NMR Structure of DREAM:  Implications for Ca²⁺-Dependent DNA Binding and Protein Dimerization^,

Abstract

DREAM (calsenilin/KChIP3) is an EF-hand calcium-binding protein that binds to specific DNA sequences and regulates Ca2+-induced transcription of prodynorphin and c-fos genes. Here, we present the atomic-resolution structure of Ca2+-bound DREAM in solution determined by nuclear magnetic resonance (NMR) spectroscopy. Pulsed-field gradient NMR diffusion experiments and 15N NMR relaxation analysis indicate that Ca2+-bound DREAM forms a stable dimer in solution. The structure of the first 77 residues from the N-terminus could not be determined by our NMR analysis. The C-terminal DREAM structure (residues 78-256) contains four EF-hand motifs arranged in a tandem linear array, similar to that seen in KChIP1, recoverin, and other structures of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily. Mg2+ is bound at the second EF-hand, whereas Ca2+ is bound functionally at the third and fourth sites. The first and second EF-hands form an exposed hydrophobic groove on the protein surface lined by side-chain atoms of L96, F100, F114, I117, Y118, F121, F122, Y151, L155, L158, and L159 that are highly conserved in all NCS proteins. An exposed leucine near the C-terminus (L251) is suggested to form intermolecular contacts with leucine residues in the hydrophobic groove (L155, L158, and L159). Positively charged side chains of Arg and Lys (Lys87, Lys90, Lys91, Arg98, Lys101, Arg160, and Lys166) are clustered on one side of the protein surface and may mediate electrostatic contacts with DNA targets. We propose that Ca2+-induced dimerization of DREAM may partially block the putative DNA-binding site, which may suggest as to how Ca2+ abolishes DREAM binding to DNA to activate the transcription of prodynorphin and other downstream genes in pain control.

Kv channel-interacting proteins as neuronal and non-neuronal calcium sensors

“…Taken together, these findings suggest that in KChIPs EF2 mediates low affinity Ca 2+ binding and is occupied by Mg 2+ under physiological conditions, whereas EF3 and EF4 mediate high affinity Ca 2+ binding [34–37,39]. The data are in accordance with crystal structure and NMR analysis, which showed only two bound Ca 2+ ions per KChIP subunit residing in EF3 and EF4 (Figure 2) [30–33]. Obviously, EF3 and EF4 are critical sites for a putative calcium sensor function of the KChIPs.…”

“…Even an EF3,EF4 fragment (amino acid 161 – 256) of KChIP3 showed a reduced spectral resolution in the absence of Ca 2+ , corroborating the central role of EF3 and EF4 in Ca 2+ binding and associated conformational changes [40]. KChIPs may also form oligomers in a divalent cation-dependent manner, with Ca 2+ binding to EF3 and EF4 favoring dimer formation [33,39,41]. KChIP3 has even been reported to oligomerize with calcineurin and calmodulin [42].…”

“…The conserved NCS core domain contains four EF-hand motifs (EF1, EF2, EF3 and EF4), a feature known from calmodulin, but in all NCS proteins the most N-terminal EF-hand motif (EF1) is degenerated and cannot bind Ca 2+ [14]. Recoverin [26], KChIP1 [30–32] and KChIP3 [33] were even isolated with only two Ca 2+ ions bound (see Figure 2 and next section). The Ca 2+ sensitivity of the NCS proteins is very high.…”

Ca2+ and Mg2+ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

“…22 Interestingly, the C-terminal loop (residues 242-244) connecting a-helix 9 and ahelix10 in the C-terminal domain exhibits high flexibility in the Ca 21 bound structure. As expected, smaller changes in protein flexibility due to Ca 21 binding to DREAM are observed for the N-terminal domain of the protein.…”

“…20,21 The role of Ca 21 in regulating DREAM functions and the structural basis of DREAM interactions with multiple partners are not fully understood. Although the solution structure of Ca 21 bound DREAM (residues 76-256) is known 22 as well as the solution structure of the C-terminal domain (residues 161-256) of Ca 21 bound DREAM, 23 it is unclear how the Ca 21 / Mg 21 association to the EF hands alters the structural properties of DREAM. Considering the large number of DREAM interacting proteins, understanding the structural diversity could provide important insight into the relationship between the sequence, structure, and the target diversity of DREAM and NCS proteins in general.…”