Secondary structural analysis of the carboxyl-terminal domain from different connexin isoforms

Abstract

The connexin carboxyl-terminal (CxCT) domain plays a role in the trafficking, localization, and turnover of gap junction channels, as well as the level of gap junction intercellular communication via numerous post-translational modifications and protein-protein interactions. As a key player in the regulation of gap junctions, the CT presents itself as a target for manipulation intended to modify function. Specific to intrinsically disordered proteins, identifying residues whose secondary structure can be manipulated will be critical toward unlocking the therapeutic potential of the CxCT domain. To accomplish this goal, we used biophysical methods to characterize CxCT domains attached to their fourth transmembrane domain (TM4). Circular dichroism and nuclear magnetic resonance were complementary in demonstrating the connexin isoforms that form the greatest amount of α-helical structure in their CT domain (Cx45 > Cx43 > Cx32 > Cx50 > Cx37 ≈ Cx40 ≈ Cx26). Studies compared the influence of 2,2,2-trifluoroethanol, pH, phosphorylation, and mutations (Cx32, X-linked Charcot-Marie Tooth disease; Cx26, hearing loss) on the TM4-CxCT structure. While pH modestly influences the CT structure, a major structural change was associated with phosphomimetic substitutions. Since most connexin CT domains are phosphorylated throughout their life cycle, studies of phospho-TM4-CxCT isoforms will be critical toward understanding the role that structure plays in regulating gap junction function.

Keywords: circular dichroism; gap junctions; intrinsically disordered; nuclear magnetic resonance.

FIGURE 1

Secondary structural analysis of soluble connexin CT domains. CD spectra of the soluble Cx26CT, Cx32CT, and Cx50CT domains in 1× PBS at pH 5.8 and pH 7.5.

FIGURE 2

Secondary structural analysis of soluble connexin CT domains in 2,2,2-trifluoroethanol (TFE) at pH 7.5. CD spectra of the soluble Cx26CT, Cx32CT, Cx37CT, Cx40CT, Cx43CT, and Cx50CT domains in 0%, 5%, 15%, and 30% TFE, 1× PBS, and at pH 7.5.

FIGURE 3

Secondary structural analysis of soluble connexin CT domains in 2,2,2-trifluoroethanol (TFE) at pH 5.8. CD spectra of the soluble Cx26CT, Cx32CT, Cx37CT, and Cx50CT domains in 0%, 5%, 15%, and 30% TFE, 1× PBS, and at pH 5.8.

FIGURE 4

Identification of Cx32CT regions that have α-helical propensity. (A) NMR control spectrum, Cx32CT-only (black), has been overlapped with spectra obtained when the Cx32CT was in the presence of 5% (red), 15% (blue), and 25% (green) 2,2,2-trifluoroethanol (TFE). Spectra for the Cx32CT in the presence of 10%, 20%, and 30% TFE were also collected but not shown due to peak overlap. (B) From (A), each residue was plotted against their change in chemical shift (Δσ = √((Δδ_HN) + (Δδ_N/5))) as a function of TFE concentration. The three regions that form α-helical structure in the presence of TFE are indicated with bars. The horizontal line indicates the chemical shift change cut-off at 0.4 ppm.

FIGURE 5

Secondary structural analysis of TM4-CxCT domains. CD spectra of TM4-Cx26CT, -Cx32CT, -Cx37CT, -Cx40CT, and -Cx50CT domains in MES buffer, 8% LPPG, and at pH 5.8 and pH 7.5.

FIGURE 6

Secondary structural analysis of TM4-CxCT domains in 2,2,2-trifluoroethanol (TFE) at pH 7.5. CD spectra of TM4-Cx26CT, -Cx32CT, -Cx37CT, -Cx40CT, -Cx43CT, -Cx45CT, and -Cx50CT domains in 0%, 5%, 10%, 15%, and 30% TFE, MES buffer, 8% LPPG, and at pH 7.5.

FIGURE 7

Secondary structural analysis of TM4-CxCT domains in 2,2,2-trifluoroethanol (TFE) at pH 5.8. CD spectra of TM4-Cx26CT, -Cx32CT, -Cx37CT, -Cx40CT, -Cx45CT, and -Cx50CT domains in 0%, 5%, 10%, 15%, and 30% TFE, MES buffer, 8% LPPG, and at pH 5.8.

FIGURE 8

Effect of 2,2,2-trifluoroethanol (TFE) on the linker and TM4 domains. CD spectra of the (A) Cx43 linker, (B) Cx43 TM4 domain, and (C) Cx45 TM4 domain in 0%, 5%, 10%, 15%, and 30% TFE, MES buffer, 8% LPPG, and at pH 5.8.

FIGURE 9

Secondary structural analysis of TM4-tethered Cx32CT CMTX mutants. CD spectra of TM4-Cx32CT mutants: (A) R219F, (B) F235C, and (C) R230C alone and in the presence of 0%, 5%, 10%, 15%, and 30% TFE, MES buffer, 8% LPPG, and at pH 7.5 and pH 5.8.

FIGURE 10

Secondary structural analysis of TM4-tethered Cx26CT nonsyndromic recessive hearing loss mutants. CD spectra of TM4-Cx26CT mutants (A) L214P and (B) K224Q alone and in the presence of 0%, 5%, 10%, 15%, and 30% TFE, MES buffer, 8% LPPG, and at pH 7.5 and pH 5.8. (C) CD Spectra of the Cx26 TM4 WT and L214P at pH 7.5 and 5.8.

FIGURE 11

Structural analysis of TM4-CxCT domains by NMR. ¹⁵N-HSQC spectra were collected for the TM4-Cx26CT, -Cx32CT, -Cx37CT, -Cx40CT, and -Cx50CT domains in 10% TFE, MES buffer, 8% LPPG, and at pH 5.8. The black circles highlight the glycine residues.

FIGURE 12

Structural analysis of multi-phosphorylated TM4-Cx43CT domains. A) CD and ¹⁵N-HSQC spectra of the (B) TM4-Cx43CT assembly_{(S325,328,330,365D)} and (C) disassembly_{(Y247,265;S255,262,279,282,368D)} phosphomimetic constructs in 10% TFE, MES buffer, 8% LPPG, and at pH 5.8. Black circles highlight the glycine residues. From the ¹⁵N-HSQC data, each (D) TM4-Cx43CT assembly_{(S325,328,330,365D)} and (E) disassembly_{(Y247,265;S255,262,279,282,368D)} residue was plotted against their change in chemical shift (Δσ =√((Δδ_HN)² + (Δδ_N/5))²) as compared to TM4-Cx43CT wild-type. The asterisks denote the serine/tyrosine residues substituted for an aspartic acid.