Abstract
Calbindin-D28K is a Ca2+-binding protein, performing roles as both a calcium buffer and calcium sensor. The NMR solution structure of Ca2+-loaded calbindin-D28K reveals a single, globular fold consisting of six distinct EF-hand subdomains, which coordinate Ca2+ in loops on EF1, EF3, EF4 and EF5. Target peptides from Ran-binding protein M and myo-inositol monophosphatase, along with a new target from procaspase-3, are shown to interact with the protein on a surface comprised of α5 (EF3), α8 (EF4) and the EF2-EF3 and EF4-EF5 loops. Fluorescence experiments reveal that calbindin-D28K adopts discrete hydrophobic states as it binds Ca2+. The structure, binding interface and hydrophobic characteristics of Ca2+-loaded calbindin-D28K provide the first detailed insights into how this essential protein may function. This structure is one of the largest high-resolution NMR structures and the largest monomeric EF-hand protein to be solved to date.
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Acknowledgements
The Duke University NMR Center and the North Carolina State University NMR Facility were established with grants from the US National Institutes of Health, US National Science Foundation and North Carolina Biotechnology Center. We acknowledge support of this work by grants from the US National Institutes of Health (to R.K. and J.C.), the Kenan Institute for Engineering, Technology & Science (to J.C.) and the American Foundation for Aging Research (to D.J.K and D.R.K.).
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D.J.K. contributed to assignments, structure calculations and manuscript preparation; R.A.V. to assignments, structure calculations, NMR data collection and analysis, and manuscript preparation; D.R.K. to sample preparations, fluorescence experiments and peptide-binding titrations; R.J.T. to sample preparations and NMR data collection; R.K. to RanBPM studies; and J.C. to NMR data collection and analysis, peptide-binding titrations and manuscript preparation.
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Supplementary information
Supplementary Fig. 1
Secondary structure and NOE statistics form Ca2+-loaded calbindin-D28K (PDF 5832 kb)
Supplementary Fig. 2
Measuring hydrophobic surface content via ANS fluorescence as calbindin-D28K binds Ca2+ (PDF 3759 kb)
Supplementary Table 1
NMR experiments used in the analysis of Ca2+-loaded calbindin-D28K (PDF 86 kb)
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Kojetin, D., Venters, R., Kordys, D. et al. Structure, binding interface and hydrophobic transitions of Ca2+-loaded calbindin-D28K. Nat Struct Mol Biol 13, 641–647 (2006). https://doi.org/10.1038/nsmb1112
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DOI: https://doi.org/10.1038/nsmb1112
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