Immunocytochemical Localization and Crystal Structure of Human Frequenin (Neuronal Calcium Sensor 1)

Abstract

Frequenin, a member of a large family of myristoylswitch calcium-binding proteins, functions as a calciumion sensor to modulate synaptic activity and secretion. We show that human frequenin colocalizes with ARF1 GTPase in COS-7 cells and occurs in similar cellular compartments as the phosphatidylinositol-4-OH kinase PI4K␤, the mammalian homolog of the yeast kinase PIK1. In addition, the crystal structure of unmyristoylated, calcium-bound human frequenin has been determined and refined to 1.9 Å resolution. The overall fold of frequenin resembles those of neurocalcin and the photoreceptor, recoverin, of the same family, with two pairs of calcium-binding EF hands and three bound calcium ions. Despite the similarities, however, frequenin displays significant structural differences. A large conformational shift of the C-terminal region creates a wide hydrophobic crevice at the surface of frequenin. This crevice, which is unique to frequenin and distinct from the myristoyl-binding box of recoverin, may accommodate a yet unknown protein ligand.

Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli

“…Furthermore, the NCS-1 E120Q protein is a considerably less efficient binder of calcium since a much higher protein concentration (110 µM) is required to decrease this ratio. These results are consistent with a number of previous studies of the calcium binding properties for these variants [5,[12][13]17], and demonstrate the presence of the C-terminal 6X His-tag does not obscure calcium binding properties of the protein. Finally, even at concentrations of 220 µM the NCS-1 EF construct shows no capacity to affect the fluorescent properties of indo-1 in the presence of calcium, indicating that under these conditions it does not appreciably bind calcium.…”

“…Consistent with the loss of calcium binding capacity, a smaller decrease was observed with the E120Q NCS-1 mutant protein (Figure 3c). Although the integrity of the EF-3 domain is interrupted by the E120Q mutation, the EF-2 and EF-4 domains remain intact, and the mutant protein retains some affinity for calcium, as previously suggested [13]. These data also suggest the conformational change of NCS-1 that occurs in the presence of calcium involves additional regions of the protein outside of the EF-2 domain.…”

“…The far-UV CD profile of each construct shows characteristic local minima at 208 nm and 220 nm, consistent with a protein of highhelical character [29]. The degree of fractional helicity predicted by this analysis is in good agreement with the helical content observed in X-ray crystallography experiments [13]. Furthermore, the CD spectra for NCS-1 variants containing a C terminal 6X His-tag track with those previously reported for NCS-1 proteins not containing the 6X His-tag [16,19], consistent with the affinity tag not grossly affecting the protein structure.…”

“…In the three functional EFhands of NCS-1, conserved glutamic acidic residues at positions 84, 120, and 168 provide a bidentate ligand necessary to coordinate calcium [11]. Binding of calcium at these three sites induces conformational changes in the protein structure that expose a hydrophobic pocket that has been proposed to switch the protein to its active state [12][13]. The calcium binding properties of NCS-1 are incompletely characterized, however, and many details of the structural changes that accompany binding also remain unclear.…”

The Diversity of Calcium Sensor Proteins in the Regulation of Neuronal Function

“…The residues involved in the interaction of the myristoyl group with the hydrophobic pocket are also conserved in the other members of the NCS family, however not all of the other family members display this Ca 2þ /myristoyl switch (O'Callaghan et al 2002;Stephen et al 2007). NCS-1 and KChIP1 expose a similar hydrophobic surface upon Ca 2þ -binding, which could be similarly important for target interactions (Bourne et al 2001;Scannevin et al 2004;Zhou et al 2004b;Pioletti et al 2006). In contrast, other NCS proteins are able to interact with certain binding proteins in the absence of Ca 2þ and therefore Ca 2þ -driven exposure of a hydrophobic surface cannot be the sole mechanism by which these proteins bind to effectors.…”