Solution structure of TbCentrin4 from Trypanosoma brucei and its interactions with Ca2+ and other centrins

2018 ◽  
Vol 475 (23) ◽  
pp. 3763-3778 ◽  
Author(s):  
Fangzhen Shan ◽  
Kaiqin Ye ◽  
Jiahai Zhang ◽  
Shanhui Liao ◽  
Xuecheng Zhang ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Calcium Binding ◽  
Solution Structure ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Resonance Spectroscopy ◽  
Basal Bodies ◽  
High Concentration ◽  
Ef Hand

Centrin is a conserved calcium-binding protein that plays an important role in diverse cellular biological processes such as ciliogenesis, gene expression, DNA repair and signal transduction. In Trypanosoma brucei, TbCentrin4 is mainly localized in basal bodies and bi-lobe structure, and is involved in the processes coordinating karyokinesis and cytokinesis. In the present study, we solved the solution structure of TbCentrin4 using NMR (nuclear magnetic resonance) spectroscopy. TbCentrin4 contains four EF-hand motifs consisting of eight α-helices. Isothermal titration calorimetry experiment showed that TbCentrin4 has a strong Ca2+ binding ability. NMR chemical shift perturbation indicated that TbCentrin4 binds to Ca2+ through its C-terminal domain composed of EF-hand 3 and 4. Meanwhile, we revealed that TbCentrin4 undergoes a conformational change and self-assembly induced by high concentration of Ca2+. Intriguingly, localization of TbCentrin4 was dispersed or disappeared from basal bodies and the bi-lobe structure when the cells were treated with Ca2+in vivo, implying the influence of Ca2+ on the cellular functions of TbCentrin4. Besides, we observed the interactions between TbCentrin4 and other Tbcentrins and revealed that the interactions are Ca2+ dependent. Our findings provide a structural basis for better understanding the biological functions of TbCentrin4 in the relevant cellular processes.

NMR Derived Solution Structure of an EF-Hand Calcium-Binding Protein fromEntamoeba Histolytica†

Biochemistry ◽  
2001 ◽  
Vol 40 (48) ◽  
pp. 14392-14403 ◽  
Author(s):  
H. S. Atreya ◽  
S. C. Sahu ◽  
A. Bhattacharya ◽  
Girjesh Govil
Keyword(s):  
Calcium Binding ◽  
Solution Structure ◽  
Binding Protein ◽  
Calcium Binding Protein ◽  
Ef Hand

scholarly journals NMR solution structure of calerythrin, an EF-hand calcium-binding protein from Saccharopolyspora erythraea

2003 ◽  
Vol 270 (11) ◽  
pp. 2505-2512 ◽  
Author(s):  
Helena Tossavainen ◽  
Perttu Permi ◽  
Arto Annila ◽  
Ilkka Kilpelainen ◽  
Torbjorn Drakenberg
Keyword(s):  
Calcium Binding ◽  
Solution Structure ◽  
Binding Protein ◽  
Calcium Binding Protein ◽  
Saccharopolyspora Erythraea ◽  
Nmr Solution Structure ◽  
Ef Hand

scholarly journals A mysterious family of calcium-binding proteins from parasitic worms

2016 ◽  
Vol 44 (4) ◽  
pp. 1005-1010 ◽  
Author(s):  
Charlotte M. Thomas ◽  
David J. Timson
Keyword(s):  
Calcium Ions ◽  
Calcium Binding ◽  
Cell Biology ◽  
Fasciola Hepatica ◽  
Family Members ◽  
Ion Concentration ◽  
Pharmacological Significance ◽  
Ef Hand ◽  
Parasitic Worms

There is a family of proteins from parasitic worms which combine N-terminal EF-hand domains with C-terminal dynein light chain-like domains. Data are accumulating on the biochemistry and cell biology of these proteins. However, little is known about their functions in vivo. Schistosoma mansoni expresses 13 family members (SmTAL1–SmTAL13). Three of these (SmTAL1, SmTAL2 and SmTAL3) have been subjected to biochemical analysis which demonstrated that they have different molecular properties. Although their overall folds are predicted to be similar, small changes in the EF-hand domains result in differences in their ion binding properties. Whereas SmTAL1 and SmTAL2 are able to bind calcium (and some other) ions, SmTAL3 appears to be unable to bind any divalent cations. Similar biochemical diversity has been seen in the CaBP proteins from Fasciola hepatica. Four family members are known (FhCaBP1–4). All of these bind to calcium ions. However, FhCaBP4 dimerizes in the presence of calcium ions, FhCaBP3 dimerizes in the absence of calcium ions and FhCaBP2 dimerizes regardless of the prevailing calcium ion concentration. In both the SmTAL and FhCaBP families, the proteins also differ in their ability to bind calmodulin antagonists and related drugs. Interestingly, SmTAL1 interacts with praziquantel (the drug of choice for treating schistosomiasis). The pharmacological significance (if any) of this finding is unknown.

scholarly journals The gene family of EF-hand calcium-binding proteins from the flagellum of Trypanosoma brucei

1994 ◽  
Vol 304 (3) ◽  
pp. 833-841 ◽  
Author(s):  
Y Wu ◽  
J Deford ◽  
R Benjamin ◽  
M G Lee ◽  
L Ruben
Keyword(s):  
Amino Acid ◽  
Trypanosoma Brucei ◽  
Calcium Binding ◽  
Binding Proteins ◽  
Direct Repeat ◽  
Calcium Binding Proteins ◽  
Amino Acid Sequences ◽  
Reading Frame ◽  
Genomic Clones ◽  
Ef Hand

The flagellum of Trypanosoma brucei contains calmodulin, and a separate family of antigenically related EF-hand calcium-binding proteins which we call calflagins. The following study evaluates the structure and genomic organization of the calflagin family. Genomic Southern blots indicated that multiple copies of calflagin genes occurred in T. brucei, and that all of these copies were contained in a single 23 kb XhoI-XhoI fragment on chromosomes 15 and 16 mRNAs of 1.2 and 1.6 kb were identified in bloodstream and procyclic life-cycle stages. Genomic fragments of 2.5 and 1.7 kb were cloned that encoded calflagin sequences. The calflagin genes were arranged tandemly along the genomic fragments. Three new members of the calflagin family were sequenced from a cDNA clone and the two genomic clones. Two unrelated families of 3′ flanking sequences were downstream from the calflagin genes. An open reading frame that was unrelated to any calflagin sequence was at the 5′ end of the 2.5 kb genomic fragment. The deduced amino acid sequences of the genomic clones (called Tb-24 and Tb-1.7g) were similar to the previously described Tb-17. Each encoded an approximately 24 kDa protein which contained three EF-hand calcium-binding motifs and one degenerate EF-hand motif. The cDNA encoded a protein (called Tb-44A) which was approximately twice as large as the other calflagins. The large size resulted from a nearly direct repeat of 186 amino acids. In general, variability among the T. brucei calflagins was greater than observed for related proteins from Trypanosoma cruzi. We demonstrate that this variability resulted from amino acid substitutions at the N-terminus, C-terminal extensions, and duplication of internal segments.

scholarly journals Solution structure of the PsIAA4 oligomerization domain reveals interaction modes for transcription factors in early auxin response

2015 ◽  
Vol 112 (19) ◽  
pp. 6230-6235 ◽  
Author(s):  
Dhurvas Chandrasekaran Dinesh ◽  
Michael Kovermann ◽  
Mohanraj Gopalswamy ◽  
Antje Hellmuth ◽  
Luz Irina A. Calderón Villalobos ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Electrostatic Interactions ◽  
Solution Structure ◽  
Protein Docking ◽  
Primary Response ◽  
Titration Calorimetry ◽  
Auxin Response ◽  
Pb1 Domain

The plant hormone auxin activates primary response genes by facilitating proteolytic removal of AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA)-inducible repressors, which directly bind to transcriptional AUXIN RESPONSE FACTORS (ARF). Most AUX/IAA and ARF proteins share highly conserved C-termini mediating homotypic and heterotypic interactions within and between both protein families. The high-resolution NMR structure of C-terminal domains III and IV of the AUX/IAA protein PsIAA4 from pea (Pisum sativum) revealed a globular ubiquitin-like β-grasp fold with homologies to the Phox and Bem1p (PB1) domain. The PB1 domain of wild-type PsIAA4 features two distinct surface patches of oppositely charged amino acid residues, mediating front-to-back multimerization via electrostatic interactions. Mutations of conserved basic or acidic residues on either face suppressed PsIAA4 PB1 homo-oligomerization in vitro and confirmed directional interaction of full-length PsIAA4 in vivo (yeast two-hybrid system). Mixing of oppositely mutated PsIAA4 PB1 monomers enabled NMR mapping of the negatively charged interface of the reconstituted PsIAA4 PB1 homodimer variant, whose stoichiometry (1:1) and equilibrium binding constant (KD ∼6.4 μM) were determined by isothermal titration calorimetry. In silico protein–protein docking studies based on NMR and yeast interaction data derived a model of the PsIAA4 PB1 homodimer, which is comparable with other PB1 domain dimers, but indicated considerable differences between the homodimeric interfaces of AUX/IAA and ARF PB1 domains. Our study provides an impetus for elucidating the molecular determinants that confer specificity to complex protein–protein interaction circuits between members of the two central families of transcription factors important to the regulation of auxin-responsive gene expression.

scholarly journals Calcium-Induced Activity and Folding of a Repeat in Toxin Lipase from Antarctic Pseudomonas fluorescens Strain AMS8

Toxins ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 27 ◽  
Author(s):  
Nur Shidaa Mohd Ali ◽  
Abu Bakar Salleh ◽  
Raja Noor Zaliha Raja Abd Rahman ◽  
Thean Chor Leow ◽  
Mohd Shukuri Mohamad Ali
Keyword(s):  
Fourier Transform ◽  
Secondary Structure ◽  
Pseudomonas Fluorescens ◽  
Calcium Binding ◽  
Protein Structures ◽  
Titration Calorimetry ◽  
Ftir Analysis ◽  
High Concentration ◽  
Spectroscopy Analysis ◽  
Repeat Structure

It is hypothesized that the Ca2+ ions were involved in the activity, folding and stabilization of many protein structures. Many of these proteins contain repeat in toxin (RTX) motifs. AMS8 lipase from Antarctic Pseudomonas fluorescens strain AMS8 was found to have three RTX motifs. So, this research aimed to examine the influence of Ca2+ ion towards the activity and folding of AMS8 lipase through various biophysical characterizations. The results showed that CaCl2 increased lipase activity. The far-UV circular dichroism (CD) and Fourier-transform infrared (FTIR) analysis suggested that the secondary structure content was improved with the addition of CaCl2. Fluorescence spectroscopy analysis showed that the presence of CaCl2 increased protein folding and compactness. Dynamic light scattering (DLS) analysis suggested that AMS8 lipase became aggregated at a high concentration of CaCl2.The binding constant (Kd) value from the isothermal titration calorimetry (ITC) analysis proved that the Ca2+ ion was tightly bound to the AMS8 lipase. In conclusion, Ca2+ ions play crucial roles in the activity and folding of the AMS8 lipase. Calcium binding to RTX nonapeptide repeats sequences will induced the formation and folding of the RTX parallel β-roll motif repeat structure.

scholarly journals Calcium Binding Is Required for Calmodulin Function in Aspergillus nidulans

2002 ◽  
Vol 1 (1) ◽  
pp. 119-125 ◽  
Author(s):  
James D. Joseph ◽  
Anthony R. Means
Keyword(s):  
Amino Acid ◽  
Aspergillus Nidulans ◽  
Calcium Binding ◽  
Structural Basis ◽  
Wild Type ◽  
Endogenous Protein ◽  
Chimeric Molecules ◽  
Overlay Assay

ABSTRACT To explore the structural basis for the essential role of calmodulin (CaM) in Aspergillus nidulans, we have compared the biochemical and in vivo properties of A. nidulans CaM (AnCaM) with those of heterologous CaMs. Neither Saccharomyces cerevisiae CaM (ScCaM) nor a Ca2+ binding mutant of A. nidulans CaM (1234) interacts appreciably with A. nidulans CaM binding proteins by an overlay assay or activates two essential CaMKs, CMKA and CMKB. In contrast, although vertebrate CaM (VCaM) binds a spectrum of proteins similar to that for AnCaM, it is unable to fully activate CMKA and CMKB, displaying a higher K CaM and reduced V max for both enzymes. In correlation with the biochemical analysis, neither ScCaM nor 1234 can support A. nidulans growth in the absence of the endogenous protein, whereas VCaM only partially complements the absence of wild-type CaM. Analysis of VCaM and AnCaM chimeras demonstrates that amino acid variations in both N- and C-terminal domains contribute to the inability of VCaM to activate CMKB, but differences in the N terminus are largely responsible for the reduced activity towards CMKA. In vivo, the chimeric molecules support growth equivalently, but only to levels intermediate between those of VCaM and AnCaM, suggesting that the reduced ability to activate the CaMKs is not solely responsible for the inability of VCaM to complement the absence of the wild-type protein. Thus, not only is Ca2+ binding required for CaM function in A. nidulans, but the essential in vivo functions of A. nidulans CaM are uniquely sensitive to the subtle amino acid variations present in vertebrate CaM.

scholarly journals Structural basis for adhesion G protein-coupled receptor Gpr126 function

2019 ◽  
Author(s):  
Katherine Leon ◽  
Rebecca L. Cunningham ◽  
Joshua A. Riback ◽  
Ezra Feldman ◽  
Jingxian Li ◽  
...  
Keyword(s):  
Binding Site ◽  
G Protein ◽  
Heart Development ◽  
Calcium Binding ◽  
Structural Basis ◽  
Calcium Binding Site ◽  
Surface Receptors ◽  
Alternatively Spliced ◽  
G Protein Coupled

AbstractMany drugs target the extracellular regions (ECRs) of cell-surface receptors. The large and alternatively-spliced ECRs of adhesion G protein-coupled receptors (aGPCRs) have key functions in diverse biological processes including neurodevelopment, embryogenesis, and tumorigenesis. However, their structures and mechanisms of action remain unclear, hampering drug development. The aGPCR Gpr126/Adgrg6 regulates Schwann cell myelination, ear canal formation, and heart development; and GPR126 mutations cause myelination defects in human. Here, we determine the structure of the complete zebrafish Gpr126 ECR and reveal five domains including a previously-unknown domain. Strikingly, the Gpr126 ECR adopts a closed conformation that is stabilized by an alternatively spliced linker and a conserved calcium-binding site. Alternative splicing regulates ECR conformation and receptor signaling, while mutagenesis of the newly-characterized calcium-binding site abolishes Gpr126 function in vivo. These results demonstrate that Gpr126 ECR utilizes a multi-faceted dynamic approach to regulate receptor function and provide novel insights into ECR-targeted drug design.

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