Evidence that the conformation of unliganded human plasminogen is maintained via an intramolecular interaction between the lysine-binding site of kringle 5 and the N-terminal peptide

Human Glu-plasminogen adopts at least three conformations that provide a means for regulating the specificity of its activation in vivo. It has been proposed previously that the closed (α) conformation of human Glu-plasminogen is maintained through physical interaction of the kringle 5 domain and a lysine residue within the N-terminal peptide (NTP). To examine this hypothesis, site-directed mutagenesis was used to generate variant proteins containing substitutions either for aspartic acid residues within the anionic centre of the kringle 5 domain or for conserved lysine residues within the NTP. Size-exclusion HPLC and rates of plasminogen activation by urokinase-type plasminogen activator were used to determine the conformational states of these variants. Variants with substitutions within the kringle 5 lysine-binding site demonstrated extended conformations, as did variants with alanine substitutions for Lys50 and Lys62. In contrast, molecules in which NTP residues Lys20 or Lys33 were replaced were shown to adopt closed conformations. We conclude that the lysine-binding site of kringle 5 is involved in maintaining the closed conformation of human Glu-plasminogen via an interaction with the NTP, probably through Lys50 and/or Lys62. These conclusions advance the current model for the initial stages of fibrinolysis during which fibrin is thought to compete with the NTP for the kringle 5 lysine-binding site.

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Three monoclonal antibodies against the serpin protease nexin-1 prevent protease translocation

Thrombosis and Haemostasis ◽

10.1160/th13-04-0340 ◽

2014 ◽

Vol 111 (01) ◽

pp. 29-40 ◽

Cited By ~ 4

Author(s):

Tina Kousted ◽

Karsten Skjoedt ◽

Steen Petersen ◽

Claus Koch ◽

Lars Vitved ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Inhibitory Activity ◽

Site Directed Mutagenesis ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Reactive Centre Loop ◽

Α Helix ◽

Protease Nexin ◽

Protease Inhibitory Activity

SummaryProtease nexin-1 (PN-1) belongs to the serpin family and is an inhibitor of thrombin, plasmin, urokinase-type plasminogen activator, and matriptase. Recent studies have suggested PN-1 to play important roles in vascular-, neuro-, and tumour-biology. The serpin inhibitory mechanism consists of the serpin presenting its so-called reactive centre loop as a substrate to its target protease, resulting in a covalent complex with the inactivated enzyme. Previously, three mechanisms have been proposed for the inactivation of serpins by monoclonal antibodies: steric blockage of protease recognition, conversion to an inactive conformation or induction of serpin substrate behaviour. Until now, no inhibitory antibodies against PN-1 have been thoroughly characterised. Here we report the development of three monoclonal antibodies binding specifically and with high affinity to human PN-1. The antibodies all abolish the protease inhibitory activity of PN-1. In the presence of the antibodies, PN-1 does not form a complex with its target proteases, but is recovered in a reactive centre cleaved form. Using site-directed mutagenesis, we mapped the three overlapping epitopes to an area spanning the gap between the loop connecting α-helix F with β-strand 3A and the loop connecting α-helix A with β-strand 1B. We conclude that antibody binding causes a direct blockage of the final critical step of protease translocation, resulting in abortive inhibition and premature release of reactive centre cleaved PN-1. These new antibodies will provide a powerful tool to study the in vivo role of PN-1’s protease inhibitory activity.

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Kinetic studies on the effect of heparin and fibrin on plasminogen activators

Biochemical Journal ◽

10.1042/bj2490077 ◽

1988 ◽

Vol 249 (1) ◽

pp. 77-81 ◽

Cited By ~ 36

Author(s):

R Fears

Keyword(s):

Binding Site ◽

Chondroitin Sulphate ◽

Kinetic Studies ◽

Model Systems ◽

Tissue Type ◽

Type Plasminogen Activator ◽

Pentosan Polysulphate ◽

A Chain ◽

Activator Complex

1. Possible interactions between fibrin(ogen) and heparin in the control of plasminogen activation were studied in model systems using the thrombolytic agents tissue-type plasminogen activator (t-PA), urokinase and streptokinase.plasminogen activator complex and the substrates Glu- and Lys-plasminogen. 2. Both t-PA and urokinase activities were promoted by heparin and by pentosan polysulphate, but not by chondroitin sulphate or hyaluronic acid. The effect was on Km. 3. In the presence of soluble fibrin (and its mimic, CNBr-digested fibrinogen) the effect of heparin on t-PA was attenuated, although not abolished. In studies using a monoclonal antibody and 6-aminohexanoic acid, it was found that heparin and fibrin did not seem to share a binding site on t-PA. 4. The activity of t-PA B-chain was unaffected by heparin, so the binding site is located on the A-chain of t-PA (and urokinase). 5. Fibrin potentiated the activity of heparin on urokinase. The activity of streptokinase.plasminogen was unaffected by heparin whether or not fibrin was present. 6. If these influences of heparin and fibrin also occur in vivo, then, in the presence of heparin, the relative fibrin enhancement of t-PA will be diminished and the likelihood of systemic activation by t-PA is increased.

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Quantitative proteomics reveals extensive lysine ubiquitination in the Arabidopsis root proteome and uncovers novel transcription factor stability states

10.1101/2021.01.07.425780 ◽

2021 ◽

Author(s):

Gaoyuan Song ◽

Damilola Olatunji ◽

Christian Montes ◽

Natalie M Clark ◽

Yunting Pu ◽

...

Keyword(s):

Posttranslational Modification ◽

Site Directed Mutagenesis ◽

Biological Processes ◽

Protein Activity ◽

Helix Loop Helix ◽

Primary Roots ◽

Lysine Residues ◽

Ubiquitin Proteasome ◽

The Stability

Protein activity, abundance, and stability can be regulated by posttranslational modification including ubiquitination. Ubiquitination is conserved among eukaryotes and plays a central role in modulating cellular function and yet we lack comprehensive catalogs of proteins that are modified by ubiquitin in plants. In this study, we describe an antibody-based approach to enrich peptides containing the di-glycine (diGly) remnant of ubiquitin and coupled that with isobaric labeling to enable quantification, from up to 16-multiplexed samples, for plant tissues. Collectively, we identified 7,130 diGly-modified lysine residues sites arising from 3,178 proteins in Arabidopsis primary roots. These data include ubiquitin proteasome dependent ubiquitination events as well as ubiquitination events associated with auxin treatment. Gene Ontology analysis indicated that ubiquitinated proteins are associated with numerous biological processes including hormone signaling, plant defense, protein homeostasis, and root morphogenesis. We determined the ubiquitinated lysine residues that directly regulate the stability of the transcription factors CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX 1 (CIB1), CIB1 LIKE PROTEIN 2 (CIL2), and SENSITIVE TO PROTON RHIZOTOXICITY (STOP1) using site directed mutagenesis and in vivo degradation assays. These comprehensive site-level ubiquitinome profiles provide a wealth of data for future studies related to modulation of biological processes mediated by this posttranslational modification in plants.

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Transcriptional Repression Mediated by LysR-Type Regulator CatR Bound at Multiple Binding Sites

Journal of Bacteriology ◽

10.1128/jb.180.9.2367-2372.1998 ◽

1998 ◽

Vol 180 (9) ◽

pp. 2367-2372 ◽

Cited By ~ 28

Author(s):

Sudha A. Chugani ◽

Matthew R. Parsek ◽

A. M. Chakrabarty

Keyword(s):

Binding Site ◽

Structural Gene ◽

Binding Sites ◽

Transcriptional Start Site ◽

Dnase I ◽

Site Directed Mutagenesis ◽

Dnase I Footprinting ◽

Gel Shift

ABSTRACT The catBCA operon of Pseudomonas putidaencodes enzymes involved in the catabolism of benzoate. Transcription of this operon requires the LysR-type transcriptional regulator CatR and an inducer molecule, cis,cis-muconate. Previous gel shift assays and DNase I footprinting have demonstrated that CatR occupies two adjacent sites proximal to thecatBCA promoter in the presence of the inducer. We report the presence of an additional binding site for CatR downstream of thecatBCA promoter within the catB structural gene. This site, called the internal binding site (IBS), extends from +162 to +193 with respect to the catB transcriptional start site and lies within the catB open reading frame. Gel shift analysis and DNase I footprinting determined that CatR binds to this site with low affinity. CatR binds cooperatively with higher affinity to the IBS in the presence of the two upstream binding sites. Parallel in vivo and in vitro studies were conducted to determine the role of the internal binding site. We measured β-galactosidase activity ofcatB-lacZ transcriptional fusions in vivo. Our results suggest a probable cis-acting repressor function for the internal binding site. Site-directed mutagenesis of the IBS verified this finding. The location of the IBS within the catBstructural gene, the cooperativity observed in footprinting studies, and phasing studies suggest that the IBS likely participates in the interaction of CatR with the upstream binding sites by looping out the intervening DNA.

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The Malonate Decarboxylase Operon of Acinetobacter calcoaceticus KCCM 40902 Is Regulated by Malonate and the Transcriptional Repressor MdcY

Journal of Bacteriology ◽

10.1128/jb.182.22.6382-6390.2000 ◽

2000 ◽

Vol 182 (22) ◽

pp. 6382-6390 ◽

Cited By ~ 8

Author(s):

Jae Hyung Koo ◽

Ick Hyun Cho ◽

Yu Sam Kim

Keyword(s):

Binding Site ◽

Transcriptional Start Site ◽

Regulatory Gene ◽

Transcriptional Repressor ◽

Acinetobacter Calcoaceticus ◽

Luria Bertani ◽

Site Directed Mutagenesis ◽

Palindromic Structure

ABSTRACT A regulatory gene-like open reading frame oriented oppositely to mdcL, coined mdcY, was found upstream from the structural genes of the mdcLMACDEGBH operon in Acinetobacter calcoaceticus KCCM 40902. To elucidate the function of this gene,mdcY was expressed in Escherichia coli, and the MdcY protein was purified to homogeneity. Its DNA binding activity and binding site were examined by gel retardation and footprinting assays in vitro and by site-directed mutagenesis of the binding sites in vivo. The regulator bound target DNA regardless of the presence of malonate, and the binding site was found centered at −65 relative to themdcL transcriptional start site and contains a 12-bp palindromic structure (5′-ATTGTA/TACAAT-3′). Using a promoter fusion to the reporter gene luc, we found that the promoter P mdcY is negatively regulated by MdcY independent of malonate. However, the promoter P mdcL recovered its activity in the presence of malonate. When mdcY was introduced into A. calcoaceticus KCCM 40902 in which the gene is inactivated by an IS3 family element, malonate decarboxylase was significantly repressed in cultures growing in acetate, succinate, or Luria-Bertani medium. However, in cells growing in malonate, malonate decarboxylase was induced, indicating that MdcY is a transcriptional repressor and that malonate or a product resulting from malonate metabolism should be the intracellular inducer of the mdcoperon.

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Identification of Two Mutations (F758W and F758Y) in the N -methyl-D-aspartate Receptor Glycine-binding Site that Selectively Prevent Competitive Inhibition by Xenon without Affecting Glycine Binding

Anesthesiology ◽

10.1097/aln.0b013e31825ada2e ◽

2012 ◽

Vol 117 (1) ◽

pp. 38-47 ◽

Cited By ~ 25

Author(s):

Scott P. Armstrong ◽

Paul J. Banks ◽

Thomas J. W. McKitrick ◽

Catharine H. Geldart ◽

Christopher J. Edge ◽

...

Keyword(s):

Amino Acids ◽

Nmda Receptor ◽

Binding Site ◽

Competitive Inhibition ◽

Site Directed Mutagenesis ◽

Glycine Site ◽

General Anesthetic ◽

Hek 293

Background Xenon is a general anesthetic with neuroprotective properties. Xenon inhibition at the glycine-binding site of the N-Methyl-D-aspartate (NMDA) receptor mediates xenon neuroprotection against ischemic injury in vitro. Here we identify specific amino acids important for xenon binding to the NMDA receptor, with the aim of finding silent mutations that eliminate xenon binding but leave normal receptor function intact. Methods Site-directed mutagenesis was used to mutate specific amino-acids in the GluN1 subunit of rat NMDA receptors. Mutant GluN1/GluN2A receptors were expressed in HEK 293 cells and were assessed functionally using patch-clamp electrophysiology. The responses of the mutant receptors to glycine and anesthetics were determined. Results Mutation of phenylalanine 758 to an aromatic tryptophan or tyrosine left glycine affinity unchanged, but eliminated xenon binding without affecting the binding of sevoflurane or isoflurane. Conclusions These findings confirm xenon binds to the glycine site of the GluN1 subunit of the NMDA receptor and indicate that interactions between xenon and the aromatic ring of the phenylalanine 758 residue are important for xenon binding. Our most important finding is that we have identified two mutations, F758W and F758Y, that eliminate xenon binding to the NMDA receptor glycine site without changing the glycine affinity of the receptor or the binding of volatile anesthetics. The identification of these selective mutations will allow knock-in animals to be used to dissect the mechanism(s) of xenon's neuroprotective and anesthetic properties in vivo.

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Identification of the glycosaminoglycan-binding site on the glycoprotein Erns of bovine viral diarrhoea virus by site-directed mutagenesis

Journal of General Virology ◽

10.1099/0022-1317-83-9-2153 ◽

2002 ◽

Vol 83 (9) ◽

pp. 2153-2159 ◽

Cited By ~ 23

Author(s):

Munir Iqbal ◽

John W. McCauley

Keyword(s):

Cell Surface ◽

Binding Site ◽

Bovine Viral Diarrhoea Virus ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Bovine Viral Diarrhoea ◽

Surface Binding ◽

C Terminus ◽

Diarrhoea Virus ◽

Lysine Residues

Bovine viral diarrhoea virus (BVDV) envelope glycoprotein Erns interacts with highly sulphated heparin-like glycosaminoglycans (GAGs) located on the cell surface as an early step in virus infection of cells. Site-directed mutagenesis of recombinant Erns was undertaken and analysis of mutants by heparin-affinity chromatography and cell surface binding showed that a cluster of basic amino acids (480KKLENKSK487) near the C terminus of Erns was essential for binding. Mutants with amino acid substitutions of lysine residues 481 and 485 in Erns reduced the binding of Erns to immobilized heparin and cellular GAGs but retained ribonuclease activity. In contrast to normal Erns, Erns that was unable to bind to cells also failed to inhibit BVDV infection of cells when the cells were pre-incubated with Erns. It is proposed that the cluster of basic residues (480KKLENKSK487) localized at the C-terminal end of Erns constitutes a GAG-binding site.

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A Genetic Dissection of Aip1p's Interactions Leads to a Model for Aip1p-Cofilin Cooperative Activities

Molecular Biology of the Cell ◽

10.1091/mbc.e05-10-0956 ◽

2006 ◽

Vol 17 (4) ◽

pp. 1971-1984 ◽

Cited By ~ 31

Author(s):

Michael G. Clark ◽

Joseph Teply ◽

Brian K. Haarer ◽

Susan C. Viggiano ◽

David Sept ◽

...

Keyword(s):

Binding Site ◽

Binding Sites ◽

Actin Filaments ◽

Random Mutagenesis ◽

Site Directed Mutagenesis ◽

Actin Binding ◽

Interacting Protein ◽

Actin Binding Site

Actin interacting protein 1 (Aip1p) and cofilin cooperate to disassemble actin filaments in vitro and are thought to promote rapid turnover of actin networks in vivo. The precise method by which Aip1p participates in these activities has not been defined, although severing and barbed-end capping of actin filaments have been proposed. To better describe the mechanisms and biological consequences of Aip1p activities, we undertook an extensive mutagenesis of AIP1 aimed at disrupting and mapping Aip1p interactions. Site-directed mutagenesis suggested that Aip1p has two actin binding sites, the primary actin binding site lies on the edge of its N-terminal β-propeller and a secondary actin binding site lies in a comparable location on its C-terminal β-propeller. Random mutagenesis followed by screening for separation of function mutants led to the identification of several mutants specifically defective for interacting with cofilin but still able to interact with actin. These mutants suggested that cofilin binds across the cleft between the two propeller domains, leaving the actin binding sites exposed and flanking the cofilin binding site. Biochemical, genetic, and cell biological analyses confirmed that the actin binding- and cofilin binding-specific mutants are functionally defective, whereas the genetic analyses further suggested a role for Aip1p in an early, internalization step of endocytosis. A complementary, unbiased molecular modeling approach was used to derive putative structures for the Aip1p-cofilin complex, the most stable of which is completely consistent with the mutagenesis data. We theorize that Aip1p-severing activity may involve simultaneous binding to two actin subunits with cofilin wedged between the two actin binding sites of the N- and C-terminal propeller domains.

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Insights into the Function of theN-Acetyltransferase SatA That Detoxifies Streptothricin inBacillus subtilisandBacillus anthracis

Applied and Environmental Microbiology ◽

10.1128/aem.03029-18 ◽

2019 ◽

Vol 85 (6) ◽

Cited By ~ 4

Author(s):

Rachel M. Burckhardt ◽

Jorge C. Escalante-Semerena

Keyword(s):

Binding Site ◽

Site Directed Mutagenesis ◽

Size Exclusion ◽

Titration Calorimetry ◽

Side Chains ◽

Missense Mutations ◽

Content Type ◽

C Terminus ◽

Exclusion Chromatography ◽

Domains Of Life

ABSTRACTAcylation of epsilon amino groups of lysyl side chains is a widespread modification of proteins and small molecules in cells of all three domains of life. Recently, we showed thatBacillus subtilisandBacillus anthracisencode the GCN5-relatedN-acetyltransferase (GNAT) SatA that can acetylate and inactivate streptothricin, which is a broad-spectrum antibiotic produced by actinomycetes in the soil. To determine functionally relevant residues ofB. subtilisSatA (BsSatA), a mutational screen was performed, highlighting the importance of a conserved area near the C terminus. Upon inspection of the crystal structure of theB. anthracisAmes SatA (BaSatA; PDB entry 3PP9), this area appears to form a pocket with multiple conserved aromatic residues; we hypothesized this region contains the streptothricin-binding site. Chemical and site-directed mutagenesis was used to introduce missense mutations intosatA, and the functionality of the variants was assessed using a heterologous host (Salmonella enterica). Results of isothermal titration calorimetry experiments showed that residue Y164 ofBaSatA was important for binding streptothricin. Results of size exclusion chromatography analyses showed that residue D160 was important for dimerization. Together, these data advance our understanding of how SatA interacts with streptothricin.IMPORTANCEThis work provides insights into how an abundant antibiotic found in soil is bound to the enzyme that inactivates it. This work identifies residues for the binding of the antibiotic and probes the contributions of substituting side chains for those in the native protein, providing information regarding hydrophobicity, size, and flexibility of the antibiotic binding site.

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