scholarly journals Crystal structure and biochemical analyses reveal that the Arabidopsis triphosphate tunnel metalloenzyme AtTTM3 is a tripolyphosphatase involved in root development

2013 ◽  
Vol 76 (4) ◽  
pp. 615-626 ◽  
Author(s):  
Wolfgang Moeder ◽  
Christel Garcia-Petit ◽  
Huoi Ung ◽  
Geoffrey Fucile ◽  
Marcus A. Samuel ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Root Development ◽  
Biochemical Analyses ◽  
Triphosphate Tunnel Metalloenzyme

scholarly journals Protein polyglutamylation catalyzed by the bacterial calmodulin-dependent pseudokinase SidJ

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Alan Sulpizio ◽  
Marena E Minelli ◽  
Min Wan ◽  
Paul D Burrowes ◽  
Xiaochun Wu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mass Spectrometry ◽  
Ubiquitin Ligase ◽  
Legionella Pneumophila ◽  
Kinase Activity ◽  
Effector Protein ◽  
Dependent Manner ◽  
Biochemical Analyses ◽  
Human And Mouse

Pseudokinases are considered to be the inactive counterparts of conventional protein kinases and comprise approximately 10% of the human and mouse kinomes. Here, we report the crystal structure of the Legionella pneumophila effector protein, SidJ, in complex with the eukaryotic Ca2+-binding regulator, calmodulin (CaM). The structure reveals that SidJ contains a protein kinase-like fold domain, which retains a majority of the characteristic kinase catalytic motifs. However, SidJ fails to demonstrate kinase activity. Instead, mass spectrometry and in vitro biochemical analyses demonstrate that SidJ modifies another Legionella effector SdeA, an unconventional phosphoribosyl ubiquitin ligase, by adding glutamate molecules to a specific residue of SdeA in a CaM-dependent manner. Furthermore, we show that SidJ-mediated polyglutamylation suppresses the ADP-ribosylation activity. Our work further implies that some pseudokinases may possess ATP-dependent activities other than conventional phosphorylation.

scholarly journals Crystal Structure of F-93 from Sulfolobus Spindle-Shaped Virus 1, a Winged-Helix DNA Binding Protein

2004 ◽  
Vol 78 (21) ◽  
pp. 11544-11550 ◽  
Author(s):  
Paul Kraft ◽  
Andrea Oeckinghaus ◽  
Daniel Kümmel ◽  
George H. Gauss ◽  
John Gilmore ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Dna Binding ◽  
Hot Springs ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
Target Sequence ◽  
Winged Helix ◽  
Reading Frame ◽  
Biochemical Analyses ◽  
Reading Frames

ABSTRACT Sulfolobus spindle-shaped viruses (SSVs), or Fuselloviridae, are ubiquitous crenarchaeal viruses found in high-temperature acidic hot springs around the world (pH ≤4.0; temperature of ≥70°C). Because they are relatively easy to isolate, they represent the best studied of the crenarchaeal viruses. This is particularly true for the type virus, SSV1, which contains a double-stranded DNA genome of 15.5 kilobases, encoding 34 putative open reading frames. Interestingly, the genome shows little sequence similarity to organisms other than its SSV homologues. Together, sequence similarity and biochemical analyses have suggested functions for only 6 of the 34 open reading frames. Thus, even though SSV1 is the best-studied crenarchaeal virus, functions for most (28) of its open reading frames remain unknown. We have undertaken biochemical and structural studies for the gene product of open reading frame F-93. We find that F-93 exists as a homodimer in solution and that a tight dimer is also present in the 2.7-Å crystal structure. Further, the crystal structure reveals a fold that is homologous to the SlyA and MarR subfamilies of winged-helix DNA binding proteins. This strongly suggests that F-93 functions as a transcription factor that recognizes a (pseudo-)palindromic DNA target sequence.

scholarly journals Crystal Structure of INTS3/INTS6 complex reveals Functional Importance of INTS3 dimerization in DSB Repair

2020 ◽  
Author(s):  
Yu Jia ◽  
Zixiu Cheng ◽  
Sakshibeedu R Bharath ◽  
Qiangzu Sun ◽  
Nannan Su ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Critical Role ◽  
Repair Process ◽  
Dsb Repair ◽  
Ssdna Binding ◽  
C Terminus ◽  
Ssdna Binding Protein ◽  
Direct Binding ◽  
Biochemical Analyses

AbstractSOSS1 is a single-stranded DNA (ssDNA)-binding protein complex that plays a critical role in double-strand DNA break (DSB) repair. SOSS1 consists of three subunits: INTS3, SOSSC, and hSSB1 with INTS3 serving as a scaffold to stabilize this complex. Moreover, the integrator complex subunit 6 (INTS6) participates in the DNA damage response through direct binding to INTS3 but how INTS3 interacts with INTS6 thereby, impacting DBS repair is not clear. Here, we determined the crystal structure of the C-terminus of INTS3 (INTS3c) in complex with the C-terminus of INTS6 (INTS6c) at a resolution of 2.4 Å. Structure analysis revealed that two INTS3c subunits dimerize and interact with INTS6c via conserved residues. Subsequent biochemical analyses confirmed that INTS3c forms a stable dimer and INTS3 dimerization is important for recognizing the longer ssDNA. Perturbation of INTS3c dimerization and disruption of the INTS3c/INTS6c interaction, impair the DSB repair process. Altogether, these results unravel the underappreciated role of INTS3 dimerization and the molecular basis of INTS3/INTS6 interaction in DSB repair.

scholarly journals Crystal structure of the INTS3/INTS6 complex reveals the functional importance of INTS3 dimerization in DSB repair

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Yu Jia ◽  
Zixiu Cheng ◽  
Sakshibeedu R. Bharath ◽  
Qiangzu Sun ◽  
Nannan Su ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Critical Role ◽  
Repair Process ◽  
Dsb Repair ◽  
Ssdna Binding ◽  
C Terminus ◽  
Ssdna Binding Protein ◽  
Direct Binding ◽  
Biochemical Analyses

AbstractSOSS1 is a single-stranded DNA (ssDNA)-binding protein complex that plays a critical role in double-strand DNA break (DSB) repair. SOSS1 consists of three subunits: INTS3, SOSSC, and hSSB1, with INTS3 serving as a scaffold to stabilize this complex. Moreover, the integrator complex subunit 6 (INTS6) participates in the DNA damage response through direct binding to INTS3, but how INTS3 interacts with INTS6, thereby impacting DSB repair, is not clear. Here, we determined the crystal structure of the C-terminus of INTS3 (INTS3c) in complex with the C-terminus of INTS6 (INTS6c) at a resolution of 2.4 Å. Structural analysis revealed that two INTS3c subunits dimerize and interact with INTS6c via conserved residues. Subsequent biochemical analyses confirmed that INTS3c forms a stable dimer and INTS3 dimerization is important for recognizing the longer ssDNA. Perturbation of INTS3c dimerization and disruption of the INTS3c/INTS6c interaction impair the DSB repair process. Altogether, these results unravel the underappreciated role of INTS3 dimerization and the molecular basis of INTS3/INTS6 interaction in DSB repair.

scholarly journals Crystal structure and biochemical analyses reveal Beclin 1 as a novel membrane binding protein

Cell Research ◽  
2012 ◽  
Vol 22 (3) ◽  
pp. 473-489 ◽  
Author(s):  
Weijiao Huang ◽  
Wooyoung Choi ◽  
Wanqiu Hu ◽  
Na Mi ◽  
Qiang Guo ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Protein ◽  
Membrane Binding ◽  
Beclin 1 ◽  
Biochemical Analyses

scholarly journals Mechanism for autoinhibition and activation of the MORC3 ATPase

2019 ◽  
Vol 116 (13) ◽  
pp. 6111-6119 ◽  
Author(s):  
Yi Zhang ◽  
Brianna J. Klein ◽  
Khan L. Cox ◽  
Bianca Bertulat ◽  
Adam H. Tencer ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Catalytic Activity ◽  
Molecular Basis ◽  
Catalytic Domain ◽  
Histone H3 ◽  
Protein Fold ◽  
Atpase Domain ◽  
Dna Binding Site ◽  
Dimeric Form ◽  
Biochemical Analyses

Microrchidia 3 (MORC3) is a human protein linked to autoimmune disorders, Down syndrome, and cancer. It is a member of a newly identified family of human ATPases with an uncharacterized mechanism of action. Here, we elucidate the molecular basis for inhibition and activation of MORC3. The crystal structure of the MORC3 region encompassing the ATPase and CW domains in complex with a nonhydrolyzable ATP analog demonstrates that the two domains are directly coupled. The extensive ATPase:CW interface stabilizes the protein fold but inhibits the catalytic activity of MORC3. Enzymatic, NMR, mutational, and biochemical analyses show that in the autoinhibited, off state, the CW domain sterically impedes binding of the ATPase domain to DNA, which in turn is required for the catalytic activity. MORC3 autoinhibition is released by disrupting the intramolecular ATPase:CW coupling through the competitive interaction of CW with histone H3 tail or by mutating the interfacial residues. Binding of CW to H3 leads to a marked rearrangement in the ATPase–CW cassette, which frees the DNA-binding site in active MORC3 (on state). We show that ATP-induced dimerization of the ATPase domain is strictly required for the catalytic activity and that the dimeric form of ATPase–CW might cooperatively bind to dsDNA. Together, our findings uncovered a mechanism underlying the fine-tuned regulation of the catalytic domain of MORC3 by the epigenetic reader, CW.

Inhibition of RNA Helicase Brr2 by the C-Terminal Tail of the Spliceosomal Protein Prp8

Science ◽  
2013 ◽  
Vol 341 (6141) ◽  
pp. 80-84 ◽  
Author(s):  
Sina Mozaffari-Jovin ◽  
Traudy Wandersleben ◽  
Karine F. Santos ◽  
Cindy L. Will ◽  
Reinhard Lührmann ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Retinitis Pigmentosa ◽  
Adenosine Triphosphatase ◽  
Rna Binding ◽  
Protein A ◽  
Rna Helicase ◽  
Core Component ◽  
Spliceosomal Protein ◽  
Spliceosome Activation ◽  
Biochemical Analyses

The Ski2-like RNA helicase Brr2 is a core component of the spliceosome that must be tightly regulated to ensure correct timing of spliceosome activation. Little is known about mechanisms of regulation of Ski2-like helicases by protein cofactors. Here we show by crystal structure and biochemical analyses that the Prp8 protein, a major regulator of the spliceosome, can insert its C-terminal tail into Brr2’s RNA-binding tunnel, thereby intermittently blocking Brr2’s RNA-binding, adenosine triphosphatase, and U4/U6 unwinding activities. Inefficient Brr2 repression is the only recognizable phenotype associated with certain retinitis pigmentosa–linked Prp8 mutations that map to its C-terminal tail. Our data show how a Ski2-like RNA helicase can be reversibly inhibited by a protein cofactor that directly competes with RNA substrate binding.

scholarly journals Discovery of an ene-reductase for initiating flavone and flavonol catabolism in gut bacteria

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gaohua Yang ◽  
Sen Hong ◽  
Pengjie Yang ◽  
Yuwei Sun ◽  
Yong Wang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Basis ◽  
Molecular Mechanisms ◽  
Mechanisms Of Action ◽  
Crystal Structure Analysis ◽  
Polyphenolic Compounds ◽  
Personalized Nutrition ◽  
Biochemical Analyses ◽  
Microbial Consumption ◽  
Insight Into

AbstractGut microbial transformations of flavonoids, an enormous class of polyphenolic compounds abundant in plant-based diets, are closely associated with human health. However, the enzymes that initiate the gut microbial metabolism of flavones and flavonols, the two most abundant groups of flavonoids, as well as their underlying molecular mechanisms of action remain unclear. Here, we discovered a flavone reductase (FLR) from the gut bacterium, Flavonifractor plautii ATCC 49531 (originally assigned as Clostridium orbiscindens DSM 6740), which specifically catalyses the hydrogenation of the C2–C3 double bond of flavones/flavonols and initiates their metabolism as a key step. Crystal structure analysis revealed the molecular basis for the distinct catalytic property of FLR. Notably, FLR and its widespread homologues represent a class of ene-reductases that has not been previously identified. Genetic and biochemical analyses further indicated the importance of FLR in gut microbial consumption of dietary and medicinal flavonoids, providing broader insight into gut microbial xenobiotic transformations and possible guidance for personalized nutrition and medicine.

Structural and biochemical analyses of theStreptococcus pneumoniaeL,D-carboxypeptidase DacB

2015 ◽  
Vol 71 (2) ◽  
pp. 283-292
Author(s):  
Juan Zhang ◽  
Yi-Hu Yang ◽  
Yong-Liang Jiang ◽  
Cong-Zhao Zhou ◽  
Yuxing Chen
Keyword(s):  
Crystal Structure ◽  
Molecular Docking ◽  
Catalytic Mechanism ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Binding Properties ◽  
Biochemical Analyses ◽  
Key Residues ◽  
Structural Insights

The L,D-carboxypeptidase DacB plays a key role in the remodelling ofStreptococcus pneumoniaepeptidoglycan during cell division. In order to decipher its substrate-binding properties and catalytic mechanism, the 1.71 Å resolution crystal structure of DacB fromS. pneumoniaeTIGR4 is reported. Structural analyses in combination with comparisons with the recently reported structures of DacB fromS. pneumoniaeD39 and R6 clearly demonstrate that DacB adopts a zinc-dependent carboxypeptidase fold and belongs to the metallopeptidase M15B subfamily. In addition, enzymatic activity assays further confirm that DacB indeed acts as an L,D-carboxypeptidase towards the tetrapeptide L-Ala-D-iGln-L-Lys-D-Ala of the peptidoglycan stem, withKmandkcatvalues of 2.84 ± 0.37 mMand 91.49 ± 0.05 s−1, respectively. Subsequent molecular docking and site-directed mutagenesis enable the assignment of the key residues that bind to the tetrapeptide. Altogether, these findings provide structural insights into substrate recognition in the metallopeptidase M15B subfamily.

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