6xj5

From Proteopedia

Carboxypeptidase G2 modified with a versatile bioconjugate for metalloprotein design

Structural highlights

6xj5 is a 8 chain structure with sequence from Pseudomonas sp. RS-16. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:	X-ray diffraction, Resolution 3.11Å
Ligands:	, ,
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

CBPG_PSES6 Catalyzes the hydrolysis of reduced and non-reduced folates to pteroates and L-glutamate. This enzyme has a broad specificity.

Publication Abstract from PubMed

Precise 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

↑ Ahmad R, Tyryshkin AM, Xie L, Hansen WA, Yachnin BJ, Emge TJ, Mashrai A, Khare SD, Knapp S. A Bis(imidazole)-based cysteine labeling tool for metalloprotein assembly. J Inorg Biochem. 2023 Apr 1;244:112206. PMID:37030124 doi:10.1016/j.jinorgbio.2023.112206