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.

Protein-Cadmium Interactions in Crowded Biomolecular Environments Probed by In-cell and Lysate NMR Spectroscopy

“…Our previous work has established, using biophysical measurements and structural data, that in aqueous buffer solutions Cd 2+ binds to C2α with comparable stoichiometry and >30-fold higher affinity. [8b] Since oxygen is a harder Lewis base than sulfur, Cd 2+ affinity to all-oxygen coordination sites is generally weaker than that to all-sulfur sites. To determine if C2α-Cd 2+ interactions take place in the crowded molecular environment that contains many potential Cd 2+ chelators, we conducted experiments on lysates of cells that express isotopically enriched C2α.…”

“…Next, we prepared lysate samples of cells grown with either 50 μM Cd 2+ or Ca 2+ but no EDTA added in culture. The chemical shifts of the C2α amides in Cd 2+ -containing lysate clearly report on the formation of the C2α-Cd 2+ complex [8b] ( Figure 4C ). For instance, the chemical shift perturbations of Gly190, Asn287, and Leu183 amide resonances are similar to those detected in C2α upon Cd 2+ binding in aqueous buffer.…”

“…For instance, the chemical shift perturbations of Gly190, Asn287, and Leu183 amide resonances are similar to those detected in C2α upon Cd 2+ binding in aqueous buffer. [8b] The cross-peaks of Thr150 and Thr151 of CMBL3 that are exchange-broadened beyond detection in the metal-free state, are readily detectable in the Cd 2+ -containing lysate due to the rigidification of the CMBL region that takes place upon metal ion complexation ( Figure 4C ).…”

“…[7] Consistent with this finding, several in vitro studies have demonstrated the ability of Cd 2+ to effectively displace Zn 2+ and Ca 2+ from the thiol- and oxygen-rich sites of proteins and protein domains. [8] However, Cd 2+ interactions in cellular environments can be affected by several factors such as influx-efflux homeostasis, accessibility of target sites, nutritive metal-ion pools, and intracellular chelators. [9] To establish biological relevance, specificity and affinity of Cd 2+ towards putative protein targets need to be examined in environments reflecting these conditions.…”

“…[12] We have previously demonstrated that in buffered solutions, amide 1 H and 15 N NMR chemical shifts in proteins are exquisitely sensitive to the changes in electrostatic environment imparted by Cd 2+ binding, leading to the unambiguous identification of Cd 2+ binding sites. [8b-e]…”