Probing the interaction between the coiled coil leucine zipper of cGMP-dependent protein kinase Ialpha and the C terminus of the myosin binding subunit of the myosin light chain phosphatase

Abstract

Nitric oxide and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase I (PKG-Ialpha)-mediated activation of myosin phosphatase (MLCP). Mechanistically it has been proposed that protein-protein interactions between the N-terminal leucine zipper (LZ) domain of PKG-Ialpha ((PKG-Ialpha(1-59)) and the LZ and/or coiled coil (CC) domain of the myosin binding subunit (MBS) of MLCP are localized in the C terminus of MBS. Although recent studies have supported these interactions, the critical amino acids responsible for these interactions have not been identified. Here we present structural and biophysical data identifying that the LZ domain of PKG-Ialpha(1-59) interacts with a well defined 42-residue CC motif (MBS(CT42)) within the C terminus of MBS. Using glutathione S-transferase pulldown experiments, chemical cross-linking, size exclusion chromatography, circular dichroism, and isothermal titration calorimetry we identified a weak dimer-dimer interaction between PKG-Ialpha(1-59) and this C-terminal CC domain of MBS. The K(d) of this non-covalent complex is 178.0+/-1.5 microm. Furthermore our (1)H-(15)N heteronuclear single quantum correlation NMR data illustrate that this interaction is mediated by several PKG-Ialpha residues that are on the a, d, e, and g hydrophobic and electrostatic interface of the C-terminal heptad layers 2, 4, and 5 of PKG-Ialpha. Taken together these data support a role for the LZ domain of PKG-Ialpha and the CC domain of MBS in this requisite contractile complex.

FIGURE 1.

Schematic illustration of PKG-Iα_1-59 and its expression and purification. A, the domain structure of PKG-Iα including the N-terminal LZ (PKG-Iα_1-59), two cGMP binding sites (G domains), and a C-terminal catalytic domain (CD) is shown. Polar and non-polar residues that are in the a and d positions of each heptad layer are highlighted in blue and green, respectively. B, expression and purification of PKG-Iα_1-59 analyzed using 15% (w/v) SDS-PAGE in the presence of 50 mm DTT. Lane 1, protein standards; lane 2, cell lysate demonstrating expression of GST-PKG-Iα_1-59 fusion protein (33.4 kDa); lane 3, separation of GST tag (26 kDa) from PKG-Iα_1-59 following thrombin cleavage; lane 4, purified PKG-Iα_1-59 depicting monomer and dimer fractions, which are labeled as p and 2p, respectively.

FIGURE 2.

Characterization of MBS C-terminal domain. A, GST-PKG-Iα_1-59 incubated with Ni²⁺-NTA-linked His₆-MBS_ct100 and as a control with Ni²⁺-NTA-agarose alone (Invitrogen). Associated proteins were transferred to nitrocellulose and immunoblotted with mouse anti-GST antibody (Santa Cruz Biotechnology, Inc.). Membranes were developed with ECL (Amersham Biosciences). A band representative of a complex between His₆-MBS_ct100 and GST-PKG-Iα_1-59 is identified, supporting an interaction between these two proteins. B, CC score prediction of the C-terminal 180 residues of MBS scaled between 0 and 1.0 using the COILS server. These data were used to define the region of MBS that had the highest probability of being a truly structured CC domain within MBS_ct100. The predicted CC and LZ CC domains of MBS are labeled with and #, respectively, within the C-terminal 100 residues, MBS_ct100.

FIGURE 3.

Chemical cross-linking of PKG-Iα_1-59, MBS_ct42, and the PKG-Iα_1-59·MBS_ct42 complex. A, time course analysis of DSP-cross-linked PKG-Iα_1-59. Lane 1, protein standards. Lanes 2, 3, and 4, represent cross-linked PKG-Iα_1-59 in the presence of 50 mm DTT at 3, 8, and 15 min, respectively, with monomeric PKG-Iα_1-59 (p). Lanes 5, 6, and 7 represent cross-linked PKG-Iα_1-59 in the absence of DTT at 3, 8, and 15 min, respectively; the dimer (2p) band is seen. Lane 8, purified PKG-Iα_1-59 sample without cross-linker in the absence of DTT shows dimer (2p) conformation. B, time course analysis of DSP-cross-linked MBS_ct42. Lanes 1, 2, and 3 represent cross-linked MBS_ct42 in the presence of 50 mm DTT at 3, 8, and 15 min, respectively; monomer (m) and dimer bands (2m) are seen. Lanes 4, 5, and 6 represent cross-linked MBS_ct42 in the absence of DTT at 3, 8, and 15 min, respectively; bands corresponding to dimer (2m) and a tetramer (4m) are seen. Lane 7, purified MBS_ct42 sample without cross-linker in the absence of DTT shows monomer (m) conformation. Lane 8, protein standards. C, time course analysis of PKG-Iα_1-59·MBS_ct42 complex. Lane 1, protein standards. Lanes 2, 3, and 4 illustrate cross-linked complex in the presence of 50 mm DTT at 3, 8, and 15 min, respectively; bands corresponding to PKG-Iα_1-59 (p) and MBS_ct42 (m) monomers are seen. Lanes 5, 6, and 7 represent cross-linked complex in the absence of DTT at 3, 8, and 15 min, respectively; bands corresponding to monomer (p) PKG-Iα_1-59 and dimer (2m) and tetramer (4m) MBS_ct42 are seen, and the complex band at ∼25 kDa is labeled with ^*. Lane 8, purified complex sample without cross-linker in the absence of DTT shows the dimer (2p) of PKG-Iα_1-59 and monomer (m) of MBS_ct42. D, SEC of PKGIα_1-59 and MBS_ct42 complex. SEC of the complex sample (molar ratio of 1:2 for PKG-Iα_1-59:MBS_ct42) performed at 25 °C on a Superdex 75 (120-ml bed volume) column. The chromatogram illustrates the elution profile of the complex, which consisted of three peaks. Of these, two represent the non-interacting proteins, and the third non-Gaussian shaped peak represents the complex. These eluted peaks were identified and correspond to MBS_ct42 homodimer (10 kDa) (MBS_ct42d), PKG-Iα homodimer (14.8 kDa) (PKG-Iα_1-59D), and non-covalent complex (dimer-dimer interaction) of ∼25 kDa that is labeled with ^* in the chromatogram. D, dimer. The inset is the SDS-PAGE of this eluted complex sample (lane 2) supporting the presence of a band corresponding to its ∼25-kDa size (marked with ^*). Lane 1 shows protein standards as indicated in A. As a reference, a chromatogram of bovine aprotinin (6.5 kDa) is also shown.

FIGURE 4.

Characterization of PKG-Iα_1-59 and MBS_ct42 interaction. A, CD spectra of PKG-Iα_1-59, MBS_ct42, and its complex. An overlay of CD spectra from PKG-Iα_1-59, MBS_ct42, and complex collected at 25 °C is shown. Data points for each spectrum are highlighted in symbols as indicated at the top right of the spectra panel. deg, degree. B, ITC of the interaction between MBS_ct42 and PKG-Iα_1-59. Top panel, heat absorbed (μcal/s) (endothermic binding) for each isotherm was plotted against titration time (min). Bottom panel, integrated heats (kcal/mol) were plotted against peptide/protein molar ratio.

FIGURE 5.

Residue perturbation analysis of PKG-Iα_1-59 following the interaction with MBS_ct42. A, two-dimensional ¹H-¹⁵N HSQC superimposition of uniformly ¹⁵N-labeled PKG-Iα_1-59 (pink contours) and PKGIα_1-59 titrated with MBS_ct42 (cyan contours) in a molar ration of 1:2. PKG-Iα_1-59 residues that show significant perturbation upon titration with MBS_ct42 (either have shown a chemical shift change or a decrease in peak intensity) are highlighted in red with a single letter amino acid code followed by its position in primary sequence. B, chemical shift perturbation of PKG-Iα_1-59. ¹H and ¹⁵N weighted chemical shifts, Δδ_weighted = [(Δ¹H)² + (0.1 × Δ¹⁵N)²]_½ were plotted versus residue number. The perturbation cutoff value is shown by a horizontal line (dashed). Perturbed residues are highlighted as red bars. C, chemical shift perturbation of PKG-Iα_1-59. The peak intensity ratios (I_Complex/I_PKG-Iα1-59) for each residue were plotted against residue number. The perturbation cutoff value is shown by a horizontal line (dashed). Perturbed residues are highlighted as red bars. The amide proton of His⁴¹ was not assignable.

FIGURE 6.

CC LZ tertiary structure of parallel conformation of PKG-Iα_1-59 upon binding to MBS_ct42. A, helical wheel representation of residues 12-46 of LZ PKG-Iα_1-59. Residues in the first heptad layer (from the N terminus) are circled. The repeating heptad positions are labeled a through g with residues placed at their position. Perturbed PKG-Iα_1-59 residues following the addition of MBS_ct42 are highlighted in red. B, lateral view of PKG-Iα_1-59 along the principal axis. Side chains of the perturbed residues are highlighted in red.