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Chemical Biology Tools to Elucidate the Solution Conformation and Cellular Interactions of Cyclic Adenosine 5′‐Diphosphate Ribose
Author(s) -
Uwakwe Chiemeka David,
Grega Sarah,
Serrano Inis,
Graham Steven
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.00683
Subject(s) - cyclic adp ribose , chemistry , nad+ kinase , second messenger system , nicotinamide adenine dinucleotide , stereochemistry , biophysics , allosteric regulation , nuclear magnetic resonance spectroscopy , biochemistry , adenosine diphosphate , cd38 , enzyme , biology , microbiology and biotechnology , stem cell , cd34 , platelet , platelet aggregation , immunology
The movement of calcium ions (Ca 2+ ) is intimately involved in regulating many physiological processes, and so there is great interest in the structure of molecules, especially second messengers, that regulate this flow. One such messenger is cyclic adenosine 5′‐diphosphate ribose (cADPR), which is suspected to play a role in Ca 2+ release in cardiomyocytes. The speculated mechanism of action involves the binding of cADPR to FKBP12.6, releasing FKBP12.6 from RyR2 channels, through which Ca 2+ enters the sarcoplasm from the sarcoplasmic reticulum during ventricular systole. Guided by the principle that structure determines function, we aim to advance the investigation of regulatory cADPR interactions by synthesizing and studying cADPR and its analogs using the combined tools of liquid chromatography‐mass spectrometry (LCMS) and nuclear magnetic resonance (NMR) spectroscopy. The endogenous cyclization of nicotinamide adenine dinucleotide (NAD + ) to cADPR by CD38 is mimicked in vitro using ADP‐ribosylcyclase (ADP‐RC, sourced from Aplysia californica). The low substrate specificity of ADP‐RC conveniently permits the cyclization of various cADPR analogs. Kinetic assays were prompted by an observed feedback inhibition of ADP‐RC by nicotinamide (IC 50 ~ 1mM), and performed using LCMS. Following synthesis and purification, NMR experimental data provided insight into favored structural conformations of cADPR (Saatori et al., 2018) Photoaffinity labeling (PAL) was aimed at identifying the cognate binding partners of cADPR, some of which have been previously described by Walseth et al. (1993). Conformational analyses of cADPR analogs revealed structural trends with correlations to the biological activity of the analogs. Support or Funding Information Department of Chemistry, Saint John’s University