Trifluoperazine Binding to Mutant Calmodulins

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 EE^83–84 or SEEE^81–84, changing DEE^118–120 → 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, EEE^82–84 → KKK, E⁶⁷ → A, DEQ^6–8 → KKK, orE¹¹→K). The mutations that affect TFP binding are localized to three regions of calmodulin: The amino-terminal α-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.