Regulation of a Phage Endolysin by Disulfide Caging

ABSTRACT In contrast to canonical phage endolysins, which require holin-mediated disruption of the membrane to gain access to attack the cell wall, signal anchor release (SAR) endolysins are secreted by the host sec system, where they accumulate in an inactive form tethered to the membrane by their N-terminal SAR domains. SAR endolysins become activated by various mechanisms upon release from the membrane. In its inactive form, the prototype SAR endolysin, LyzP1, of coliphage P1, has an active-site Cys covalently blocked by a disulfide bond; activation involves a disulfide bond isomerization driven by a thiol in the newly released SAR domain, unblocking the active-site Cys. Here, we report that Lyz103, the endolysin of Erwinia phage ERA103, is also a SAR endolysin. Although Lyz103 does not have a catalytic Cys, genetic evidence suggests that it also is activated by a thiol-disulfide isomerization triggered by a thiol in the SAR domain. In this case, the inhibitory disulfide in nascent Lyz103 is formed between cysteine residues flanking a catalytic glutamate, caging the active site. Thus, LyzP1 and Lyz103 define subclasses of SAR endolysins that differ in the nature of their inhibitory disulfide, and Lyz103 is the first enzyme found to be regulated by disulfide bond caging of its active site.

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Crystal structure of an acetylesterase fromTalaromyces cellulolyticusand the importance of a disulfide bond near the active site

FEBS Letters ◽

10.1016/j.febslet.2015.03.020 ◽

2015 ◽

Vol 589 (11) ◽

pp. 1200-1206 ◽

Cited By ~ 8

Author(s):

Masahiro Watanabe ◽

Harumi Fukada ◽

Hiroyuki Inoue ◽

Kazuhiko Ishikawa

Keyword(s):

Crystal Structure ◽

Disulfide Bond ◽

Active Site

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Chemical Modification of the Active Site of the Acetylcholine Receptor

The Journal of General Physiology ◽

10.1085/jgp.54.1.245 ◽

1969 ◽

Vol 54 (1) ◽

pp. 245-264 ◽

Cited By ~ 191

Author(s):

Arthur Karlin

Keyword(s):

Chemical Modification ◽

Acetylcholine Receptor ◽

Disulfide Bond ◽

Active Site ◽

Quaternary Ammonium ◽

Competitive Inhibitor ◽

Affinity Labels ◽

Electrophorus Electricus ◽

Depolarizing Agents ◽

Monoquaternary Ammonium

The receptor for acetylcholine in the subsynaptic membrane of the electroplax of Electrophorus electricus is a protein with a disulfide bond in the vicinity of the active site. This disulfide can be reduced and reoxidized with concomitant inhibition and restoration of the response to acetylcholine and other monoquaternary ammonium-depolarizing agents. Conversely, the bisquaternary hexamethonium, normally a competitive inhibitor, causes depolarization, and the activity of decamethonium is increased following reduction of the disulfide. The reduced receptor can be alkylated by various maleimide derivatives and is then no longer reoxidizable. Some quaternary ammonium maleimide derivatives act as affinity labels of the reduced receptor, alkylating it at a rate three orders of magnitude faster then do uncharged maleimide derivatives. Other types of potential affinity labels also react only with the reduced receptor and the resulting covalently attached quaternary ammonium moieties interact with the active site, strongly activating the receptor. These results suggest a model for the active site and its transitions in which an activator such as acetylcholine bridges between a negative subsite and a hydrophobic subsite in the vicinity of the disulfide, causing an altered conformation around the negative subsite and a decrasee of a few angstroms in the distance between the two subsites.

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Structures of cell wall arabinosyltransferases with the anti-tuberculosis drug ethambutol

Science ◽

10.1126/science.aba9102 ◽

2020 ◽

Vol 368 (6496) ◽

pp. 1211-1219 ◽

Cited By ~ 7

Author(s):

Lu Zhang ◽

Yao Zhao ◽

Yan Gao ◽

Lijie Wu ◽

Ruogu Gao ◽

...

Keyword(s):

Cell Wall ◽

Active Site ◽

Structural Basis ◽

Drug Resistant ◽

Cell Wall Synthesis ◽

X Ray ◽

Cryo Electron Microscopy ◽

Donor And Acceptor ◽

Biochemical Function ◽

Glycosyl Donor

The arabinosyltransferases EmbA, EmbB, and EmbC are involved in Mycobacterium tuberculosis cell wall synthesis and are recognized as targets for the anti-tuberculosis drug ethambutol. In this study, we determined cryo–electron microscopy and x-ray crystal structures of mycobacterial EmbA-EmbB and EmbC-EmbC complexes in the presence of their glycosyl donor and acceptor substrates and with ethambutol. These structures show how the donor and acceptor substrates bind in the active site and how ethambutol inhibits arabinosyltransferases by binding to the same site as both substrates in EmbB and EmbC. Most drug-resistant mutations are located near the ethambutol binding site. Collectively, our work provides a structural basis for understanding the biochemical function and inhibition of arabinosyltransferases and the development of new anti-tuberculosis agents.

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Investigation of the Staphylococcus aureus GraSR Regulon Reveals Novel Links to Virulence, Stress Response and Cell Wall Signal Transduction Pathways

PLoS ONE ◽

10.1371/journal.pone.0021323 ◽

2011 ◽

Vol 6 (7) ◽

pp. e21323 ◽

Cited By ~ 98

Author(s):

Mélanie Falord ◽

Ulrike Mäder ◽

Aurélia Hiron ◽

Michel Débarbouillé ◽

Tarek Msadek

Keyword(s):

Signal Transduction ◽

Staphylococcus Aureus ◽

Cell Wall ◽

Stress Response ◽

Signal Transduction Pathways ◽

Wall Signal

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Inactivation of sarcoplasmic-reticulum Ca2+-ATPase in low-frequency-stimulated muscle results from a modification of the active site

Biochemical Journal ◽

10.1042/bj2850303 ◽

1992 ◽

Vol 285 (1) ◽

pp. 303-309 ◽

Cited By ~ 35

Author(s):

S Matsushita ◽

D Pette

Keyword(s):

Sarcoplasmic Reticulum ◽

Active Site ◽

Low Frequency ◽

Fluorescein Isothiocyanate ◽

Tryptophan Fluorescence ◽

Atp Binding ◽

Binding Characteristics ◽

Intrinsic Tryptophan Fluorescence ◽

Inactive Form ◽

Induced Changes

Molecular changes underlying the partial inactivation of the sarcoplasmic-reticulum (SR) Ca(2+-) ATPase in low-frequency-stimulated fast-twitch muscle were investigated in the present study. The specific Ca(2+)-ATPase activity, as well as the ATP- and acetyl phosphate-driven Ca2+ uptakes by the SR, were reduced by approx. 30% in 4-day-stimulated muscle. Phosphoprotein formation of the enzyme in the presence of ATP or Pi was also decreased to the same extent. Measurements of ATP binding revealed a 30% decrease in binding to the enzyme. These changes were accompanied by similar decreases in the ligand-induced (ATP, ADP, Pi) intrinsic tryptophan fluorescence. A decreased binding of fluorescein isothiocyanate (FITC) corresponded to the lower ATP binding and phosphorylation of the enzyme. Moreover, Pi-induced changes in fluorescence of the FITC-labelled enzyme did not differ between SR from stimulated and contralateral muscles, indicating that Ca(2+)- ATPase molecules which did not bind FITC were responsible for the decreased Pi-dependent phosphorylation, and therefore represented the inactive form of the enzyme. No differences existed between the Ca(2+)-induced changes in the intrinsic fluorescence of SR from stimulated and contralateral muscles which fit their similar Ca(2+)-binding characteristics. Taking the proposed architecture of the Ca2(+)-ATPase into consideration, our results suggest that the inactivation relates to a circumscribed structural alteration of the enzyme in sections of the active site consisting of the nucleotide-binding and phosphorylation domains.

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Furin Cleavage Potentiates the Membrane Fusion-Controlling Intersubunit Disulfide Bond Isomerization Activity of Leukemia Virus Env

Journal of Virology ◽

10.1128/jvi.01851-05 ◽

2006 ◽

Vol 80 (11) ◽

pp. 5540-5551 ◽

Cited By ~ 13

Author(s):

Mathilda Sjöberg ◽

Michael Wallin ◽

Birgitta Lindqvist ◽

Henrik Garoff

Keyword(s):

Membrane Fusion ◽

Disulfide Bond ◽

Fusion Reaction ◽

Leukemia Virus ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Furin Cleavage ◽

Membrane Fusion Protein ◽

Peptide Complexes ◽

Disulfide Bond Isomerization

ABSTRACT The membrane fusion protein of murine leukemia virus is a trimer of a disulfide-linked peripheral-transmembrane (SU-TM) subunit complex. The intersubunit disulfide bond is in SU linked to a disulfide bond isomerization motif, CXXC, with which the virus controls its fusion reaction (M. Wallin, M. Ekström, and H. Garoff, EMBO J. 23:54-65, 2004). Upon receptor binding the isomerase rearranges the intersubunit disulfide bond into a disulfide bond isomer within the motif. This facilitates SU dissociation and fusion activation in the TM subunit. In the present study we have asked whether furin cleavage of the Env precursor potentiates the isomerase to be triggered. To this end we accumulated the late form of the precursor, gp90, in the cell by incubation in the presence of a furin-inhibiting peptide. The isomerization was done by NP-40 incubation or by a heat pulse under alkylation-free conditions. The cells were lysed in the presence of alkylator, and the precursor was immunoprecipitated, gel isolated, deglycosylated, and subjected to complete trypsin digestion. Disulfide-linked peptide complexes were separated by sodium dodecyl sulfate-tricine-polyacrylamide gel electrophoresis under nonreducing conditions. This assay revealed the size of the characteristic major disulfide-linked peptide complex that differentiates the two isomers of the disulfide bond between Cys336 (or Cys339) and Cys563, i.e., the bond corresponding to the intersubunit disulfide bond. The analyses showed that the isomerase was five- to eightfold more resistant to triggering in the precursor than in the mature, cleaved form. This suggests that the isomerase becomes potentiated for triggering by a structural change in Env that is induced by furin cleavage in the cell.

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S-glutathionylation of glyceraldehyde-3-phosphate dehydrogenase induces formation of C150-C154 intrasubunit disulfide bond in the active site of the enzyme

Biochimica et Biophysica Acta (BBA) - General Subjects ◽

10.1016/j.bbagen.2017.09.008 ◽

2017 ◽

Vol 1861 (12) ◽

pp. 3167-3177 ◽

Cited By ~ 14

Author(s):

K.V. Barinova ◽

M.V. Serebryakova ◽

V.I. Muronetz ◽

E.V. Schmalhausen

Keyword(s):

Disulfide Bond ◽

Active Site

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The Sortase A Enzyme That Attaches Proteins to the Cell Wall ofBacillus anthracisContains an Unusual Active Site Architecture

Journal of Biological Chemistry ◽

10.1074/jbc.m110.135434 ◽

2010 ◽

Vol 285 (30) ◽

pp. 23433-23443 ◽

Cited By ~ 44

Author(s):

Ethan M. Weiner ◽

Scott Robson ◽

Melanie Marohn ◽

Robert T. Clubb

Keyword(s):

Cell Wall ◽

Active Site ◽

Sortase A

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Disulfide Bond Isomerization and the Assembly of MHC Class I-Peptide Complexes

Immunity ◽

10.1016/s1074-7613(02)00263-7 ◽

2002 ◽

Vol 16 (1) ◽

pp. 87-98 ◽

Cited By ~ 173

Author(s):

Tobias P Dick ◽

Naveen Bangia ◽

David R Peaper ◽

Peter Cresswell

Keyword(s):

Mhc Class I ◽

Disulfide Bond ◽

Class I ◽

Peptide Complexes ◽

Disulfide Bond Isomerization

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Detection of Allosteric Kinase Inhibitors by Displacement of Active Site Probes

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057112439889 ◽

2012 ◽

Vol 17 (6) ◽

pp. 813-821 ◽

Cited By ~ 13

Author(s):

Connie S. Lebakken ◽

Laurie J. Reichling ◽

Jason M. Ellefson ◽

Steven M. Riddle

Keyword(s):

Active Site ◽

Kinase Inhibitors ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Published Data ◽

Binding Assays ◽

Allosteric Inhibitors ◽

Time Resolved ◽

Inactive Form ◽

Competitive Inhibitors

Non–adenosine triphosphate (ATP) competitive, allosteric inhibitors provide a promising avenue to develop highly selective small-molecule kinase inhibitors. Although this class of compounds is growing, detection of such inhibitors can be challenging as standard kinase activity assays preferentially detect compounds that bind to active kinases in an ATP competitive manner. We have previously described a time-resolved fluorescence resonance energy transfer (TR-FRET)–based kinase binding assay using the competitive displacement of ATP competitive active site fluorescent probes (“tracers”). Although this format has gained acceptance, published data with this and related formats are almost entirely without examples of non-ATP competitive compounds. Thus, this study addresses whether this format is useful for non-ATP competitive inhibitors. To this end, 15 commercially available non-ATP competitive inhibitors were tested for their ability to displace ATP competitive probes. Despite the diversity of both compound structures and their respective targets, 14 of the 15 compounds displaced the tracers with IC50 values comparable to literature values. We conclude that such binding assays are well suited for the study of non-ATP competitive inhibitors. In addition, we demonstrate that allosteric inhibitors of BCR-Abl and MEK bind preferentially to the nonphosphorylated (i.e., inactive) form of the kinase, indicating that binding assays may be a preferred format in some cases.

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