin recognition by theSevere acute respiratory syndrome coronaviruspapain-like protease

2014 ◽  
Vol 70 (2) ◽  
pp. 572-581 ◽  
Author(s):  
Chi-Yuan Chou ◽  
Hsing-Yi Lai ◽  
Hung-Yi Chen ◽  
Shu-Chun Cheng ◽  
Kai-Wen Cheng ◽  
...  
Keyword(s):  
Main Chain ◽  
Kinetic Studies ◽  
Cysteine Proteases ◽  
Catalytic Triad ◽  
Proteolytic Processing ◽  
Structural Basis ◽  
Hydrophilic Interactions ◽  
C Terminus ◽  
Catalytic Cleft

Papain-like protease (PLpro) is one of two cysteine proteases involved in the proteolytic processing of the polyproteins ofSevere acute respiratory syndrome coronavirus(SARS-CoV). PLproalso shows significantin vitrodeubiquitinating and de-ISGylating activities, although the detailed mechanism is still unclear. Here, the crystal structure of SARS-CoV PLproC112S mutant in complex with ubiquitin (Ub) is reported at 1.4 Å resolution. The Ub core makes mostly hydrophilic interactions with PLpro, while the Leu-Arg-Gly-Gly C-terminus of Ub is located in the catalytic cleft of PLpro, mimicking the P4–P1 residues and providing the first atomic insights into its catalysis. One of the O atoms of the C-terminal Gly residue of Ub is located in the oxyanion hole consisting of the main-chain amides of residues 112 and 113. Mutations of residues in the PLpro–Ub interface lead to reduced catalytic activity, confirming their importance for Ub binding and/or catalysis. The structure also revealed anN-cyclohexyl-2-aminethanesulfonic acid molecule near the catalytic triad, and kinetic studies suggest that this binding site is also used by other PLproinhibitors. Overall, the structure provides a foundation for understanding the molecular basis of coronaviral PLprocatalysis.

scholarly journals Structural basis of inactivation of Ras and Rap1 small GTPases by Ras/Rap1-specific endopeptidase from the sepsis-causing pathogen Vibrio vulnificus

2018 ◽  
Vol 293 (47) ◽  
pp. 18110-18122 ◽  
Author(s):  
Song Yee Jang ◽  
Jungwon Hwang ◽  
Byoung Sik Kim ◽  
Eun-Young Lee ◽  
Byung-Ha Oh ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Vibrio Vulnificus ◽  
Bacterial Pathogen ◽  
Small Gtpases ◽  
Structural Basis ◽  
Membrane Localization ◽  
Host Cytoplasm ◽  
Biochemical Analyses ◽  
Α Helix

Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are secreted by Gram-negative bacteria and function as primary virulence-promoting macromolecules that deliver multiple cytopathic and cytotoxic effector domains into the host cytoplasm. Among these effectors, Ras/Rap1-specific endopeptidase (RRSP) catalyzes the sequence-specific cleavage of the Switch I region of the cellular substrates Ras and Rap1 that are crucial for host innate immune defenses during infection. To dissect the molecular basis underpinning RRSP-mediated substrate inactivation, we determined the crystal structure of an RRSP from the sepsis-causing bacterial pathogen Vibrio vulnificus (VvRRSP). Structural and biochemical analyses revealed that VvRRSP is a metal-independent TIKI family endopeptidase composed of an N-terminal membrane-localization and substrate-recruitment domain (N lobe) connected via an inter-lobe linker to the C-terminal active site–coordinating core β-sheet–containing domain (C lobe). Structure-based mutagenesis identified the 2His/2Glu catalytic residues in the core catalytic domain that are shared with other TIKI family enzymes and that are essential for Ras processing. In vitro KRas cleavage assays disclosed that deleting the N lobe in VvRRSP causes complete loss of enzymatic activity. Endogenous Ras cleavage assays combined with confocal microscopy analysis of HEK293T cells indicated that the N lobe functions both in membrane localization via the first α-helix and in substrate assimilation by altering the functional conformation of the C lobe to facilitate recruitment of cellular substrates. Collectively, these results indicate that RRSP is a critical virulence factor that robustly inactivates Ras and Rap1 and augments the pathogenicity of invading bacteria via the combined effects of its N and C lobes.

scholarly journals Necator americanus Ancylostoma secreted protein-2 (Na-ASP-2) selectively binds an ascaroside (ascr#3)

2020 ◽  
Author(s):  
Ola El Atab ◽  
Rabih Darwiche ◽  
Nathanyal J. Truax ◽  
Roger Schneiter ◽  
Kenneth G. Hull ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Palmitic Acid ◽  
Small Molecules ◽  
Structural Basis ◽  
Side Chain ◽  
Secreted Protein ◽  
Pathogenesis Related ◽  
Binding Cavity ◽  
Antigen 5

AbstractDuring their infective stages, hookworms release excretory-secretory (E-S) products, including small molecules and proteins, to help evade and suppress the host’s immune system. Small molecules found in E-S products of mammalian hookworms include nematode derived metabolites like ascarosides, which are composed of the sugar ascarylose linked to a fatty acid side chain. Ascarosides play vital roles in signaling, development, reproduction, and survival. The most abundant proteins found in hookworm E-S products are members of the protein family known as Ancylostoma secreted protein (ASP). ASP belongs to the SCP/TAPS (sperm-coating protein / Tpx / antigen 5 / pathogenesis related-1 / Sc7) superfamily of proteins, members of which have previously been shown to bind to eicosanoids and fatty acids. These molecules are structurally similar to the fatty acid moieties of ascarosides. The objective of this study was to determine if the hookworm ASP; N. americanus Ancylostoma secreted protein 2 (Na-ASP-2) binds to the ascarosides or their fatty acid moieties. We describe investigations of our hypothesis that there is a functional relationship between the major secreted proteins and signaling small molecules found in hookworm E-S products. To accomplish this, several ascarosides and their fatty acid moieties were synthesized and tested for in vitro binding to Na-ASP-2 using a ligand competition assay and microscale thermophoresis. Our results reveal that the fatty acid moieties of the ascarosides, bind specifically to the palmitic acid binding cavity of Na-ASP-2. Additionally, ascr#3, an ascaroside that is present in mammalian hookworm E-S products binds to the palmitic acid binding cavity of Na-ASP-2, whereas oscr#10 which is not found in hookworm E-S products does not bind. Future studies are required to determine the structural basis of ascaroside binding by Na-ASP-2 and to understand the physiological significance of these observations.

Structural basis of a novel activity of bacterial 6-pyruvoyltetrahydropterin synthase homologues distinct from mammalian 6-pyruvoyltetrahydropterin synthase activity

2014 ◽  
Vol 70 (5) ◽  
pp. 1212-1223 ◽  
Author(s):  
Kyung Hye Seo ◽  
Ningning Zhuang ◽  
Young Shik Park ◽  
Ki Hun Park ◽  
Kon Ho Lee
Keyword(s):  
Active Site ◽  
Essential Role ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Side Chain ◽  
Wild Type ◽  
Specific Enzyme ◽  
Aromatic Residues

Escherichia coli6-carboxytetrahydropterin synthase (eCTPS), a homologue of 6-pyruvoyltetrahydropterin synthase (PTPS), possesses a much stronger catalytic activity to cleave the side chain of sepiapterinin vitrocompared with genuine PTPS activity and catalyzes the conversion of dihydroneopterin triphosphate to 6-carboxy-5,6,7,8-tetrahydropterinin vivo. Crystal structures of wild-type apo eCTPS and of a Cys27Ala mutant eCTPS complexed with sepiapterin have been determined to 2.3 and 2.5 Å resolution, respectively. The structures are highly conserved at the active site and the Zn2+binding site. However, comparison of the eCTPS structures with those of mammalian PTPS homologues revealed that two specific residues, Trp51 and Phe55, that are not found in mammalian PTPS keep the substrate bound by stacking it with their side chains. Replacement of these two residues by site-directed mutagenesis to the residues Met and Leu, which are only found in mammalian PTPS, converted eCTPS to the mammalian PTPS activity. These studies confirm that these two aromatic residues in eCTPS play an essential role in stabilizing the substrate and in the specific enzyme activity that differs from the original PTPS activity. These aromatic residues Trp51 and Phe55 are a key signature of bacterial PTPS enzymes that distinguish them from mammalian PTPS homologues.

A Physiologic Regulator of Collagen-Induced Platelet Aggregation: Inhibition by Clq

1981 ◽  
Vol 45 (02) ◽  
pp. 110-115 ◽  
Author(s):  
György Csákó ◽  
Eva A Suba
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Platelet Reactivity ◽  
High Specificity ◽  
Turbidimetric Method ◽  
Specific Manner ◽  
Aggregation Inhibition ◽  
Different Tissues

SummaryPlatelet aggregations were studied by a turbidimetric method in citrated human platelet-rich plasmas (PRP) in vitro. Human Clq inhibited the aggregations caused by collagens derived from different tissues and species. Clq was needed by weight in comparable quantities to collagen for neutralizing the aggregating effect. The dependence of the inhibitory reaction on the preincubation of platelets with Clq and the differences in the occurrence of aggregating substances in supernatants of PRP triggered with collagen in the presence or absence of Clq, confirmed that Clq exerts its effect by preventing fixation of collagen to platelets. In addition, the high specificity of the inhibitory action of Clq for collagen-induced platelet aggregation was demonstrated by results obtained for testing a variety of aggregating agents in combination with Clq and/or collagen.Since normal concentrations of Clq in the blood are in the range of inhibitory doses of Clq for collagen-induced platelet aggregations in vitro and upon activation of complement Clq is known to dissociate from Cl, it is proposed that Clq may participate in a highly specific manner in regulating platelet reactivity to collagen in vivo.

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