scholarly journals Machine learning-based modulation of Ca2+-binding affinity in EFhand proteins and comparative structural insights into site-specific cooperative binding

2021 ◽  
Author(s):  
Mohit Mazumder ◽  
Sanjeev Kumar ◽  
Devbrat Kumar ◽  
Samudrala Gourinath
Keyword(s):  
Calcium Binding ◽  
Site Directed Mutagenesis ◽  
Binding Affinities ◽  
Dynamic Correlation ◽  
Loop Sequence ◽  
Binding Isotherms ◽  
Ef Hand ◽  
Coordination Spheres ◽  
Living Organisms ◽  
Almost All

Ca2+-binding proteins are present in almost all living organisms and different types display different levels of binding affinities for the cation. Here, we applied two new scoring schemes enabling the user to manipulate the binding affinities of such proteins. We specifically designed a unique EF-hand loop capable of binding calcium with high affinity by altering five residues of the loop based on the scoring scheme. We worked on the N-terminal domain of Entamoeba histolytica calcium-binding protein1 (NtEhCaBP1), and used site-directed mutagenesis to incorporate the designed loop sequence into the second EF hand motif of this protein. The binding isotherms calculated using ITC calorimetry showed a ~500-fold greater association constant (Ka) for the mutant. The crystal structure of the mutant was also determined, and displayed more compact Ca2+-coordination spheres in both of its EF loops than did the structure of the wildtype protein, consistent with the greater calcium-binding affinities of the mutant. The NtEhCaBP1 mutant was also shown to form a hexamer rather than just a trimer, and this hexamer formation was attributed to the position of the last helix of the mutant having been changed as a result of the strong calcium coordination. Further dynamic correlation analysis revealed that the mutation in the second EF loop changed the entire residue network of the monomer, resulting in a stronger coordination of Ca2+.

scholarly journals Structural elements within the methylation loop (residues 112–117) and EF hands III and IV of calmodulin are required for Lys115 trimethylation

1999 ◽  
Vol 340 (2) ◽  
pp. 417-424 ◽  
Author(s):  
Jennifer A. COBB ◽  
Chang-Hoon HAN ◽  
David M. WILLS ◽  
Daniel M. ROBERTS
Keyword(s):  
Single Point ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Structural Elements ◽  
Nad Kinase ◽  
Wild Type ◽  
Loop Sequence ◽  
Ef Hand ◽  
Single Point Mutations ◽  
Ef Hands

Calmodulin is trimethylated by a specific methyltransferase on Lys115, a residue located in a six amino acid loop (LGEKLT) between EF hands III and IV. To investigate the structural requirements for methylation, domain exchange mutants as well as single point mutations of conserved methylation loop residues (E114A, Glu114 → Ala; L116T, Leu116 → Thr) were generated. E114A and L116T activated cyclic nucleotide phosphodiesterase (PDE) and NAD+ kinase (NADK) similar to wild-type calmodulin, but lost their ability to be methylated. Domain exchange mutants in which EF hand III or IV was replaced by EF hand I or II respectively (CaM1214 and CaM1232 respectively) showed a modest effect on PDE and NADK activation (50 to 100% of wild-type), but calmodulin methylation was abolished. A third domain exchange mutant, CaMEKL, has the methylation loop sequence placed at a symmetrical position between EF hands I and II in the N-terminal lobe [residues QNP(41-43) replaced by EKL]. CaMEKL activated PDE normally, but did not activate NADK. However, CaMEKL retained the ability to bind to NADK and inhibited activation by wild-type calmodulin. Site-directed mutagenesis of single residues showed that Gln41 and Pro43 substitutions had the strongest effect on NADK activation. Additionally, CaMEKL was not methylated, suggesting that the introduction of the methylation loop between EF hands I and II is not adequate for methyltransferase recognition. Overall the data indicate that residues in the methylation loop are essential but not sufficient for methyltransferase recognition, and that additional residues unique to EF hands III and IV are required. Secondly, the QNP sequence in the loop between EF hands I and II is necessary for NADK activation.

Obtaining Site-Specific Calcium-Binding Affinities Of Calmodulin

2003 ◽  
Vol 10 (4) ◽  
pp. 331-345 ◽  
Author(s):  
Jenny Yang ◽  
Amy Gawthrop ◽  
Yiming Ye
Keyword(s):  
Calcium Binding ◽  
Binding Affinities ◽  
Site Specific

scholarly journals Changes in the Population Size of Calbindin D-28k-Immunoreactive Enteric Neurons in the Porcine Caecum under the Influence of Bisphenol A: A Preliminary Study

Toxics ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 1
Author(s):  
Ignacy Gonkowski ◽  
Slawomir Gonkowski ◽  
Ewa Dzika ◽  
Joanna Wojtkiewicz
Keyword(s):  
Bisphenol A ◽  
Calcium Binding ◽  
Neuronal Cells ◽  
Enteric Neurons ◽  
Toxic Activity ◽  
Protective Properties ◽  
Calcium Buffer ◽  
Preliminary Study ◽  
Living Organisms ◽  
Calbindin D

Calbindin D-28k (CB) is a calcium-binding protein widely distributed in living organisms that may act as a calcium buffer and sensory protein. CB is present in the enteric nervous system (ENS) situated in the gastrointestinal tract, which controls the majority of activities of the stomach and intestine. The influence of various doses of bisphenol A (BPA)—a chemical compound widely used in plastics production—on the number and distribution of CB-positive enteric neuronal cells in the porcine caecum was investigated with an immunofluorescence technique. The obtained results showed that low dosages of BPA resulted in an increase in the number of CB-positive neuronal cells in the myenteric (MP) and inner submucous (ISP) plexuses, whereas it did not alter the number of such neuronal cells in the outer submucous plexus (OSP). High dosages of BPA caused the increase in the amount of CB-positive perikarya in all the above-mentioned kinds of the caecal neuronal plexuses. These observations strongly suggest that CB in the ENS participates in the processes connected with the toxic activity of BPA. Most likely, the changes noted in this experiment result from the adaptive and protective properties of CB.

scholarly journals The Diverse Calpain Family in Trypanosomatidae: Functional Proteins Devoid of Proteolytic Activity?

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 299
Author(s):  
Vítor Ennes-Vidal ◽  
Marta Helena Branquinha ◽  
André Luis Souza dos Santos ◽  
Claudia Masini d’Avila-Levy
Keyword(s):  
Proteolytic Activity ◽  
Current Knowledge ◽  
Current Therapy ◽  
Calcium Dependent ◽  
Administration Routes ◽  
Life Threatening ◽  
Living Organisms ◽  
Almost All ◽  
Open Question ◽  
Calpain Inhibitors

Calpains are calcium-dependent cysteine peptidases that were originally described in mammals and, thereafter, their homologues were identified in almost all known living organisms. The deregulated activity of these peptidases is associated with several pathologies and, consequently, huge efforts have been made to identify selective inhibitors. Trypanosomatids, responsible for life-threatening human diseases, possess a large and diverse family of calpain sequences in their genomes. Considering that the current therapy to treat trypanosomatid diseases is limited to a handful of drugs that suffer from unacceptable toxicity, tough administration routes, like parenteral, and increasing treatment failures, a repurposed approach with calpain inhibitors could be a shortcut to successful chemotherapy. However, there is a general lack of knowledge about calpain functions in these parasites and, currently, the proteolytic activity of these proteins is still an open question. Here, we highlight the current research and perspectives on trypanosomatid calpains, overview calpain description in these organisms, and explore the potential of targeting the calpain system as a therapeutic strategy. This review gathers the current knowledge about this fascinating family of peptidases as well as insights into the puzzle: are we unable to measure calpain activity in trypanosomatids, or are the functions of these proteins devoid of proteolytic activity in these parasites?

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