scholarly journals Expression, purification, and structural analysis of the trimeric form of the catalytic domain of the Escherichia coli dihydrolipoamide succinyltransferase

2008 ◽  
Vol 9 (1) ◽  
pp. 37-48 ◽  
Author(s):  
James E. Knapp ◽  
Donald Carroll ◽  
Janet E. Lawson ◽  
Stephen R. Ernst ◽  
Lester J. Reed ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Structural Analysis ◽  
Catalytic Domain ◽  
Dihydrolipoamide Succinyltransferase

scholarly journals Structure of the dihydrolipoamide succinyltransferase catalytic domain from Escherichia coli in a novel crystal form: a tale of a common protein crystallization contaminant

2019 ◽  
Vol 75 (9) ◽  
pp. 616-624
Author(s):  
Babak Andi ◽  
Alexei S. Soares ◽  
Wuxian Shi ◽  
Martin R. Fuchs ◽  
Sean McSweeney ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Protein Crystallization ◽  
Crystal Form ◽  
Molecular Replacement ◽  
Biosynthesis Pathway ◽  
Unit Cell Parameters ◽  
Cell Parameters ◽  
Search Models ◽  
Dihydrolipoamide Succinyltransferase

The crystallization of amidase, the ultimate enzyme in the Trp-dependent auxin-biosynthesis pathway, from Arabidopsis thaliana was attempted using protein samples with at least 95% purity. Cube-shaped crystals that were assumed to be amidase crystals that belonged to space group I4 (unit-cell parameters a = b = 128.6, c = 249.7 Å) were obtained and diffracted to 3.0 Å resolution. Molecular replacement using structures from the PDB containing the amidase signature fold as search models was unsuccessful in yielding a convincing solution. Using the Sequence-Independent Molecular replacement Based on Available Databases (SIMBAD) program, it was discovered that the structure corresponded to dihydrolipoamide succinyltransferase from Escherichia coli (PDB entry 1c4t), which is considered to be a common crystallization contaminant protein. The structure was refined to an R work of 23.0% and an R free of 27.2% at 3.0 Å resolution. The structure was compared with others of the same protein deposited in the PDB. This is the first report of the structure of dihydrolipoamide succinyltransferase isolated without an expression tag and in this novel crystal form.

scholarly journals Cryo-EM structural analysis of FADD:Caspase-8 complexes defines the catalytic dimer architecture for co-ordinated control of cell fate

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joanna L. Fox ◽  
Michelle A. Hughes ◽  
Xin Meng ◽  
Nikola A. Sarnowska ◽  
Ian R. Powley ◽  
...  
Keyword(s):  
Cell Death ◽  
Structural Analysis ◽  
Cell Fate ◽  
Catalytic Domain ◽  
Caspase 8 ◽  
Type I ◽  
Signaling Complex ◽  
Catalytic Domains ◽  
Regulated Cell Death ◽  
Death Effector

AbstractRegulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.

Mutational and Structural Analysis of the RNA Binding Site for Escherichia coli Ribosomal Protein S7

1994 ◽  
Vol 244 (1) ◽  
pp. 74-85 ◽  
Author(s):  
François Dragon ◽  
Catherine Payant ◽  
Léa Brakier-Gingras
Keyword(s):  
Escherichia Coli ◽  
Structural Analysis ◽  
Ribosomal Protein ◽  
Binding Site ◽  
Rna Binding

scholarly journals Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System

2006 ◽  
Vol 188 (6) ◽  
pp. 2163-2172 ◽  
Author(s):  
Paul W. King ◽  
Matthew C. Posewitz ◽  
Maria L. Ghirardi ◽  
Michael Seibert
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Expression System ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Functional Studies ◽  
Hydrogenase Maturation ◽  
Iron Sulfur ◽  
And Function

ABSTRACT Maturation of [FeFe] hydrogenases requires the biosynthesis and insertion of the catalytic iron-sulfur cluster, the H cluster. Two radical S-adenosylmethionine (SAM) proteins proposed to function in H cluster biosynthesis, HydEF and HydG, were recently identified in the hydEF-1 mutant of the green alga Chlamydomonas reinhardtii (M. C. Posewitz, P. W. King, S. L. Smolinski, L. Zhang, M. Seibert, and M. L. Ghirardi, J. Biol. Chem. 279:25711-25720, 2004). Previous efforts to study [FeFe] hydrogenase maturation in Escherichia coli by coexpression of C. reinhardtii HydEF and HydG and the HydA1 [FeFe] hydrogenase were hindered by instability of the hydEF and hydG expression clones. A more stable [FeFe] hydrogenase expression system has been achieved in E. coli by cloning and coexpression of hydE, hydF, and hydG from the bacterium Clostridium acetobutylicum. Coexpression of the C. acetobutylicum maturation proteins with various algal and bacterial [FeFe] hydrogenases in E. coli resulted in purified enzymes with specific activities that were similar to those of the enzymes purified from native sources. In the case of structurally complex [FeFe] hydrogenases, maturation of the catalytic sites could occur in the absence of an accessory iron-sulfur cluster domain. Initial investigations of the structure and function of the maturation proteins HydE, HydF, and HydG showed that the highly conserved radical-SAM domains of both HydE and HydG and the GTPase domain of HydF were essential for achieving biosynthesis of active [FeFe] hydrogenases. Together, these results demonstrate that the catalytic domain and a functionally complete set of Hyd maturation proteins are fundamental to achieving biosynthesis of catalytic [FeFe] hydrogenases.

scholarly journals Characterization of a beta-galactosidase hybrid protein carrying the catalytic domain of Escherichia coli adenylate cyclase

1986 ◽  
Vol 159 (3) ◽  
pp. 605-609 ◽  
Author(s):  
Isabella CRENON ◽  
Daniel LADANT ◽  
Nicole GUISO ◽  
Anne-Marie GILLES ◽  
Octavian BARZU
Keyword(s):  
Escherichia Coli ◽  
Adenylate Cyclase ◽  
Catalytic Domain ◽  
Hybrid Protein ◽  
Beta Galactosidase

scholarly journals The extent of B-form structure in a 6S RNA variant that mimics DNA to bind to the Escherichia coli RNA Polymerase

2018 ◽  
Author(s):  
Nilesh K. Banavali
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Structural Analysis ◽  
Rna Polymerase ◽  
Rna Structure ◽  
Recent Article ◽  
Single Strand ◽  
Bacterial Rna ◽  
6S Rna

AbstractIn a recent article by Darst and coworkers, it was found that a non-coding 6S RNA variant regulates a bacterial RNA polymerase by mimicking B-Form DNA, and a few different nucleic acid duplex parameters were analyzed to understand the extent of B-form RNA structure. In this manuscript, a different structural analysis based on conformational distance from canonical A-form and B-form single-strand structures is presented. This analysis addresses the occurrence and extent of both local and global B-form structure in the published 6S RNA variant model.

scholarly journals Regulation of Escherichia coli RelA Requires Oligomerization of the C-Terminal Domain

2001 ◽  
Vol 183 (2) ◽  
pp. 570-579 ◽  
Author(s):  
Michal Gropp ◽  
Yael Strausz ◽  
Miriam Gross ◽  
Gad Glaser
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Catalytic Domain ◽  
Mutational Analysis ◽  
Amino Acid Starvation ◽  
E Coli ◽  
Terminal Domain ◽  
Negative Effect ◽  
And Control ◽  
Two Hybrid

ABSTRACT The E. coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD inrelA + cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length relA gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752–5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.

scholarly journals A protein architecture guided screen for modification dependent restriction endonucleases

2019 ◽  
Vol 47 (18) ◽  
pp. 9761-9776 ◽  
Author(s):  
Thomas Lutz ◽  
Kiersten Flodman ◽  
Alyssa Copelas ◽  
Honorata Czapinska ◽  
Megumu Mabuchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fusion Proteins ◽  
Dna Methyltransferase ◽  
Catalytic Domain ◽  
Dna Methyltransferases ◽  
Restriction Endonucleases ◽  
Endonuclease Activity ◽  
Protein Architecture ◽  
Escherichia Coli Cells ◽  
Dependent Activity

Abstract Modification dependent restriction endonucleases (MDREs) often have separate catalytic and modification dependent domains. We systematically looked for previously uncharacterized fusion proteins featuring a PUA or DUF3427 domain and HNH or PD-(D/E)XK catalytic domain. The enzymes were clustered by similarity of their putative modification sensing domains into several groups. The TspA15I (VcaM4I, CmeDI), ScoA3IV (MsiJI, VcaCI) and YenY4I groups, all featuring a PUA superfamily domain, preferentially cleaved DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. ScoA3V, also featuring a PUA superfamily domain, but of a different clade, exhibited 6-methyladenine stimulated nicking activity. With few exceptions, ORFs for PUA-superfamily domain containing endonucleases were not close to DNA methyltransferase ORFs, strongly supporting modification dependent activity of the endonucleases. DUF3427 domain containing fusion proteins had very little or no endonuclease activity, despite the presence of a putative PD-(D/E)XK catalytic domain. However, their expression potently restricted phage T4gt in Escherichia coli cells. In contrast to the ORFs for PUA domain containing endonucleases, the ORFs for DUF3427 fusion proteins were frequently found in defense islands, often also featuring DNA methyltransferases.

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