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.

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.

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.

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.

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.

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.