Reactivity of Thiol-Rich Zn Sites in Diacylglycerol-Sensing PKC C1 Domain Probed by NMR Spectroscopy

Abstract

Conserved homology 1 (C1) domains are peripheral zinc finger domains that are responsible for recruiting their host signaling proteins, including Protein Kinase C (PKC) isoenzymes, to diacylglycerol-containing lipid membranes. In this work, we investigated the reactivity of the C1 structural zinc sites, using the cysteine-rich C1B regulatory region of the PKCα isoform as a paradigm. The choice of Cd²⁺ as a probe was prompted by previous findings that xenobiotic metal ions modulate PKC activity. Using solution NMR and UV-vis spectroscopy, we found that Cd²⁺ spontaneously replaced Zn²⁺ in both structural sites of the C1B domain, with the formation of all-Cd and mixed Zn/Cd protein species. The Cd²⁺ substitution for Zn²⁺ preserved the C1B fold and function, as probed by its ability to interact with a potent tumor-promoting agent. Both Cys₃His metal-ion sites of C1B have higher affinity to Cd²⁺ than Zn²⁺, but are thermodynamically and kinetically inequivalent with respect to the metal ion replacement, despite the identical coordination spheres. We find that even in the presence of the oxygen-rich sites presented by the neighboring peripheral membrane-binding C2 domain, the thiol-rich sites can successfully compete for the available Cd²⁺. Our results indicate that Cd²⁺ can target the entire membrane-binding regulatory region of PKCs, and that the competition between the thiol- and oxygen-rich sites will likely determine the activation pattern of PKCs.

Keywords: C1 domain; NMR spectroscopy; cadmium; cysteine reactivity; metal ion toxicity; protein kinase C; thiol-rich sites; zinc finger.

FIGURE 1

Cd²⁺ replaces Zn²⁺ in the C1B domain. (A) Ribbon diagrams of C1B (2ELI) and C2 (4L1L) highlighting the metal-ion ligands. CMBLs stand for Ca²⁺- and membrane-binding loop loops. (B) UV-vis absorption spectra for the Cd²⁺ titration of 25 μM C1B-C2. Inset: UV-vis absorption spectra for the Cd²⁺ titration of 25 μM isolated C2 domain. The spectrum of free Cd²⁺ served as the reference and was subtracted from each spectrum. (C) Difference UV-vis absorption spectra between C1B-C2 and C2 obtained at increasing molar equivalents of Cd²⁺. The position of the absorption shoulder is consistent with the formation of the Cd²⁺-thiolate bonds. (D) Cd²⁺-stimulated Zn²⁺ release from the C1B-C2 domain monitored using fluorescence intensity of FluoZin-3 (Pubchem CID 101165894) at λ = 516 nm. The no-Cd²⁺ control is shown in blue.

FIGURE 2

Cd²⁺ treatment results in the formation of fully Cd-bound and Zn/Cd mixed C1B species. (A) [¹⁵N- ¹H] HSQC of 0.1 mM [U-¹⁵N] C1B^Zn by itself (red) and in the presence of 2 molar-equivalents of Cd²⁺ (black). Addition of Cd²⁺ results in an appearance of a new subset of cross-peaks. Arrows indicate the residue-specific changes in chemical shifts associated with Cd²⁺ binding to C1B. Zn²⁺-coordinating residues are highlighted in blue. (B) Expansions of the [¹⁵N-¹H] HSQC spectra for three residues, His140, Ile145, and Val147 that show four distinct cross-peaks upon treatment of C1B^Zn with Cd²⁺. His140 is a Zn²⁺-coordinating residue; Ile145 and Val147 reside on the C-terminal α helix. The four Zn/Cd C1B species are shown in cartoon representation.

FIGURE 3

Cd²⁺ simultaneously populates thiol- and oxygen-rich sites in C1B-C2. (A) Overlays of the expansions of the [¹⁵N-¹H] HSQC spectra of C1B^Zn-C2 (red) and Cd²⁺-treated C1B-C2 (black). The N-H cross-peaks of Cd²⁺ and Zn²⁺ containing species are connected with blue lines. (B) Chemical shift perturbations (CSPs) for backbone N-H groups as a function of C1B-C2 primary structure. The CSP values were calculated between the C1B^Zn-C2 and Cd²⁺-treated C1B-C2. Cys and His residues that coordinate Zn²⁺ in sites 1 and 2 are labeled accordingly. The C1B and C2 membrane-binding loops are highlighted in orange and tan, respectively. Inset: Correlation of C1B CSPs in the presence of 2 molar equivalents Cd ²⁺ in the isolated domain and in C1B-C2.

FIGURE 4

Cd²⁺ supports the PKC agonist-binding function of C1B. (A) Schematic representation of the experimental setup that involves C1B^Cd, mixed micelles, and the PKC agonist PDBu. (B) [¹⁵N-¹H] HSQC spectra of isolated native C1B^Zn (red), C1B^Cd (black), and C1B^Cd complexed to PDBu and mixed micelles (blue). (C) Chemical shift perturbations (CSPs) upon micelle/PDBu binding for the backbone N-H groups as a function of C1B primary structure. The membrane binding loops of C1B are highlighted in orange.

FIGURE 5

Site-resolved kinetics of Cd²⁺ binding to C1B. (A) The build-up of the Cd²⁺-bound C1B is plotted for the Cys₃His sites 1 (orange) and 2 (purple). The error bars represent the standard deviations of the I_Cd/I₀ values within a given residue subset. (B) The residues that form sites 1 and 2 are highlighted on the ribbon diagram of C1B (2ELI). (C) WebLogo representations of the sequence alignment of 31 DAG-sensitive C1 domains found in DAG effector proteins (R. norvegicus). The sequence homology values are all between 52 and 92%. Cys₃His motifs are strictly conserved. β12 and β34 denote the membrane-binding loops.