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Carboxypeptidase G2 modified with a versatile bioconjugate for metalloprotein design
Structural highlights
FunctionCBPG_PSES6 Catalyzes the hydrolysis of reduced and non-reduced folates to pteroates and L-glutamate. This enzyme has a broad specificity. Publication Abstract from PubMedPrecise metal-protein coordination by design remains a considerable challenge. Polydentate, high-metal-affinity protein modifications, both chemical and recombinant, can enable metal localization. However, these constructs are often bulky, conformationally and stereochemically ill-defined, or coordinately saturated. Here, we expand the biomolecular metal-coordination toolbox with the irreversible attachment to cysteine of bis(1-methylimidazol-2-yl)ethene ("BMIE"), which generates a compact imidazole-based metal-coordinating ligand. Conjugate additions of small-molecule thiols (thiocresol and N-Boc-Cys) with BMIE confirm general thiol reactivity. The BMIE adducts are shown to complex the divalent metal ions Cu(++) and Zn(++) in bidentate (N(2)) and tridentate (N(2)S*) coordination geometries. Cysteine-targeted BMIE modification (>90% yield at pH 8.0) of a model protein, the S203C variant of carboxypeptidase G2 (CPG2), measured with ESI-MS, confirms its utility as a site-selective bioconjugation method. ICP-MS analysis confirms mono-metallation of the BMIE-modified CPG2 protein with Zn(++), Cu(++), and Co(++). EPR characterization of the BMIE-modified CPG2 protein reveals the structural details of the site selective 1:1 BMIE-Cu(++) coordination and symmetric tetragonal geometry under physiological conditions and in the presence of various competing and exchangeable ligands (H(2)O/HO(-), tris, and phenanthroline). An X-ray protein crystal structure of BMIE-modified CPG2-S203C demonstrates that the BMIE modification is minimally disruptive to the overall protein structure, including the carboxypeptidase active sites, although Zn(++) metalation could not be conclusively discerned at the resolution obtained. The carboxypeptidase catalytic activity of BMIE-modified CPG2-S203C was also assayed and found to be minimally affected. These features, combined with ease of attachment, define the new BMIE-based ligation as a versatile metalloprotein design tool, and enable future catalytic and structural applications. A Bis(imidazole)-based cysteine labeling tool for metalloprotein assembly.,Ahmad R, Tyryshkin AM, Xie L, Hansen WA, Yachnin BJ, Emge TJ, Mashrai A, Khare SD, Knapp S J Inorg Biochem. 2023 Apr 1;244:112206. doi: 10.1016/j.jinorgbio.2023.112206. PMID:37030124[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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