A Competitive Chemical-Proteomic Platform To Identify Zinc-Binding Cysteines

Abstract

Zinc ions (Zn(2+)) play vital catalytic, structural, and regulatory roles in protein function and are commonly chelated to cysteine residues within the protein framework. Current methods to identify Zn(2+)-binding cysteines rely on computational studies based on known Zn(2+)-chelating motifs, as well as high-resolution structural data. These available approaches preclude the global identification of putative Zn(2+)-chelating cysteines, particularly on poorly characterized proteins in the proteome. Herein, we describe an experimental platform that identifies metal-binding cysteines on the basis of their reduced nucleophilicity upon treatment with metal ions. As validation of our platform, we utilize a peptide-based cysteine-reactive probe to show that the known Zn(2+)-chelating cysteine in sorbitol dehydrogenase (SORD) demonstrates an expected loss in nucleophilicity in the presence of Zn(2+) ions and a gain in nucleophilicity upon treatment with a Zn(2+) chelator. We also identified the active-site cysteine in glutathione S-transferase omega-1 (GSTO1) as a potential Zn(2+)-chelation site, albeit with lower metal affinity relative to SORD. Treatment of recombinant GSTO1 with Zn(2+) ions results in a dose-dependent decrease in GSTO1 activity. Furthermore, we apply a promiscuous cysteine-reactive probe to globally identify putative Zn(2+)-binding cysteines across ∼900 cysteines in the human proteome. This proteomic study identified several well-characterized Zn(2+)-binding proteins, as well as numerous uncharacterized proteins from functionally distinct classes. This platform is highly versatile and provides an experimental tool that complements existing computational and structural methods to identify metal-binding cysteine residues.

Zinc-Binding Cysteines: Diverse Functions and Structural Motifs

“…With the ever-increasing number of recognized Zn 2+ -chelating proteins the ability to globally evaluate Zn 2+ -binding within a complex proteome has become paramount. Toward this end, a recent study developed a chemical-proteomic platform that serves as a valuable complement to previous approaches (Figure 5a) [49]. This platform exploits the reduced nucleophilicity of cysteine residues upon metal-binding by utilizing cysteine-reactive chemical probes that preferentially bind the more nucleophilic apo-form.…”

Covalent protein modification: the current landscape of residue-specific electrophiles

“…The more reactive IA covalently modifies 1000 cysteines in the proteome [7], enabling the global profiling of changes in cysteine reactivity. This highly reactive electrophile has been instrumental in identifying bacterial redox modulators [41], cellular targets of electrophilic lipids [42], and zinc-binding cysteines within complex proteomes [43]. These studies highlight the utility of a highly-reactive, yet residue-selective electrophile to gain insight into modulators of amino-acid nucleophilicity.…”

Chemoproteomic Strategy to Quantitatively Monitor Transnitrosation Uncovers Functionally Relevant S -Nitrosation Sites on Cathepsin D and HADH2

“…This platform is derived from the previously reported isotopic tandem orthogonal proteolysis-ABPP (isoTOP-ABPP) method (Qian et al, 2013; Weerapana et al, 2010), which reliably and quantitatively monitors the intrinsic reactivity of 500–1000 cysteines concurrently within cell and tissue proteomes generated without protein denaturation. To date, the isoTOP-ABPP method and derivatives thereof have been successfully applied to quantitatively measure the loss in cysteine reactivity upon oxidation (Deng et al, 2013), reaction with lipid-derived electrophiles (Wang et al, 2014), and metal binding (Pace and Weerapana, 2014). In this approach (Figure 1), a cell lysate is exposed to GSNO or a buffer control.…”