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Characterization of the S100A1 protein binding site on TRPC6 C-terminus.

Bily J, Grycova L, Holendova B, Jirku M, Janouskova H, Bousova K, Teisinger J - PLoS ONE (2013)

Bottom Line: Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6.The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6.This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6.

View Article: PubMed Central - PubMed

Affiliation: Department of Protein Structures, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

ABSTRACT
The transient receptor potential (TRP) protein superfamily consists of seven major groups, among them the "canonical TRP" family. The TRPC proteins are calcium-permeable nonselective cation channels activated after the emptying of intracellular calcium stores and appear to be gated by various types of messengers. The TRPC6 channel has been shown to be expressed in various tissues and cells, where it modulates the calcium level in response to external signals. Calcium binding proteins such as Calmodulin or the family of S100A proteins are regulators of TRPC channels. Here we characterized the overlapping integrative binding site for S100A1 at the C-tail of TRPC6, which is also able to accomodate various ligands such as Calmodulin and phosphatidyl-inositol-(4,5)-bisphosphate. Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6. The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6. This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6.

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Amino acid senquence of TRPC6 fusion protein.Native rat TRPC6 801–878 amino acid sequence containing integrative binding site was investigated. Predicted important basic amino acids that were replaced by alanine are in red.
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pone-0062677-g003: Amino acid senquence of TRPC6 fusion protein.Native rat TRPC6 801–878 amino acid sequence containing integrative binding site was investigated. Predicted important basic amino acids that were replaced by alanine are in red.

Mentions: The protein samples were used for steady-state fluorescence anisotropy measurement to characterize the binding ability of the S100A1 protein to TRPC6 C-terminal region 801–878. Increasing amounts of the TRPC6 protein construct were titrated into the DNS-S100A1 solution. The equilibrium dissociation constant of the TRPC6(801–878)/Ca2+-S100A1 complex was estimated to be 0.31+/−0.04 µM (Fig. 2). The value of the equilibrium dissociation constant is nearly the same as was estimated for CaM binding to be 0.320+/−0.019 µM [8]. As the amino acid residues important for CaM binding are known (Fig. 3), we tested their role in S100A1 binding (Fig. 2). The single substitution of R852A and triple substitution of K859A/R860A/R864A had almost no effect on its binding. (Fig. 2, Tab. 2). Interestingly, in comparison to the CaM binding, where the neutralization of this residue had the most striking effect [8], the R852 residue did not influence the interaction with S100A1 at all. Although the TRPC6 WT binding affinities to both ligands CaM and S100A1 are almost the same, according to the results we obtained it seems that different residues are involved in the binding. The mutations of K856A and R864A lead to an up to 3-fold decrease in the binding affinity to S100A1. These results are in a good agreement with TRPC6/CaM binding data. The R852A/K859A/R860A triple mutation caused the most significant decrease in binding ability (Tab. 2).


Characterization of the S100A1 protein binding site on TRPC6 C-terminus.

Bily J, Grycova L, Holendova B, Jirku M, Janouskova H, Bousova K, Teisinger J - PLoS ONE (2013)

Amino acid senquence of TRPC6 fusion protein.Native rat TRPC6 801–878 amino acid sequence containing integrative binding site was investigated. Predicted important basic amino acids that were replaced by alanine are in red.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643951&req=5

pone-0062677-g003: Amino acid senquence of TRPC6 fusion protein.Native rat TRPC6 801–878 amino acid sequence containing integrative binding site was investigated. Predicted important basic amino acids that were replaced by alanine are in red.
Mentions: The protein samples were used for steady-state fluorescence anisotropy measurement to characterize the binding ability of the S100A1 protein to TRPC6 C-terminal region 801–878. Increasing amounts of the TRPC6 protein construct were titrated into the DNS-S100A1 solution. The equilibrium dissociation constant of the TRPC6(801–878)/Ca2+-S100A1 complex was estimated to be 0.31+/−0.04 µM (Fig. 2). The value of the equilibrium dissociation constant is nearly the same as was estimated for CaM binding to be 0.320+/−0.019 µM [8]. As the amino acid residues important for CaM binding are known (Fig. 3), we tested their role in S100A1 binding (Fig. 2). The single substitution of R852A and triple substitution of K859A/R860A/R864A had almost no effect on its binding. (Fig. 2, Tab. 2). Interestingly, in comparison to the CaM binding, where the neutralization of this residue had the most striking effect [8], the R852 residue did not influence the interaction with S100A1 at all. Although the TRPC6 WT binding affinities to both ligands CaM and S100A1 are almost the same, according to the results we obtained it seems that different residues are involved in the binding. The mutations of K856A and R864A lead to an up to 3-fold decrease in the binding affinity to S100A1. These results are in a good agreement with TRPC6/CaM binding data. The R852A/K859A/R860A triple mutation caused the most significant decrease in binding ability (Tab. 2).

Bottom Line: Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6.The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6.This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6.

View Article: PubMed Central - PubMed

Affiliation: Department of Protein Structures, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

ABSTRACT
The transient receptor potential (TRP) protein superfamily consists of seven major groups, among them the "canonical TRP" family. The TRPC proteins are calcium-permeable nonselective cation channels activated after the emptying of intracellular calcium stores and appear to be gated by various types of messengers. The TRPC6 channel has been shown to be expressed in various tissues and cells, where it modulates the calcium level in response to external signals. Calcium binding proteins such as Calmodulin or the family of S100A proteins are regulators of TRPC channels. Here we characterized the overlapping integrative binding site for S100A1 at the C-tail of TRPC6, which is also able to accomodate various ligands such as Calmodulin and phosphatidyl-inositol-(4,5)-bisphosphate. Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6. The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6. This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6.

Show MeSH