scholarly journals Molecular characterization of CHAD domains as inorganic polyphosphate binding modules

2019 ◽  
Author(s):  
Laura Lorenzo-Orts ◽  
Ulrich Hohmann ◽  
Jinsheng Zhu ◽  
Michael Hothorn
Keyword(s):  
Mutational Analysis ◽  
Dissociation Constants ◽  
Complex Structure ◽  
High Specificity ◽  
Eukaryotic Cells ◽  
Inorganic Polyphosphates ◽  
Inorganic Polyphosphate ◽  
Surface Patches ◽  
C Terminus

AbstractInorganic polyphosphates (polyPs) are long polymers of orthophosphate units (Pi), linked by energy-rich phosphoanhydride bonds. Conserved histidine α-helical (CHAD) domains of unknown biochemical function are often located at the C-terminus of polyP-metabolizing triphosphate tunnel metalloenzymes (TTMs), or can be found as stand-alone proteins in bacterial operons harboring polyP kinases or phosphatases. Here we report that bacterial, archaeal and eukaryotic CHAD domains are specific polyP binding modules. Crystal structures reveal that CHAD domains are formed by two four-helix bundles, giving rise to a central cavity surrounded by two conserved basic surface patches. Different CHAD domains bind polyPs with dissociation constants ranging from the nano-to mid-micromolar range, but not DNA or other Pi-containing ligands. A 2.1 Å CHAD - polyP complex structure reveals the phosphate polymer binding across a central pore and along the two basic patches. Mutational analysis of CHAD – polyP interface residues validates the complex structure and reveals that CHAD domains evolved to bind long-chain polyPs. The presence of a CHAD domain in the polyPase ygiF enhances its enzymatic activity. In plants, CHAD domains bind polyP in vivo and localize to the nucleus and nucleolus, suggesting that plants harbor polyP stores in these compartments. We propose that CHAD domains may be used to engineer the properties of polyP-metabolizing enzymes and to specifically localize polyP stores in eukaryotic cells and tissues.SignificanceA domain of unknown function termed CHAD, present in all kingdoms of life, is characterized as a specific inorganic polyphosphate binding domain. The small size of the domain and its high specificity for inorganic polyphosphates suggest that it could be used as a tool to locate inorganic polyphosphate stores in pro- and eukaryotic cells and tissues.

scholarly journals Molecular characterization of CHAD domains as inorganic polyphosphate-binding modules

2019 ◽  
Vol 2 (3) ◽  
pp. e201900385 ◽  
Author(s):  
Laura Lorenzo-Orts ◽  
Ulrich Hohmann ◽  
Jinsheng Zhu ◽  
Michael Hothorn
Keyword(s):  
Mutational Analysis ◽  
Dissociation Constants ◽  
Ricinus Communis ◽  
Complex Structure ◽  
Linear Polymers ◽  
Inorganic Polyphosphate ◽  
Metabolizing Enzymes ◽  
Surface Patches ◽  
Central Pore

Inorganic polyphosphates (polyPs) are linear polymers of orthophosphate units linked by phosphoanhydride bonds. Here, we report that bacterial, archaeal, and eukaryotic conserved histidine α-helical (CHAD) domains are specific polyP-binding modules. Crystal structures reveal that CHAD domains are formed by two four-helix bundles, giving rise to a central pore surrounded by conserved basic surface patches. Different CHAD domains bind polyPs with dissociation constants ranging from the nano- to mid-micromolar range, but not nucleic acids. A CHAD—polyP complex structure reveals the phosphate polymer binding across the central pore and along the two basic patches. Mutational analysis of CHAD—polyP interface residues validates the complex structure. The presence of a CHAD domain in the polyPase ygiF enhances its enzymatic activity. The only known CHAD protein from the plant Ricinus communis localizes to the nucleus/nucleolus when expressed in Arabidopsis and tobacco, suggesting that plants may harbor polyPs in these compartments. We propose that CHAD domains may be used to engineer the properties of polyP-metabolizing enzymes and to specifically localize polyP stores in eukaryotic cells and tissues.

SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana

2020 ◽  
Vol 477 (1) ◽  
pp. 173-189 ◽  
Author(s):  
Marco Pedretti ◽  
Carolina Conter ◽  
Paola Dominici ◽  
Alessandra Astegno
Keyword(s):  
Excision Repair ◽  
Complex Structure ◽  
Binding Motif ◽  
C Terminus ◽  
Repair Protein ◽  
Nucleotide Excision ◽  
Ef Hand ◽  
Molecular Determinants ◽  
Structure In Solution

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp–CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein–peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

scholarly journals Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Mutational Analysis ◽  
Wild Type ◽  
Content Type ◽  
Growth Defects ◽  
C Terminus ◽  
K 12 ◽  
And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

scholarly journals Mutational analysis of yeast profilin.

1993 ◽  
Vol 13 (12) ◽  
pp. 7864-7873 ◽  
Author(s):  
B K Haarer ◽  
A S Petzold ◽  
S S Brown
Keyword(s):  
Amino Acids ◽  
Adenylate Cyclase ◽  
Mutational Analysis ◽  
Actin Binding ◽  
In Vitro Assays ◽  
C Terminus ◽  
Physiological Relevance ◽  
In Vivo Effects

We have mutated two regions within the yeast profilin gene in an effort to functionally dissect the roles of actin and phosphatidylinositol 4,5-bisphosphate (PIP2) binding in profilin function. A series of truncations was carried out at the C terminus of profilin, a region that has been implicated in actin binding. Removal of the last three amino acids nearly eliminated the ability of profilin to bind polyproline in vitro but had no dramatic in vivo effects. Thus, the extreme C terminus is implicated in polyproline binding, but the physiological relevance of this interaction is called into question. More extensive truncation, of up to eight amino acids, had in vivo effects of increasing severity and resulted in changes in conformation and expression level of the mutant profilins. However, the ability of these mutants to bind actin in vitro was not eliminated, suggesting that this region cannot be solely responsible for actin binding. We also mutagenized a region of profilin that we hypothesized might be involved in PIP2 binding. Alteration of basic amino acids in this region produced mutant profilins that functioned well in vivo. Many of these mutants, however, were unable to suppress the loss of adenylate cyclase-associated protein (Cap/Srv2p [A. Vojtek, B. Haarer, J. Field, J. Gerst, T. D. Pollard, S. S. Brown, and M. Wigler, Cell 66:497-505, 1991]), indicating that a defect could be demonstrated in vivo. In vitro assays demonstrated that the inability to suppress loss of Cap/Srv2p correlated with a defect in the interaction with actin, independently of whether PIP2 binding was reduced. Since our earlier studies of Acanthamoeba profilins suggested the importance of PIP2 binding for suppression, we conclude that both activities are implicated and that an interplay between PIP2 binding and actin binding may be important for profilin function.

scholarly journals In vivo import of firefly luciferase into the glycosomes of Trypanosoma brucei and mutational analysis of the C-terminal targeting signal.

1992 ◽  
Vol 3 (7) ◽  
pp. 749-759 ◽  
Author(s):  
J M Sommer ◽  
Q L Cheng ◽  
G A Keller ◽  
C C Wang
Keyword(s):  
Amino Acid ◽  
Trypanosoma Brucei ◽  
Protein Import ◽  
Mutational Analysis ◽  
Firefly Luciferase ◽  
C Terminus ◽  
Peroxisomal Targeting ◽  
Hydrophobic Amino Acids ◽  
Targeting Signal

The compartmentalization of glycolytic enzymes into specialized organelles, the glycosomes, allows the bloodstream form of Trypanosoma brucei to rely solely on glycolysis for its energy production. The biogenesis of glycosomes in these parasites has been studied intensively as a potential target for chemotherapy. We have adapted the recently developed methods for stable transformation of T. brucei to the in vivo analysis of glycosomal protein import. Firefly luciferase, a peroxisomal protein in the lantern of the insect, was expressed in stable transformants of the procyclic form of T. brucei, where it was found to accumulate inside the glycosomes. Mutational analysis of the peroxisomal targeting signal serine-lysine-leucine (SKL) located at the C-terminus of luciferase showed that replacement of the serine residue (Serine548) with a small neutral amino acid (A, C, G, H, N, P, T) still resulted in an import efficiency of 50-100% of the wild-type luciferase. Lysine549 could be substituted with an amino acid capable of hydrogen bonding (H, M, N, Q, R, S), whereas the C-terminal leucine550 could be replaced with a subset of hydrophobic amino acids (I, M, Y). Thus, a peroxisome-like C-terminal SKL-dependent targeting mechanism may function in T. brucei to import luciferase into the glycosomes. However, a few significant differences exist between the glycosomal targeting signals identified here and the tripeptide sequences that direct proteins to mammalian or yeast peroxisomes.

scholarly journals Bromodomain Factor 1 (Bdf1) Is Phosphorylated by Protein Kinase CK2

2004 ◽  
Vol 24 (11) ◽  
pp. 4734-4742 ◽  
Author(s):  
Chika Sawa ◽  
Eduard Nedea ◽  
Nevan Krogan ◽  
Tadashi Wada ◽  
Hiroshi Handa ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Rna Polymerase Ii ◽  
Protein Kinase Ck2 ◽  
Mutational Analysis ◽  
Histone H4 ◽  
C Terminus ◽  
Bromodomain Factor 1 ◽  
C Complex ◽  
Kinase Ck2

ABSTRACT Bromodomain factor 1 (Bdf1) associates with Saccharomyces cerevisiae TFIID and corresponds to the C-terminal half of higher eukaryotic TAF1. It also associates with the SWR-C complex, which is important for Htz1 deposition. Bdf1 binds preferentially to acetylated histone H4. Bdf1 is phosphorylated, but the mechanism and significance of this modification have been unclear. Two distinct regions within Bdf1 are phosphorylated; one is just C terminal to the bromodomains and the other is near the C terminus. Mutational analysis shows that phosphorylation is necessary for Bdf1 function in vivo. Endogenous protein kinase CK2 purifies with Bdf1 and phosphorylates both domains. A similar mechanism may be responsible for phosphorylation of the C-terminal region of mammalian TAF1. These findings suggest that CK2 phosphorylation of Bdf1 may regulate RNA polymerase II transcription and/or chromatin structure.

Functions of inorganic polyphosphates in eukaryotic cells: a coat of many colours

2014 ◽  
Vol 42 (1) ◽  
pp. 98-102 ◽  
Author(s):  
Cristina Azevedo ◽  
Adolfo Saiardi
Keyword(s):  
High Energy ◽  
Linear Polymer ◽  
Detection Methods ◽  
Eukaryotic Cells ◽  
Inorganic Polyphosphates ◽  
Inorganic Polyphosphate ◽  
Structural Functions ◽  
Higher Eukaryotes ◽  
Living Organisms ◽  
And Function

PolyP (inorganic polyphosphate) is a linear polymer of tens to hundreds of orthophosphate residues linked by high-energy phosphoanhydride bonds. This polymer is present in all living organisms from bacteria to mammals. Until recently, most of the studies on polyP have focused on its function in prokaryotes. In prokaryotes, polyP has been implicated in many unrelated processes ranging from basic metabolism to structural functions. However, polyP analysis and function in higher eukaryotes has been gaining momentum recently. In the present review, we mainly aim to discuss the proposed intracellular functions of polyP in higher eukaryotes and its detection methods.

Mutational analysis of yeast profilin

1993 ◽  
Vol 13 (12) ◽  
pp. 7864-7873
Author(s):  
B K Haarer ◽  
A S Petzold ◽  
S S Brown
Keyword(s):  
Amino Acids ◽  
Adenylate Cyclase ◽  
Mutational Analysis ◽  
Actin Binding ◽  
In Vitro Assays ◽  
C Terminus ◽  
Physiological Relevance ◽  
In Vivo Effects

We have mutated two regions within the yeast profilin gene in an effort to functionally dissect the roles of actin and phosphatidylinositol 4,5-bisphosphate (PIP2) binding in profilin function. A series of truncations was carried out at the C terminus of profilin, a region that has been implicated in actin binding. Removal of the last three amino acids nearly eliminated the ability of profilin to bind polyproline in vitro but had no dramatic in vivo effects. Thus, the extreme C terminus is implicated in polyproline binding, but the physiological relevance of this interaction is called into question. More extensive truncation, of up to eight amino acids, had in vivo effects of increasing severity and resulted in changes in conformation and expression level of the mutant profilins. However, the ability of these mutants to bind actin in vitro was not eliminated, suggesting that this region cannot be solely responsible for actin binding. We also mutagenized a region of profilin that we hypothesized might be involved in PIP2 binding. Alteration of basic amino acids in this region produced mutant profilins that functioned well in vivo. Many of these mutants, however, were unable to suppress the loss of adenylate cyclase-associated protein (Cap/Srv2p [A. Vojtek, B. Haarer, J. Field, J. Gerst, T. D. Pollard, S. S. Brown, and M. Wigler, Cell 66:497-505, 1991]), indicating that a defect could be demonstrated in vivo. In vitro assays demonstrated that the inability to suppress loss of Cap/Srv2p correlated with a defect in the interaction with actin, independently of whether PIP2 binding was reduced. Since our earlier studies of Acanthamoeba profilins suggested the importance of PIP2 binding for suppression, we conclude that both activities are implicated and that an interplay between PIP2 binding and actin binding may be important for profilin function.

scholarly journals A systematic mutational analysis identifies a 5-residue proline tag that enhances the in vivo immunogenicity of a non-immunogenic model protein 240 folds

2020 ◽  
Author(s):  
Nafsoon Rahman ◽  
Mohammad Monirul Islam ◽  
Md Golam Kibria ◽  
Satoru Unzai ◽  
Yutaka Kuroda
Keyword(s):  
Mutational Analysis ◽  
Peptide Vaccines ◽  
Tertiary Structures ◽  
Small Proteins ◽  
C Terminus ◽  
Pancreatic Trypsin Inhibitor ◽  
Model Protein ◽  
Soluble Aggregates ◽  
Igg Level

AbstractSmall proteins are generally non-immunogenic, which can be a major hurdle in developing protein and peptide vaccines or producing antibodies for biopharmaceutical usage. For improving a protein’s immunogenicity, we previously proposed to use short Solubility Controlling Peptide (SCP) tags that oligomerize proteins into soluble aggregates. Here, we systematically analyzed the effect of SCP-tags that do not induce oligomerization on the immunogenicity of a small, non-immunogenic, model protein, Bovine Pancreatic Trypsin Inhibitor (BPTI-19A; 6 kDa). We assessed the effect of the following ten SCP-tags: Six tags made of five consecutive Arg, Lys, His, Asp, Asn, Pro; one made of seven Pro; two tags made of consecutive Arg-lle and Asn-Ile, all attached at the C-terminus of BPTI-19A; and a 5-proline tag attached at the N-terminus. Circular dichroism, fluorescence, dynamic light scattering measurements, and analytical ultra-centrifugation indicated that the addition of the SCP-tags did not change the secondary structure content nor the tertiary structures of the protein nor its monomeric state. On the other hand, the C-terminus 5-proline (C5P) tag unexpectedly increased the immunogenicity (IgG level) of BPTI-19A by up to 240 fold as assessed by ELISA. Additionally, the 5-arginine tag (C5R) increased the titer by up to 73 fold. The titer increase lasted for several weeks, and the effect was cumulative to that of the Freund’s adjuvant, which is commonly used to boost a protein’s immunogenicity. Altogether, SCP-tags that do not oligomerize proteins substantially increased the immunogenicity of a non-immunogenic protein, suggesting that the 5-proline and the 5-arginine SCP-tags may provide a novel tool for facilitating the production of antibodies or improving the effectiveness of protein-based vaccines.

scholarly journals A Conserved Mechanism of Synaptogyrin Localization

2001 ◽  
Vol 12 (8) ◽  
pp. 2275-2289 ◽  
Author(s):  
Hongjuan Zhao ◽  
Michael L. Nonet
Keyword(s):  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
C Elegans ◽  
C Terminus ◽  
Green Fluorescent ◽  
Cultured Hippocampal Neurons

We have studied the localization of synaptogyrin family members in vivo. Both native and green fluorescent protein (GFP)-taggedCaenorhabditis elegans synaptogyrin (SNG-1) are expressed in neurons and synaptically localized. Deletion and mutational analysis with the use of GFP-tagged SNG-1 has defined a 38 amino acid sequence within the C terminus of SNG-1 and a single arginine in the cytoplasmic loop between transmembrane domain 2 and 3 that are required for SNG-1 localization. These domains may represent components of signals that target synaptogyrin for endocytosis from the plasma membrane and direct synaptogyrin to synaptic vesicles, respectively. In chimeric studies, these regions were sufficient to relocalize cellugyrin, a nonneuronal form of synaptogyrin, from nonsynaptic regions such as the sensory dendrites and the cell body to synaptic vesicles. Furthermore, GFP-tagged rat synaptogyrin is synaptically localized in neurons of C. elegans and in cultured hippocampal neurons. Similarly, the C-terminal domain of rat synaptogyrin is necessary for localization in hippocampal neurons. Our study suggests that the mechanisms for synaptogyrin localization are likely to be conserved from C. elegans to vertebrates.

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