Mode of action of cGMP-dependent protein kinase-specific inhibitors probed by photoaffinity cross-linking mass spectrometry

Abstract

The inhibitor peptide DT-2 (YGRKKRRQRRRPPLRKKKKKH) is the most potent and selective inhibitor of the cGMP-dependent protein kinase (PKG) known today. DT-2 is a construct of a PKG tight binding sequence (W45, LRKKKKKH, KI=0.8 microM) and a membrane translocating sequence (DT-6, YGRKKRRQRRRPP, KI=1.1 microM), that combined strongly inhibits PKG catalyzed phosphorylation (KI=12.5 nM) with approximately 1000-fold selectivity toward PKG over protein kinase A, the closest relative of PKG. However, the molecular mechanism behind this inhibition is not entirely understood. Using a combination of photoaffinity labeling, stable isotope labeling, and mass spectrometry, we have located the binding sites of PKG-specific substrate and inhibitor peptides. Covalent linkage of a PKG-specific substrate analogue was localized in the catalytic core on residues 356-372, also known as the glycine-rich loop, essential for ATP binding. By analogy, the individual inhibitor peptides W45 and DT-6 were also found to cross-link near the glycine-rich loop, suggesting these are both substrate competitive inhibitors. A bifunctional photoreactive analogue of DT-2 was found to generate dimers of PKG. This cross-linking induced covalent PKG dimerization was not observed for an N-terminal deletion mutant of PKG, which lacks the dimerization domain. In addition, non-covalent mass spectrometry was used to determine binding stoichiometry and binding order of the inhibitor peptides. Dimeric PKG binds two W45 and DT-6 peptides, whereas only one DT-2 molecule was observed to bind to the dimeric PKG. Taken together, these findings imply that (i) the two individual components making up DT-2 are both targeted against the substrate-binding site and (ii) binding of a single DT-2 molecule inactivates both PKG monomers simultaneously, which is an indication that (iii) in cGMP-activated PKG the catalytic centers of both subunits may be in each other's proximity.

FIGURE 1.

Linear arrangement of the functional domains of the regulatory and catalytic subunit of PKA (A) and PKG (B) type I and schematic representation of the current working models of the activation process of PKA (C) and PKG (D) type 1. Binding of cAMP to the PKA induces a conformational change that results in the dissociation of the catalytic subunits. Binding of cGMP to PKG also induces a conformational change, which exposes the catalytic domains, but both catalytic domains remain near each other via the N-terminal dimerization domain. (Images adapted from Scholten et al. (4).)

FIGURE 2.

Nanoflow ESI-TOF mass spectra of PKG that was irradiated in the presence of the photolabel W64-[Phe^(Tmd)-12] for 10 min at 366 nm in the absence of cGMP (A) and the presence of 100 μm cGMP (B). Shown are on the left are the m/z spectra containing the charge state envelope of dimeric PKG, and shown are on the right are the corresponding deconvoluted mass spectra. These spectra clearly indicate that cGMP activation is a prerequisite for the binding of peptides substrates.

FIGURE 3.

Analysis of a tryptic digest of the PKG/W64-[Phe^(Bz)-12] cross-link product. A, base peak intensity ion chromatogram of the trypsin digest. B, peptide display of the LC-MS chromatogram. Inset, enlarged view of the peptide display around 41 min shows the presence of an ion doublet. C, three-dimensional image of the [M+4H]⁴⁺ confirms the presence of ion doublet with 7 Da mass difference due to the 50% ¹³C₆,¹⁵N₁-leucine in the photoaffinity substrate.

FIGURE 4.

Tandem mass spectrometry (MS/MS) analysis of the tryptic cross-linked peptide derived from PKG/W64-[Phe^(Bz)-12]. A, the [M+4H]⁴⁺ precursor ion that was selected by the quadrupole using a broad selection window (∼5 m/z units) to allow transmission and subsequent simultaneous fragmentation of the light and heavy leucine labeled cross-linked products. B, MS/MS spectrum of the [M+4H]⁴⁺ at m/z 675–676. C, primary sequence of the cross-linked product and identified fragment ions. The b and y″ ions corresponding the primary sequence of PKG, without covalently attached substrate are annotated by b_n or y_n, whereas b or y″ ions of the primary sequence of PKG that carries the covalent attached substrate are labeled *b_n or *y_n.

FIGURE 5.

MS/MS analysis of a cross-linked product derived from a GluC-digest of PKG, which was cross-linked with W45-[Phe^(Bz)-6]. A, the [M+5H]⁵⁺ precursor ion at m/z 590–593 that was selected by the quadrupole using a broad selection window (∼5 m/z units). The characteristic doublet is indicative for the presence of the isotopically labeled leucine from the photoaffinity labeled peptide. B, MS/MS spectrum of the [M+5H]⁵⁺ at m/z 590–593. C, primary sequence of the cross-linked product and identified fragment ions. The b and y″ ions corresponding the primary sequence of PKG, without covalently attached inhibitor are annotated by b_n or y_n, whereas b or y″ ions of the primary sequence of PKG that carries the covalent attached photolabel are labeled *b_n or *y_n.

FIGURE 6.

MS/MS analysis of a cross-linked product derived from a trypsin-digest of PKG, which was cross-linked with W45-[Phe^(Bz)-2]. A, the [M+3H]³⁺ precursor ion at m/z 649–652 that was selected by the quadrupole using a broad selection window (∼5 m/z units). The characteristic doublet is indicative for the presence of the isotopically labeled leucine from the photoaffinity-labeled peptide. B, MS/MS spectrum of the [M+3H]³⁺ at m/z 649–652. C, primary sequence of the cross-linked product and identified fragment ions. The b and y″ ions corresponding the primary sequence of PKG, without covalently attached inhibitor are annotated by b_n or y_n, whereas b or y″ ions of the primary sequence of PKG that carries the covalent attached photolabel are labeled *b_n or *y_n. The cysteine was found to carry a dithiothreitol adduct that was subsequently modified by iodoacetamide.

FIGURE 7.

SDS-PAGE analysis of reduced PKG DT-2-[Phe^(Bz)-6,14] cross-link product shows dimerization of monomer. A, PKG wild type was cross-linked in the presence of 100 μm cGMP at three different DT-2-[Phe^(Bz)-6,14] concentrations (1, 10, and 100 μm). At 100 μm DT-2-[Phe^(Bz)-6,14] a high molecular weight band is clearly visible. The DT-2-photoaffinity label is unable to form covalent dimers of the constitutively active monomer PKG Δ77. B, the formation of covalent PKG dimers is specifically seen with the dual DT-2-photolabel and not with regular DT-2 or with the W45 or DT-6-based photoaffinity labels.

FIGURE 8.

Nanoflow positive ESI-MS of ∼1.5 μm PKG (A), ∼1.5 μm PKG in the presence of 5 μm DT-2 (B), ∼1.5 μm PKG in the presence of 20 μm cGMP (C), ∼1.5 μm PKG and in the presence of 5 μm DT-2 and 20 μm cGMP (D). On the left, the raw m/z spectra are shown, and each multiply charge ion is labeled with a single letter, followed by the charge state and the centroid m/z value. On the right, the corresponding deconvoluted mass spectra are shown. Each peak is labeled with a single-letter code as in the raw spectra, followed by the calculated mass and determined complex composition.

FIGURE 9.

Ribbon diagrams of the catalytic subunit of PKA co-crystallized with MnATP and the inhibitor peptide PKI 5–24 (TTYADFIASGRTGRRNAIHD) (PDB entry code 1APM). The glycine-rich loop (shown in blue) participates in ATP binding and/or catalysis. The inhibitor peptide (shown in green) largely docks on the large lobe of the catalytic subunit. The P0 site, representing the phosphate-accepting residues in the substrate, is shown in cyan. The two arginines at the P3 and P2 positions, important for substrate recognition, are shown in red. Site of cross-linking of PKA-specific substrate are shown in yellow, and the sites of cross-linking of PKG are shown in pink. The ribbon diagram on the right (II) is a 90-degree clockwise rotation of the diagram on the left (I).

FIGURE 10.

Ribbon diagrams of the PKA Type I holoenzyme (PDB entry code 3FHI) (A) and PKA Type IIα (PDB entry code 2QVS) (B). The catalytic subunit is shown in brown, and the glycine-rich loop is highlighted in blue. The site of cross-linkage between PKG and the substrate and W45 and DT-6 analogues is highlighted in green. The regulatory subunit in type I (A) and in type IIα (B) are both shown in gray. The homologous part in both regulatory subunits represents the second site of cross-linking of W45 in the regulatory subunit of PKG, and is colored in red. C, sequence alignment of the glycine-rich loop in the catalytic domain of PKA and PKG type I and type II and sequence alignment of the b/c helix in the regulatory subunit of PKA (PKA-RI and PKA-RII) and the corresponding primary sequence in PKG type I and type II.