Mutational analysis of Thermococcus kodakarensis Endonuclease III reveals the roles of evolutionarily conserved residues

ABSTRACT The Spo0A protein of Bacillus subtilis is a DNA-binding protein that is required for the expression of genes involved in the initiation of sporulation. Spo0A binds directly to and both activates and represses transcription from the promoters of several genes required during the onset of endospore formation. The C-terminal 113 residues are known to contain the DNA-binding activity of Spo0A. Previous studies identified a region of the C-terminal half of Spo0A that is highly conserved among species of endospore-formingBacillus and Clostridium and which encodes a putative helix-turn-helix DNA-binding domain. To test the functional significance of this region and determine if this motif is involved in DNA binding, we changed three conserved residues, S210, E213, and R214, to Gly and/or Ala by site-directed mutagenesis. We then isolated and analyzed the five substitution-containing Spo0A proteins for DNA binding and sporulation-specific gene activation. The S210A Spo0A mutant exhibited no change from wild-type binding, although it was defective in spoIIA and spoIIE promoter activation. In contrast, both the E213G and E213A Spo0A variants showed decreased binding and completely abolished transcriptional activation of spoIIA and spoIIE, while the R214G and R214A variants completely abolished both DNA binding and transcriptional activation. These data suggest that these conserved residues are important for transcriptional activation and that the E213 residue is involved in DNA binding.

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Evolutionarily Conserved Residues at Glucagon-like Peptide-1 (GLP-1) Receptor Core Confer Ligand-induced Receptor Activation

Journal of Biological Chemistry ◽

10.1074/jbc.m111.276808 ◽

2011 ◽

Vol 287 (6) ◽

pp. 3873-3884 ◽

Cited By ~ 17

Author(s):

Mi Jin Moon ◽

Hee Young Kim ◽

Sumi Park ◽

Dong-Kyu Kim ◽

Eun Bee Cho ◽

...

Keyword(s):

Receptor Activation ◽

Conserved Residues ◽

Evolutionarily Conserved ◽

Glucagon Like Peptide ◽

Glucagon Like Peptide 1

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Analysis of YfgL and YaeT Interactions through Bioinformatics, Mutagenesis, and Biochemistry

Journal of Bacteriology ◽

10.1128/jb.01477-07 ◽

2007 ◽

Vol 190 (5) ◽

pp. 1507-1517 ◽

Cited By ~ 77

Author(s):

Phu Vuong ◽

Drew Bennion ◽

Jeremy Mantei ◽

Danielle Frost ◽

Rajeev Misra

Keyword(s):

Escherichia Coli ◽

Steady State ◽

Membrane Protein ◽

Direct Contact ◽

Mutational Analysis ◽

Cross Linking ◽

Conserved Residues ◽

Protein Biogenesis ◽

Steady State Levels

ABSTRACT In Escherichia coli, YaeT, together with four lipoproteins, YfgL, YfiO, NlpB, and SmpA, forms a complex that is essential for β-barrel outer membrane protein biogenesis. Data suggest that YfgL and YfiO make direct but independent physical contacts with YaeT. Whereas the YaeT-YfiO interaction needs NlpB and SmpA for complex stabilization, the YaeT-YfgL interaction does not. Using bioinformatics, genetics, and biochemical approaches, we have identified three residues, L173, L175, and R176, in the mature YfgL protein that are critical for both function and interactions with YaeT. A single substitution at any of these sites produces no phenotypic defect, but two or three simultaneous alterations produce mild or yfgL-null phenotypes, respectively. Interestingly, biochemical data show that all YfgL variants, including those with single substitutions, have weakened in vivo YaeT-YfgL interaction. These defects are not due to mislocalization or low steady-state levels of YfgL. Cysteine-directed cross-linking data show that the region encompassing L173, L175, and R176 makes direct contact with YaeT. Using the same genetic and biochemical strategies, it was found that altering residues D227 and D229 in another region of YfgL from E221 to D229 resulted in defective YaeT bindings. In contrast, mutational analysis of conserved residues V319 to H328 of YfgL shows that they are important for YfgL biogenesis but not YfgL-YaeT interactions. The five YfgL mutants defective in YaeT associations and the yfgL background were used to show that SurA binds to YaeT (or another complex member) without going through YfgL.

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Biochemistry and site-directed mutational analysis of Δ7-sterol-C5(6)-desaturase

Biochemical Society Transactions ◽

10.1042/bst0280799 ◽

2000 ◽

Vol 28 (6) ◽

pp. 799-803 ◽

Cited By ~ 6

Author(s):

A. Rahier ◽

P. Benveniste ◽

T. Husselstein ◽

M. Taton

Keyword(s):

Biochemical Characterization ◽

Mutational Analysis ◽

Enzyme System ◽

Conserved Residues ◽

Catalytic Role ◽

Membrane Bound ◽

Haem Iron ◽

Oxygen Site

This report describes recent work on the process of desaturation at C5(6) of sterol precursors in plants. Biochemical characterization of the plant Δ7-sterol C5(6)-desaturase (5-DES) indicates that the enzyme system involved shows important similarities to the soluble and membrane-bound non-haem iron desaturases found in eukaryotes, including cyanide and hydrophobic chelators sensitivity, CO resistance and a requirement for exogenous reductant and molecular oxygen. Site-directed mutational analysis of highly conserved residues in 5-DES indicated that eight histidine residues from three histidine-rich motifs were essential for the catalysis, possibly by providing the ligands for a putative Fe centre. This mutational analysis also revealed the catalytic role of the functionally conserved Thr-114.

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Mutational analysis of Mdm2 C-terminal tail suggests an evolutionarily conserved role of its length in Mdm2 activity toward p53 and indicates structural differences between Mdm2 homodimers and Mdm2/MdmX heterodimers

Cell Cycle ◽

10.4161/cc.11.5.19445 ◽

2012 ◽

Vol 11 (5) ◽

pp. 953-962 ◽

Cited By ~ 16

Author(s):

Pavlina Dolezelova ◽

Katerina Cetkovska ◽

Karen H Vousden ◽

Stjepan Uldrijan

Keyword(s):

Mutational Analysis ◽

Evolutionarily Conserved ◽

Structural Differences

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TRPA1 modulation by piperidine carboxamides suggests an evolutionarily conserved binding site and gating mechanism

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1913929116 ◽

2019 ◽

Vol 116 (51) ◽

pp. 26008-26019 ◽

Cited By ~ 6

Author(s):

Tania Chernov-Rogan ◽

Eleonora Gianti ◽

Chang Liu ◽

Elisia Villemure ◽

Andrew P. Cridland ◽

...

Keyword(s):

Binding Site ◽

Transient Receptor Potential ◽

Trp Channels ◽

Mutational Analysis ◽

Receptor Potential ◽

Covalent Modification ◽

Rational Drug Design ◽

Binding Modes ◽

Gating Mechanism ◽

Evolutionarily Conserved

The transient receptor potential ankyrin 1 (TRPA1) channel functions as an irritant sensor and is a therapeutic target for treating pain, itch, and respiratory diseases. As a ligand-gated channel, TRPA1 can be activated by electrophilic compounds such as allyl isothiocyanate (AITC) through covalent modification or activated by noncovalent agonists through ligand binding. However, how covalent modification leads to channel opening and, importantly, how noncovalent binding activates TRPA1 are not well-understood. Here we report a class of piperidine carboxamides (PIPCs) as potent, noncovalent agonists of human TRPA1. Based on their species-specific effects on human and rat channels, we identified residues critical for channel activation; we then generated binding modes for TRPA1–PIPC interactions using structural modeling, molecular docking, and mutational analysis. We show that PIPCs bind to a hydrophobic site located at the interface of the pore helix 1 (PH1) and S5 and S6 transmembrane segments. Interestingly, this binding site overlaps with that of known allosteric modulators, such as A-967079 and propofol. Similar binding sites, involving π-helix rearrangements on S6, have been recently reported for other TRP channels, suggesting an evolutionarily conserved mechanism. Finally, we show that for PIPC analogs, predictions from computational modeling are consistent with experimental structure–activity studies, thereby suggesting strategies for rational drug design.

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Mutational Analysis of Narrow Pores at the Fivefold Symmetry Axes of Adeno-Associated Virus Type 2 Capsids Reveals a Dual Role in Genome Packaging and Activation of Phospholipase A2 Activity

Journal of Virology ◽

10.1128/jvi.79.4.2528-2540.2005 ◽

2005 ◽

Vol 79 (4) ◽

pp. 2528-2540 ◽

Cited By ~ 102

Author(s):

Svenja Bleker ◽

Florian Sonntag ◽

Jürgen A. Kleinschmidt

Keyword(s):

Phospholipase A2 ◽

Virus Type ◽

Mutational Analysis ◽

Pla2 Activity ◽

Genome Packaging ◽

Adeno Associated Virus ◽

Conserved Residues ◽

Axes Of Symmetry ◽

Phospholipase A2 Activity

ABSTRACT Adeno-associated virus type 2 (AAV2) capsids show 12 pores at the fivefold axes of symmetry. We mutated amino acids which constitute these pores to investigate possible functions of these structures within the AAV2 life cycle. Mutants with alterations in conserved residues were impaired mainly in genome packaging or infectivity, whereas few mutants were affected in capsid assembly. The packaging phenotype was characterized by increased capsid-per-genome ratios. Analysis of capsid-associated DNA versus encapsidated DNA revealed that this observation was due to reduced and not partial DNA encapsidation. Most mutants with impaired infectivity showed a decreased capability to expose their VP1 N termini. As a consequence, the activation of phospholipase A2 (PLA2) activity, which is essential for efficient infection, was affected on intact capsids. In a few mutants, the exposure of VP1 N termini and the development of PLA2 activity were associated with enhanced capsid instability, which is obviously also deleterious for virus infection. Therefore, PLA2 activity seems to be required on intact capsids for efficient infection. In conclusion, these results suggest that the pores at the fivefold axes function not only as portals for AAV2 single-stranded DNA packaging but also as channels for presentation of the PLA2 domain on AAV2 virions during infection.

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Phosphatidylinositol 4,5-Bisphosphate Mediates the Targeting of the Exocyst to the Plasma Membrane for Exocytosis in Mammalian Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e07-05-0461 ◽

2007 ◽

Vol 18 (11) ◽

pp. 4483-4492 ◽

Cited By ~ 127

Author(s):

Jianglan Liu ◽

Xiaofeng Zuo ◽

Peng Yue ◽

Wei Guo

Keyword(s):

Plasma Membrane ◽

Mammalian Cells ◽

Direct Interaction ◽

Secretory Vesicles ◽

Vesicular Stomatitis Virus Glycoprotein ◽

Conserved Residues ◽

Vesicle Tethering ◽

C Terminus ◽

Evolutionarily Conserved ◽

Vesicular Stomatitis

The exocyst is an evolutionarily conserved octameric protein complex that tethers post-Golgi secretory vesicles at the plasma membrane for exocytosis. To elucidate the mechanism of vesicle tethering, it is important to understand how the exocyst physically associates with the plasma membrane (PM). In this study, we report that the mammalian exocyst subunit Exo70 associates with the PM through its direct interaction with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Furthermore, we have identified key conserved residues at the C-terminus of Exo70 that are crucial for the interaction of Exo70 with PI(4,5)P2. Disrupting Exo70-PI(4,5)P2 interaction abolished the membrane association of Exo70. We have also found that wild-type Exo70 but not the PI(4,5)P2-binding–deficient Exo70 mutant is capable of recruiting other exocyst components to the PM. Using the ts045 vesicular stomatitis virus glycoprotein trafficking assay, we demonstrate that Exo70-PI(4,5)P2 interaction is critical for the docking and fusion of post-Golgi secretory vesicles, but not for their transport to the PM.

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Mutational Analysis of Highly Conserved Residues in the Phage PhiC31 Integrase Reveals Key Amino Acids Necessary for the DNA Recombination

PLoS ONE ◽

10.1371/journal.pone.0008863 ◽

2010 ◽

Vol 5 (1) ◽

pp. e8863 ◽

Cited By ~ 13

Author(s):

Shaohui Liu ◽

Jinfang Ma ◽

Wei Wang ◽

Maoxiang Zhang ◽

Qingting Xin ◽

...

Keyword(s):

Amino Acids ◽

Mutational Analysis ◽

Dna Recombination ◽

Phic31 Integrase ◽

Conserved Residues

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Cancer genome datamining and functional genetic analysis implicate mechanisms of ATM/ATR dysfunction underpinning carcinogenesis

Communications Biology ◽

10.1038/s42003-021-01884-x ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Erik Waskiewicz ◽

Michalis Vasiliou ◽

Isaac Corcoles-Saez ◽

Rita S. Cha

Keyword(s):

Genetic Analysis ◽

Dna Damage Response ◽

Kinase Activity ◽

Cancer Genome ◽

Conserved Residues ◽

Protein Partner ◽

Evolutionarily Conserved ◽

Damage Response ◽

Critical Residues ◽

Enzyme Complexes

AbstractATM and ATR are conserved regulators of the DNA damage response linked to cancer. Comprehensive DNA sequencing efforts identified ~4,000 cancer-associated mutations in ATM/ATR; however, their cancer implications remain largely unknown. To gain insights, we identify functionally important conserved residues in ATM, ATR and budding yeast Mec1ATR via cancer genome datamining and a functional genetic analysis, respectively. Surprisingly, only a small fraction of the critical residues is in the active site of the respective enzyme complexes, implying that loss of the intrinsic kinase activity is infrequent in carcinogenesis. A number of residues are solvent accessible, suggestive of their involvement in interacting with a protein-partner(s). The majority, buried inside the respective enzyme complexes, might play a structural or regulatory role. Together, these findings identify evolutionarily conserved ATM, ATR, and Mec1ATR residues involved in diverse aspects of the enzyme function and provide fresh insights into the elusive genotype-phenotype relationships in ATM/ATR and their cancer-associated variants.

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