Role of Lys Residue at Position 87 of DREAM in Allosteric Regulation of DREAM's Interactions with KV Channel

Abstract

Downstream regulatory element antagonist modulator (DREAM) is a 29kDa protein, which interacts with diverse intracellular partners and is involved in many biological processes, namely, pain sensation, gene apoptosis, and modulation of Kv4 voltage channels. Previous research in our group and elsewhere demonstrated that DREAM interacts with the helix-9 of presenilin-1 (PS1HL9), the residue 2-22 (site-1) peptide of Kv4.3, and the residue 70-90 (site-2) peptide of Kv4.3 in a calcium-dependent manner. Molecular dynamics data suggests that Lys at the position 87 forms a salt bridge with Asp 165 and directly involved in the propagation of calcium-triggered structural changes between the C- terminal and N- terminal domain. To determine the impact of Lys 87 on the interdomain communication as well as to characterize its contribution to DREAM stability, Lys 87 was mutated to Ala, and the effects of the mutation on DREAM's interaction with “PS1HL9”, “site-1” peptide, and “site-2” were determined. The results show that Trp residue in DREAM(K87A) is more solvent exposed compared with wild-type suggesting that the absence of the salt bridge destabilizes the protein structure. The emission maximum of 1,8-ANS in the presence of DREAM(K87A) is about 10 nm redshifted indicating that the mutation influences the hydrophobic cavity between the N- and C-terminal domain. The lifetime of Trp and 1,8-ANS are significantly altered in DREAM(K87A) compared with wild-type form, which in agreement with the steady-state data. Fluorescence anisotropy titration data suggest that mutation of Lys to Ala at position 87 of DREAM completely inhibit DREAM's interaction with site-2. We do plan to investigate DREAM(K87A)'s interactions with site-1 and PS1HL9. Data presented here provide insight into the role of Lys residue at position 87 of DREAM regulating DREAM's interaction with intracellular partners.