Site-Specific Histidine Aza-Michael Addition in Proteins Enabled by a Ferritin-Based Metalloenzyme

Abstract

Histidine modifications of proteins are broadly based on chemical methods triggering N-substitution reactions such as aza-Michael addition at histidine's moderately nucleophilic imidazole side chain. While recent studies have demonstrated chemoselective, histidine-specific modifications by further exploiting imidazole's electrophilic reactivity to overcome interference from the more nucleophilic lysine and cysteine, achieving site-specific histidine modifications remains a major challenge due to the absence of spatial control over chemical processes. Herein, through X-ray crystallography and cryo-electron microscopy structural studies, we describe the rational design of a nature-inspired, noncanonical amino-acid-incorporated, human ferritin-based metalloenzyme that is capable of introducing site-specific post-translational modifications (PTMs) to histidine in peptides and proteins. Specifically, chemoenzymatic aza-Michael additions on single histidine residues were carried out on eight protein substrates ranging from 10 to 607 amino acids including the insulin peptide hormone. By introducing an insulin-targeting peptide into our metalloenzyme, we further directed modifications to be carried out site-specifically on insulin's B-chain histidine 5. The success of this biocatalysis platform outlines a novel approach in introducing residue- and, moreover, site-specific post-translational modifications to peptides and proteins, which may further enable reactions to be carried out in vivo.

Scheme 1. Protein Histidine PTMs, ncAAs and α,β-Unsaturated Chemicals, and Rational Design of a Ferritin-Based aza-Michael Ligase for Site-Specific Protein Histidine Modifications

Figure 1

(A) R63 and E67 of wt-Ftn (PDB code: 2FHA) are targeted for histidine analogs 1–3 (Scheme 1). Incorporation at the C₂ interface, as shown between two FTH1 monomers (red and blue). (B) 4–12% native PAGE analyses of the six ncAA-incorporated Ftn variants, with (C) ESI-MS analyses of the six ncAA-incorporated Ftn variants and (D) their Fe(II) and Cu(II) contents measured by ICP-MS alongside those of apoferritin, equine spleen ferritin, and wt-Ftn.

Figure 2

Cryo-EM structures of metal binding sites at the C2 engineered interface and monomer of (A, B) Ftn-2x-2 (PDB code 9JQB), (C, D) Ftn-2x-2-Cu(II) (PDB code: 9JQC), (E, F) Ftn-2x-3 (PDB code: 9JQD), and (G, H) Ftn-2x-3-Cu(II) (PDB code: 9JQE). Protein residues are depicted as light blue sticks, sodium ions in purple, copper in yellow, and iron in orange. Cryo-EM density maps are colored transparent gray.

Figure 3

MALDI-TOF-MS analyses of the insulin protein either (A) unmodified or modified with a single DEEM adduct (+186 m/z or Da) by CuCl₂-treated (B) Ftn-1x-3 (CVR = 20%, turnover number (TON) = 4.0 h^–1 ball^–1), (C) Ftn-2x-2 (CVR = 33%, TON = 6.6 h^–1 ball^–1), (D) Ftn-α-1x-3 (CVR = 46%, TON = 9.2 h^–1 ball^–1), and (E) Ftn-α-2x-2 (CVR = ∼100%, TON = 24 h^–1 ball^–1).

Figure 4

(A) Yield of Ftn-2x-2’s α,β-unsaturated chemical moiety-modified insulin products; (B) Amino acid, histidine and cysteine count, and yield of Ftn-2x-2’s DEEM-modified peptide and protein products.

Figure 5

(A) Ftn-2x-2’s DEEM-modified insulin product is treated with DTT to be digested into (B) A- and (C) B-chains, which are then separately analyzed by MALDI-TOF-MS to determine DEEM adduct’s localization. (D) MALDI-TOF-MS/MS analysis of Ftn-α-2x-2’s DEEM-modified insulin product revealed the site-selective modification to be carried out on only insulin’s B-chain H5.