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Trifluoperazine binding to mutant calmodulins
L R Massom1, T J Lukas, A Persechini
1Department of Biochemistry, University of Tennessee, Memphis 38168.
Abstract:
Trifluoperazine (TFP) binding by 14 calmodulins, including 12 produced by site-directed mutagenesis, was determined. While vertebrate calmodulin binds 4.2 +/- 0.2 equiv of TFP, Escherichia coli expressed but unmutated calmodulins bind about 5.0 +/- 0.5 equiv of TFP. The cause for this difference is not known. The E. coli expressed proteins consist of two different series expressed from different calmodulin genes, CaMI and SYNCAM. The wild-type genes code for proteins that differ by nine conservative amino acid substitutions. Both these calmodulins bind 5 equiv of TFP with similar affinities, thus none of these conservative substitutions has any additional effect on TFP binding. Some altered calmodulins (deletion of EE83-84 or SEEE81-84, changing DEE118-120----KKK, M124----I,E120----K, or E82----K) have no appreciable effect on TFP binding. Other mutations affect either the binding of one TFP (deletion of E84) or about two TFP (changing E84----K, EEE82-84----KKK, E67----A, DEQ6-8----KKK, or E11----K). The mutations that affect TFP binding are localized to three regions of calmodulin: The amino-terminal alpha-helix, the central helix between the two globular ends of calmodulin, and a calcium-binding site in the second calcium-binding domain. The results are consistent with each of these regions either directly participating in drug binding or involved structurally in maintaining or inducing the correct conformation for TFP binding in the amino-terminal half of calmodulin.
Insights
Trifluoperazine (TFP) binding differs between vertebrate and bacterial calmodulin. Mutations in specific calmodulin regions significantly alter TFP binding capacity, revealing key structural roles in drug interaction.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Calmodulin (CaM) is a crucial calcium-binding protein involved in numerous cellular processes.
- Trifluoperazine (TFP) is a drug known to bind calmodulin, with its binding stoichiometry varying across species.
- Understanding TFP-CaM interactions provides insights into CaM structure-function relationships and drug mechanisms.
Purpose of the Study:
- To investigate the binding of trifluoperazine (TFP) to various calmodulin (CaM) variants.
- To identify specific regions and mutations in CaM that influence TFP binding affinity and stoichiometry.
- To elucidate the structural basis of TFP binding to CaM.
Main Methods:
- Site-directed mutagenesis was used to create 12 altered calmodulin variants.
- Trifluoperazine (TFP) binding was quantified for wild-type and mutant calmodulins (CaMs).
- Binding affinities and stoichiometries were determined for TFP-CaM interactions.
Main Results:
- Escherichia coli-expressed wild-type calmodulins bind more TFP (approx. 5.0 equiv) than vertebrate calmodulin (approx. 4.2 equiv).
- Conservative amino acid substitutions in wild-type E. coli calmodulins did not significantly alter TFP binding.
- Specific mutations, particularly in the N-terminal helix, central helix, and a calcium-binding site, markedly affected TFP binding stoichiometry.
Conclusions:
- The N-terminal helix, central helix, and a calcium-binding site in calmodulin are critical for trifluoperazine (TFP) binding.
- Structural integrity in these regions is essential for maintaining or inducing the correct conformation for TFP binding.
- Differences in TFP binding between vertebrate and bacterial calmodulins may stem from variations in these key structural regions.