Probing the DNA Interface of theEcoRV DNA-(Adenine-N6)-methyltransferase by Site-Directed Mutagenesis, Fluorescence Spectroscopy, and UV Cross-Linking†

Abstract A hallmark of inflammasome activation is the ASC speck, a micrometre-sized structure formed by the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), which consists of a pyrin domain (PYD) and a caspase recruitment domain (CARD). Here we show that assembly of the ASC speck involves oligomerization of ASCPYD into filaments and cross-linking of these filaments by ASCCARD. ASC mutants with a non-functional CARD only assemble filaments but not specks, and moreover disrupt endogenous specks in primary macrophages. Systematic site-directed mutagenesis of ASCPYD is used to identify oligomerization-deficient ASC mutants and demonstrate that ASC speck formation is required for efficient processing of IL-1β, but dispensable for gasdermin-D cleavage and pyroptosis induction. Our results suggest that the oligomerization of ASC creates a multitude of potential caspase-1 activation sites, thus serving as a signal amplification mechanism for inflammasome-mediated cytokine production.

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Mapping the Contact Sites of the Escherichia coli Division-Initiating Proteins FtsZ and ZapA by BAMG Cross-Linking and Site-Directed Mutagenesis

International Journal of Molecular Sciences ◽

10.3390/ijms19102928 ◽

2018 ◽

Vol 19 (10) ◽

pp. 2928 ◽

Cited By ~ 7

Author(s):

Winfried Roseboom ◽

Madhvi Nazir ◽

Nils Meiresonne ◽

Tamimount Mohammadi ◽

Jolanda Verheul ◽

...

Keyword(s):

Site Directed Mutagenesis ◽

Cross Linking ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Binding Interface ◽

Tetrameric Structure ◽

Z Ring ◽

Cross Links ◽

Chemical Cross Linking

Cell division in bacteria is initiated by the polymerization of FtsZ at midcell in a ring-like structure called the Z-ring. ZapA and other proteins assist Z-ring formation and ZapA binds ZapB, which senses the presence of the nucleoids. The FtsZ–ZapA binding interface was analyzed by chemical cross-linking mass spectrometry (CXMS) under in vitro FtsZ-polymerizing conditions in the presence of GTP. Amino acids residue K42 from ZapA was cross-linked to amino acid residues K51 and K66 from FtsZ, close to the interphase between FtsZ molecules in protofilaments. Five different cross-links confirmed the tetrameric structure of ZapA. A number of FtsZ cross-links suggests that its C-terminal domain of 55 residues, thought to be largely disordered, has a limited freedom to move in space. Site-directed mutagenesis of ZapA reveals an interaction site in the globular head of the protein close to K42. Using the information on the cross-links and the mutants that lost the ability to interact with FtsZ, a model of the FtsZ protofilament–ZapA tetramer complex was obtained by information-driven docking with the HADDOCK2.2 webserver.

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An intersubunit interaction at the active site of D-ribulose-1,5-bisphosphate carboxylase/oxygenase as revealed by cross-linking and site-directed mutagenesis

Biochemistry ◽

10.1021/bi00389a001 ◽

1987 ◽

Vol 26 (15) ◽

pp. 4599-4604 ◽

Cited By ~ 19

Author(s):

Eva H. Lee ◽

Thomas S. Soper ◽

Richard J. Mural ◽

Claude D. Stringer ◽

Fred C. Hartman

Keyword(s):

Active Site ◽

Site Directed Mutagenesis ◽

Cross Linking ◽

Directed Mutagenesis ◽

Bisphosphate Carboxylase

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Probing the Dynamics of a Mobile Loop above the Active Site of L1, a Metallo-β-lactamase fromStenotrophomonas maltophilia, via Site-directed Mutagenesis and Stopped-flow Fluorescence Spectroscopy

Journal of Biological Chemistry ◽

10.1074/jbc.m406826200 ◽

2004 ◽

Vol 279 (38) ◽

pp. 39663-39670 ◽

Cited By ~ 16

Author(s):

James D. Garrity ◽

James M. Pauff ◽

Michael W. Crowder

Keyword(s):

Fluorescence Spectroscopy ◽

Active Site ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Stopped Flow

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Mammalian inositol monophosphatase: the identification of residues important for the binding of Mg2+ and Li+ ions using fluorescence spectroscopy and site-directed mutagenesis

Biochemical Journal ◽

10.1042/bj2960811 ◽

1993 ◽

Vol 296 (3) ◽

pp. 811-815 ◽

Cited By ~ 11

Author(s):

M G Gore ◽

P Greasley ◽

G McAllister ◽

C I Ragan

Keyword(s):

Fluorescence Spectroscopy ◽

Specific Activity ◽

Fluorescence Emission ◽

Site Directed Mutagenesis ◽

Native Enzyme ◽

Mutant Enzyme ◽

Directed Mutagenesis ◽

Inositol Monophosphatase ◽

Fluorescence Emission Intensity ◽

Ph Dependent

The fluorescence properties of residue Trp-219 in inositol monophosphatase are sensitive to the ionization of neighbouring groups. The pH-dependent changes in the fluorescence emission intensity and wavelength of maximum emission appear to arise as the result of two separate ionizations in the proximity of Trp-219, namely due to the ionization of His-217 and Cys-218. By studying the curve of fluorescence intensity against pH, given by the mutants Cys-218→Ala or His-217→Gln, the pK of His-217 was determined to be 7.54 and the pK of Cys-218 was estimated to be about 8.2. These mutants have altered kinetic parameters for catalytic Mg2+ ions and inhibitory Mg2+ and Li+ ions. The Cys-218→Ala mutant enzyme is not subject to inhibition by concentrations of Mg2+ ions up to 400 mM and has a specific activity of 156% of the maximum obtainable activity of the native enzyme. The His-217→Gln mutant enzyme shows reduced sensitivity to inhibition by Mg2+ and Li+ ions, and has a specific activity of 110% of that obtainable for the native enzyme.

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