Fig. 3: Assessing the ability of EF-hand pairs of dynein–dynactin adaptors to bind Ca²⁺.

a ITC titrations of CaCl₂ into EF-hand pairs from human CRACR2a, FIP3, and NIN (as indicated). ITC data shown are representative of n = 2 independent experiments, with similar results. Listed for each titration are the experimental conditions and fitting parameters, including the dissociation constant (K_D) and binding stoichiometry (N) derived from fitting to a binding isotherm (black line). The K_D and N values are given as mean ± s.d. of the fits. Open symbols correspond to a control titration into buffer. b Comparison of the sequences of EF-hand pairs of CRACR2a, FIP3, NIN, and Rab45 with those of classical Ca²⁺-binding EF-hands from calmodulin (four EF-hands), skeletal muscle troponin C (four EF-hands), and S100A1 (one high-affinity Ca²⁺-binding EF-hand). The sequences are all human and UniProt accession codes are listed with the name of each sequence. The adaptors are separated into Ca²⁺-binding (CRACR2a and Rab45) and Non-Ca²⁺-binding (FIP3 and NIN). This classification is based on the experimental results shown in part a and the conservation of key amino acids involved in the coordination of Ca²⁺, with per-residue conservation scores ≥50% among 3071 sequences of known Ca²⁺-binding EF-hands (middle graph), selected using PROSITE (http://prosite.expasy.org/) with matrix PS50222 and pattern PS00018. According to the classical nomenclature²⁵, amino acids implicated in the pentagonal bipyramidal coordination of Ca²⁺ occupy positions X, Y, Z, −Y, −X, and −Z (as indicated). See main text for why certain Ca²⁺ coordinating positions are highly conserved whereas others can vary.