Khoa Pham

Modulation of the Voltage-gated Potassium Channel (Kv4.3) and the Auxiliary Protein (KChIP3) Interactions by the Current Activator NS5806

Background: KChIP3 association with Kv4 channels regulate the K+ current gating. Results: NS5806 binds to KChIP3 and stabilizes the KChIP3-Kv4 complex. Conclusion: Ca2+ or NS5806 binding on KChIP3 decreases the dissociation rate of Kv4.3. Significance: The role of the hydrophobic cavity on KChIP3 in drug and protein association could lead to a better drug design for treatment of heart conditions. KChIP3 (potassium channel interacting protein 3) is a calcium-binding protein that binds at the N terminus of the Kv4 voltage-gated potassium channel through interactions at two contact sites and has been shown to regulate potassium current gating kinetics as well as channel trafficking in cardiac and neuronal cells. Using fluorescence spectroscopy, isothermal calorimetry, and docking simulations we show that the novel potassium current activator, NS5806, binds at a hydrophobic site on the C terminus of KChIP3 in a calcium-dependent manner, with an equilibrium dissociation constant of 2–5 μm in the calcium-bound form. We further determined that the association between KChIP3 and the hydrophobic N terminus of Kv4.3 is calcium-dependent, with an equilibrium dissociation constant in the apo-state of 70 ± 3 μm and 2.7 ± 0.1 μm in the calcium-bound form. NS5806 increases the affinity between KChIP3 and the N terminus of Kv4.3 (Kd = 1.9 ± 0.1 μm) in the presence and absence of calcium. Mutation of Tyr-174 or Phe-218 on KChIP3 abolished the enhancement of Kv4.3 site 1 binding in the apo-state, highlighting the role of these residues in drug and K4.3 binding. Kinetic studies show that NS5806 decreases the rate of dissociation between KChIP3 and the N terminus of KV4.3. Overall, these studies support the idea that NS5806 directly interacts with KChIP3 and modulates the interactions between this calcium-binding protein and the T1 domain of the Kv4.3 channels through reorientation of helix 10 on KChIP3.

Characterization of Intramolecular Interactions of Cytochrome c Using Hydrogen–Deuterium Exchange-Trapped Ion Mobility Spectrometry–Mass Spectrometry and Molecular Dynamics

Globular proteins, such as cytochrome c (cyt c), display an organized native conformation, maintained by a hydrogen bond interaction network. In the present work, the structural interrogation of kinetically trapped intermediates of cyt c was performed by correlating the ion-neutral collision cross section (CCS) and charge state with the starting solution conditions and time after desolvation using collision induced activation (CIA), time-resolved hydrogen/deuterium back exchange (HDX) and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). The high ion mobility resolving power of the TIMS analyzer allowed the identification of new ion mobility bands, yielding a total of 63 mobility bands over the +6 to +21 charge states and 20 mobility bands over the -5 to -10 charge states. Mobility selected HDX rates showed that for the same charge state, conformers with larger CCS present faster HDX rates in both positive and negative ion mode, suggesting that the charge sites and neighboring exchange sites on the accessible surface area define the exchange rate regardless of the charge state. Complementary molecular dynamic simulations permitted the generation of candidate structures and a mechanistic model of the folding transitions from native (N) to molten globule (MG) to kinetic intermediates (U) pathways. Our results suggest that cyt c major structural unfolding is associated with the distancing of the N- and C-terminal helices and subsequent solvent exposure of the hydrophobic, heme-containing cavity.

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

Downstream Regulatory Element Antagonist Modulator (DREAM) belongs to the family of neuronal calcium sensors (NCS) that transduce the intracellular changes in Ca2+ concentration into a variety of responses including gene expression, regulation of Kv channel activity, and calcium homeostasis. Despite the significant sequence and structural similarities with other NCS members, DREAM shows several features unique among NCS such as formation of a tetramer in the apo‐state, and interactions with various intracellular biomacromolecules including DNA, presenilin, Kv channels, and calmodulin. Here we use spectroscopic techniques in combination with molecular dynamics simulation to study conformational changes induced by Ca2+/Mg2+ association to DREAM. Our data indicate a minor impact of Ca2+ association on the overall structure of the N‐ and C‐terminal domains, although Ca2+ binding decreases the conformational heterogeneity as evident from the decrease in the fluorescence lifetime distribution in the Ca2+ bound forms of the protein. Time‐resolved fluorescence data indicate that Ca2+binding triggers a conformational transition that is characterized by more efficient quenching of Trp residue. The unfolding of DREAM occurs through an partially unfolded intermediate that is stabilized by Ca2+ association to EF‐hand 3 and EF‐hand 4. The native state is stabilized with respect to the partially unfolded state only in the presence of both Ca2+ and Mg2+ suggesting that, under physiological conditions, Ca2+ free DREAM exhibits a high conformational flexibility that may facilitate its physiological functions.

Molecular insight of DREAM and presenilin 1 C-terminal fragment interactions

Interactions between downstream regulatory element antagonist modulator (DREAM) and presenilin 1 (PS1) are related to numerous neuronal processes. We demonstrate that association of PS1 carboxyl peptide (residues 445–467, HL9) with DREAM is calcium dependent and stabilized by a cluster of three aromatic residues: F462 and F465 from PS1 and F252 from DREAM. Additional stabilization is provided by residues in a loop connecting α helices 7 and 8 in DREAM and residues of PS1, namely cation–π interactions between R200 in DREAM and F465 in PS1 and the salt bridges formed by R207 in DREAM and D450 and D458 in PS1.

Characterization of the Role of Individual EF-Hands in Dream in Modulating Conformational Dynamics, Oligomeric States, and Interaction with DNA

Downstream Regulatory Element Antagonistic Modulator (DREAM) carries four calcium binding motifs (EF-hand) of which EF-3 and EF-4 bind to Ca2+ with high affinity, whereas EF-2 preferentially binds to Mg2+ and EF-1 is inactive. In neuronal cells, DREAM serves as a gene transcriptional repressor of dynorphin, an endogenous ligand, to regulate pain transmission by controlling kappa receptor activation. Recent findings have shown that tetrameric ApoDREAM binds to DNA with a high affinity, whereas association of Ca2+ abolishes the interaction with DNA. Despite mechanisms of interactions between DREAM and DNA have been proposed, it remains unclear which EF-hand in DREAM triggers tetramer dissociation and thus controls DNA affinity. In this study, a combination of fluorescence steady-state, time-resolved anisotropy, circular dichroism (CD), and isothermal titration calorimetry (ITC) techniques were employed to investigate the role of individual EF-hands. Here, we showed that Mg2+ binding to EF-2 does not affect the tertiary and secondary structures of DREAM. Ca2+ binding to EF-3 induces conformational changes in DREAM evidenced by a blue-shift and a decrease in emission intensity of the single tryptophan, whereas the association of Ca2+ to EF-4 does not disturb the DREAM structure. We also showed that elimination of Mg2+ binding to DREAM results in dimerization in Apo form and increase of the dimeric fraction in Ca2+ form. In contrast, abolishment of Ca2+ binding to EF-4 results in a high fraction of DREAM tetramer (60-75%) in Apo, Mg2+, Ca2+, and Ca2+Mg2+ bound forms. Finally, ITC results showed that ApoDREAM wild-type endothermically (ΔH=+32 kcal/mol) binds to prodynorphin DRE oligomer with a Kd of 0.5 µM, whereas a weak interaction was observed in the Ca2+ form (Kd∼200 µM). Interactions of DREAM mutants with DNA will also be discussed.

Mg2+/Ca2+ Induced Changes in Structure, Dynamics and Stability of Dream Protein

Downstream Regulatory Element Antagonist Modulator (DREAM) is a member of the neuronal calcium sensor family that controls activity of potassium voltage channels and regulates c-fos and prodynorphine gene transcription in a Ca2+ dependent manner. Here, we have investigated the impact of Mg2+ and Ca2+ binding on the structure, stability and dynamics of DREAM and DREAM C-terminal domain (DREAM-C). Mg2+ binding to the apoDREAM does not alter the secondary or tertiary structure as based on CD and Trp emission spectra whereas the association of Mg2+ to either EF-3 or EF-4 in apoDREAM-C results in an increase in protein secondary structure and alteration of the tertiary structure. Ca2+ binding to either apo- or Mg2+DREAM and apo- or Mg2+DREAM-C triggers larger conformational changes as evident from the blue-shift in emission spectra and the decrease of Stern-Volmer constant. Ca2+ triggered changes in DREAM conformational dynamics were characterized by time-resolved fluorescence. In apoDREAM, single tryptophan residue exhibits two lifetimes (τ1=3.38 ns, f1=73% and τ2=7.72 ns, f2=27%). Ca2+ binding to apo- and Mg2+DREAM leads to the shortening of the first lifetime and decrease of the fractional contribution (τ1 = 1.75 ns, f1 = 47% and τ2 = 7. ns, f2 = 53%). Furthermore, the impact of Ca2+/Mg2+ on DREAM stability was determined in equilibrium folding studies. The binding of Ca2+ increase the protein stability by ∼ 7 kcal mol−1 whereas the impact of Mg2+ on DREAM stability is significantly smaller, ∼ 1 kcal mol−1. The stability of the C terminal domain in both apo and Ca2+ bound form is significantly smaller compared to the full length protein, suggesting that the inter-domain interactions significantly contribute to the structural and functional properties of DREAM.