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. 2013 Aug 23;288(34):24857-70.
doi: 10.1074/jbc.M113.454389. Epub 2013 Jul 4.

Effects of phosphorylation on the structure and backbone dynamics of the intrinsically disordered connexin43 C-terminal domain

Rosslyn Grosely  1 , Jennifer L Kopanic, Sarah Nabors, Fabien Kieken, Gaëlle Spagnol, Mona Al-Mugotir, Sydney Zach, Paul L Sorgen
Affiliations

Effects of phosphorylation on the structure and backbone dynamics of the intrinsically disordered connexin43 C-terminal domain

Rosslyn Grosely et al. J Biol Chem. .

Abstract

Phosphorylation of the connexin43 C-terminal (Cx43CT) domain regulates gap junction intercellular communication. However, an understanding of the mechanisms by which phosphorylation exerts its effects is lacking. Here, we test the hypothesis that phosphorylation regulates Cx43 gap junction intercellular communication by mediating structural changes in the C-terminal domain. Circular dichroism and nuclear magnetic resonance were used to characterize the effects of phosphorylation on the secondary structure and backbone dynamics of soluble and membrane-tethered Cx43CT domains. Cx43CT phospho-mimetic isoforms, which have Asp substitutions at specific Ser/Tyr sites, revealed phosphorylation alters the α-helical content of the Cx43CT domain only when attached to the membrane. The changes in secondary structure are due to variations in the conformational preference and backbone flexibility of residues adjacent and distal to the site(s) of modification. In addition to the known direct effects of phosphorylation on molecular partner interactions, the data presented here suggest phosphorylation may also indirectly regulate binding affinity by altering the conformational preference of the Cx43CT domain.

Keywords: Circular Dichroism (CD); Gap Junctions; Nuclear Magnetic Resonance; Phosphorylation; Protein Dynamics.

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Figures

FIGURE 1.
FIGURE 1.
Electrophoretic mobility of Cx43. SDS-PAGE analysis of WT and phospho-mimetic isoforms of the Cx43CT indicating shifts in electrophoretic mobility. Substitutions are listed along with the kinase responsible for the phosphorylation of Cx43. The soluble Cx43CT is shown in A along with a summary of the soluble CT results in B. The membrane-tethered TM4-Cx43CT is shown in C. P0, P1, and P2 SDS-PAGE mobility shifts are typical of full-length, endogenously phosphorylated Cx43.
FIGURE 2.
FIGURE 2.
15N HSQC spectra of the soluble Cx43CT (Val-236–Ile-382). A, peak assignments for the backbone amides are indicated with numbering corresponding to the full-length Cx43 protein. B, 15N HSQC spectrum of wild-type Cx43CT (black) overlaid with the CK1 phospho-mimetic isoform spectrum (red). Residues affected by the amino acid substitutions are numbered in the spectra.
FIGURE 3.
FIGURE 3.
Secondary structure the Cx43CT. The CD spectra for the soluble Cx43CT and the phospho-mimetic isoforms at pH 5.8 (A) and pH 7.5 (B) (WT, black line; isoform name for kinase responsible for the endogenous phosphorylation, grayscale). C, CD spectra of the Cx43CT attached to the 4th transmembrane domain (TM4-Cx43CT; black lines) and the TM4 (Asp-197–Val-240; gray lines), which consists of the fourth transmembrane domain and lacks the C-terminal domain. Spectra were collected at pH 7.5 (solid lines) and pH 5.8 (dotted lines). Units are in MRE.
FIGURE 4.
FIGURE 4.
Global effect of phosphorylation on the secondary structure of the TM4-Cx43CT. CD spectra of the TM4-Cx43CT phospho-mimetic isoforms (gray lines) compared with wild type (WT; black lines) at pH 7.5 (solid lines) and pH 5.8 (dotted lines) are given in A–F. Isoforms are named based on kinase responsible for the endogenous phosphorylation. Units are in MRE. Changes in α-helical content are determined by evaluation of the MRE at 222 nm (vertical solid line).
FIGURE 5.
FIGURE 5.
Residue-specific effects of phosphorylation on the TM4-Cx43CT. The 15N HSQC spectra of the TM4-Cx43CT (black) overlaid with 15N HSQC spectra of each of the phospho-mimetic isoforms (red; A–F). Phospho-mimetic isoform chemical shifts that were different from WT are mapped to the Cx43CT sequences given in G. Blue letters, residues with minor changes in chemical shift (peaks are still slightly overlapped); red letters, residues with large changes in chemical shift or peaks that disappeared; green, underlined* letters, sites of Asp substitution; black lines indicate the seven regions previously predicted to be α-helical. Phospho-mimetic isoforms are named based on kinase responsible for the endogenous phosphorylation.
FIGURE 6.
FIGURE 6.
Effects of phosphorylation on TM4-Cx43CT inter-residue NOEs. 15N NOESY-HSQC spectra, WT TM4-Cx43CT (left panel) and CK1 isoform (middle) shown at level = 1×; the right panel is the spectrum of the CK1 isoform at level = 3×.
FIGURE 7.
FIGURE 7.
TM4-Cx43CT backbone dynamics. 15N relaxation parameters R1 (A), R2 (B), and 15N heteronuclear NOE (C) were measured at 600 MHz. Bars indicate the curve-fit root mean square deviation for each point. The residue specific R2/R1 and R2R1 are given in D and E, respectively. Peaks with chemical shift overlap were excluded from the analysis. Mean values (solid line) and standard deviations (dotted) are indicated.
FIGURE 8.
FIGURE 8.
Effects of phosphorylation on TM4-Cx43CT R2R1 backbone dynamics. 15N relaxation parameters (R1 and R2) for the phospho-mimetic isoforms were measured at 600 MHz. The R2R1 values are given for each of the isoforms in A–F. Residues with chemical shift overlap or ambiguous assignments (including * sites of substitution for the cdc2 and Src isoforms) were excluded from the analysis. Vertical lines indicate sites of Cx43CT phosphorylation; mean values (solid line) and standard deviations (horizontal dotted lines) are provided.
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