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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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Circular dichroism spectroscopy measurement of TRPC6(801–878)WT, S100A1 and the complex of S100A1 and TRPC6 (801–878)WT.Examples of CD spectra of TRPC6(801–878)WT, S100A1 and the complex of S100A1 and TRPC6 (801–878)WT expressed as a molar elipticity Q (deg·cm2·dmol–1) per residue.
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pone-0062677-g001: Circular dichroism spectroscopy measurement of TRPC6(801–878)WT, S100A1 and the complex of S100A1 and TRPC6 (801–878)WT.Examples of CD spectra of TRPC6(801–878)WT, S100A1 and the complex of S100A1 and TRPC6 (801–878)WT expressed as a molar elipticity Q (deg·cm2·dmol–1) per residue.

Mentions: The TRPC6 C-terminal protein construct (801–878) and its mutants were expressed as fusion proteins with a 6x His-tag on their C-termini in E. coli Rosetta cells. The S100A1 protein was expressed in E.coli BL21 cells. All proteins were purified by a two-step purification process. The proteins were soluble and in sufficient amount to perform the binding experiments (Fig. S1 and S2). The integrity of the proteins was verified by circular dichroism spectroscopy measurement. Numerical analysis of the experimental spectra enabled estimation of the relative abundance of the various secondary structure elements. (Fig. 1, Tab. 1). The α-helical conformation (66%) was found to be the major component of the S100A1 protein, which is in good agreement with the conformation found in its native state. The structure of the TRPC6 C-terminus is unknown. According to the theoretical prediction of the secondary structural elements using computational tools, the region was predicted to be mostly unordered. The CD spectra analysis confirmed the theoretical prediction, suggesting that the TRPC6 protein construct was adopting its native form. The experiment was also used to observe changes in the secondary structural elements during the creation of the TRPC6/S100A1 complex (Fig 1, Tab 1). We compared the CD spectrum of the complex with the CD spectra of the proteins alone. Because the CD spectra of the mixture are the sum of the TRPC6 protein construct and S100A1 individually, we suggest that the changes in the secondary structure of TRPC6 (801–878) have no significant effect on its binding to S100A1.


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)

Circular dichroism spectroscopy measurement of TRPC6(801–878)WT, S100A1 and the complex of S100A1 and TRPC6 (801–878)WT.Examples of CD spectra of TRPC6(801–878)WT, S100A1 and the complex of S100A1 and TRPC6 (801–878)WT expressed as a molar elipticity Q (deg·cm2·dmol–1) per residue.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0062677-g001: Circular dichroism spectroscopy measurement of TRPC6(801–878)WT, S100A1 and the complex of S100A1 and TRPC6 (801–878)WT.Examples of CD spectra of TRPC6(801–878)WT, S100A1 and the complex of S100A1 and TRPC6 (801–878)WT expressed as a molar elipticity Q (deg·cm2·dmol–1) per residue.
Mentions: The TRPC6 C-terminal protein construct (801–878) and its mutants were expressed as fusion proteins with a 6x His-tag on their C-termini in E. coli Rosetta cells. The S100A1 protein was expressed in E.coli BL21 cells. All proteins were purified by a two-step purification process. The proteins were soluble and in sufficient amount to perform the binding experiments (Fig. S1 and S2). The integrity of the proteins was verified by circular dichroism spectroscopy measurement. Numerical analysis of the experimental spectra enabled estimation of the relative abundance of the various secondary structure elements. (Fig. 1, Tab. 1). The α-helical conformation (66%) was found to be the major component of the S100A1 protein, which is in good agreement with the conformation found in its native state. The structure of the TRPC6 C-terminus is unknown. According to the theoretical prediction of the secondary structural elements using computational tools, the region was predicted to be mostly unordered. The CD spectra analysis confirmed the theoretical prediction, suggesting that the TRPC6 protein construct was adopting its native form. The experiment was also used to observe changes in the secondary structural elements during the creation of the TRPC6/S100A1 complex (Fig 1, Tab 1). We compared the CD spectrum of the complex with the CD spectra of the proteins alone. Because the CD spectra of the mixture are the sum of the TRPC6 protein construct and S100A1 individually, we suggest that the changes in the secondary structure of TRPC6 (801–878) have no significant effect on its binding to S100A1.

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