Binding by calmodulin is coupled to transient unfolding of the third FF domain of Prp40A

Abstract

Human pre-mRNA processing protein 40 homolog A (hPrp40A) is a splicing factor that interacts with the Huntington's disease protein huntingtin (Htt). Evidence has accumulated that both Htt and hPrp40A are modulated by the intracellular Ca²⁺ sensor calmodulin (CaM). Here we report characterization of the interaction of human CM with the third FF domain (FF₃ ) of hPrp40A using calorimetric, fluorescence and structural approaches. Homology modeling, differential scanning calorimetry and small angle X-ray scattering (SAXS) data show FF₃ forms a folded globular domain. CaM was found to bind FF₃ in a Ca²⁺ -dependent manner with a 1:1 stoichiometry and a dissociation constant (K_d ) of 25 ± 3 μM at 25°C. NMR studies showed that both domains of CaM are engaged in binding and SAXS analysis of the FF₃ -CaM complex revealed CaM occupies an extended configuration. Analysis of the FF₃ sequence showed that the anchors for CaM binding must be buried in its hydrophobic core, suggesting that binding to CaM requires unfolding of FF₃ . Trp anchors were proposed based on sequence analysis and confirmed by intrinsic Trp fluorescence of FF₃ upon binding of CaM and substantial reductions in affinity for Trp-Ala FF₃ mutants. The consensus model of the complex showed that binding to CaM binding occurs to an extended, non-globular state of the FF₃ , consistent with coupling to transient unfolding of the domain. The implications of these results are discussed in the context of the complex interplay of Ca²⁺ signaling and Ca²⁺ sensor proteins in modulating Prp40A-Htt function.

Keywords: FF domain; NMR; Prp40; calmodulin; isothermal titration calorimetry; small angle X-ray scattering.