Session: Current Topics in Biological Chemistry:

Neuronal Calcium Sensor 1 (NCS1), a member of calcium binding protein superfamily, contains 190 highly conserved amino acid residues. Ca²⁺ binding to NCS1 protein causes a large positional shift in C-terminal EF-hands leading to an exposure of the hydrophobic crevice on protein surface that facilitates interaction with the target molecules. Previously, it has been shown that non-physiological metals such as Pb²⁺, bind to EF-hands in calmodulin and modulate interactions with its binding partners, leading to the Pb²⁺-induced neurotoxicity. Mn is a highly abundant metal in nature which has been found in human brain and associated with the several diseases like multiple sclerosis, Alzheimer’s and Parkinson’s disease and is considered as potential toxic element. Previous studies from our group showed Pb²⁺ and Cd²⁺ binding to DREAM protein that can contribute to Pb²⁺/Cd²⁺ induced neurotoxicity. Here we show that Pb²⁺ binds to EF-hands in apo-NCS1with a high affinity (K_d = 7.0±1.5 nM) whereas Cd²⁺ did not show any binding with NCS1. For the first time we investigated the Mn²⁺ association to NCS1 protein. This cation binds with an apparent equilibrium dissociation constant of 340±67 µM. Spectroscopic data show that Pb²⁺ and Mn²⁺binding trigger secondary and tertiary structural rearrangements of NCS1 that are analogous to those observed for Ca²⁺ association. In addition, Pb²⁺ as well as Mn²⁺promote NCS1 interaction with hydrophobic molecule 1,8-ANS. Interestingly, Ca²⁺NCS1:1,8-ANS complex exhibits a co-operative binding of Pb²⁺ with K_d = 103±23 µM and n=1.57±0.06 whereas Mn²⁺association to Ca²⁺NCS1:1,8-ANS complex is non-cooperative, with apparent K_d value of 924±185 µM. These results suggest allosteric interactions between the 1,8-ANS binding site and Pb²⁺ binding EF-hands. Data provided here points towards ability of EF-hands in NCS-1 to interact with various metals and a possible role of NCS1 in Pb²⁺/Mn²⁺ neurotoxicity.

Jaroslava Miksovska

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