Structural and Biophysical Investigation of Calmodulin Binding to the Third Domain of Human Prp40A

Abstract

Human Pre-mRNA-processing factor 40 homolog A (hPrp40A) is a splicing factor that interacts with the Huntington’s Disease (HD) protein huntingtin (HTT). Evidence has accumulated that both HTT and hPrp40A are modulated by the intracellular Ca2+-sensor calmodulin (CaM). However, the molecular basis and functional role of these interactions is not known. Here we report an investigation of the interaction of human CaM with the third FF domain (FF3) of hPrp40A using a combination of biophysical and structural approaches. Isothermal titration calorimetry revealed that CaM binds FF3 in a Ca2+-dependent manner with a 1:1 stoichiometry and a dissociation constant (Kd) of 25 ± 3 µM at 25 °C. A five-fold increase in affinity (Kd = 4.9 ± 0.3 µM) is observed when increasing the temperature to 37 °C. SAXS and NMR analysis showed that CaM occupies an extended configuration with both domains engaged in binding the FF3 domain. Analysis of the FF3 sequence and structure suggested that the anchors in the FF3 must be buried in a hydrophobic core in the folded domain, suggesting that binding to CaM requires unfolding of FF3. Intrinsic tryptophan fluorescence monitored as CaM was titrated into a solution of the FF3 domain suggested that two residues buried in the hydrophobic core, Trp531 and Trp550, serve as the hydrophobic anchors into the two CaM domains. To further test this hypothesis, the effects on CaM binding from mutation of the FF3 tryptophan residues to alanine alone and in tandem were assayed by isothermal titration calorimetry and NMR. Both single site mutants exhibited substantially weaker binding to CaM and a complete loss of binding was observed for the double mutant. The implications of these results are discussed in the context of the complex interplay of Ca2+ signaling and Ca2+ sensor proteins in modulating hPrp40A-HTT interactions.