Designing artificial metalloenzymes exploiting the Spy technology: study of a Cu(II)-Spy construct and perspectives

Metalloproteins promote several among the most complex biomolecular processes in Nature. The expanding demand for synthetic catalysts displaying enzyme-like activity is moving toward the development of ARTificial metalloenZYMES (ARTZYMES).[1] Protein redesign and de novo design are the two major strategies for the development of Artzymes, both having potentiality and drawbacks concerning the introduction of metal binding sites in a construct. Within the redesign approach, the so called ‘Trojan horse’ strategy involves the covalent or supramolecular grafting of a metal complex to a biomolecule,[2] to obtain the introduction of a metal ion into a known protein scaffold. We present here the original incorporation of a transition-metal binding site specifically designed on a small peptide component (SpyTag), onto a Spy-protein construct, thereby providing the Spy protein with a selective binding site for metal ions (Scheme 1). The Spy protein, redesigned by Howarth and colleagues,[3] comprises two components: a small beta-sandwich SpyCatcher (SC) protein component (ca. 110 residue), and the SpyTag (ST) peptide (13-16 residues). SC and ST incubated together in buffered aqueous solutions at neutral pH interact rapidly and selectively to form an isopeptide bond between a Lys (SC) and an Asp residue (ST). We redesigned the ST sequence by introducing an ATCUN sequence onto the Tag peptides. The ATCUN (Amino Terminal Cu and Ni binding site) fragment, characterized by a H2N-Xxx-Xxx-His sequence at the N-terminus, is known for its ability to bind Cu(II) and Ni(II) ions.[4] Additionally, the SC sequence was modified mutating two His residues into Gln to reduce the possibility of copper binding while preserving hydrogen bond interactions between residues. We were able to demonstrate that three different SpyTag-ATCUN peptides exhibit high binding affinity for Cu(II). Additionally, we confirm that the specific binding of Cu(II) at the ATCUN site of the ST sequence occurs in the presence of one equivalent of copper when SC is bound to ST. We observed this behaviour both with short ST peptides and when the ST sequence is expressed at the N-terminus of a natural protein, such as Thioredoxin. This engineered construct serves as an interesting proof-of-concept for novel metalloproteins, wherein the SpyTag peptide establishes the initial metal coordination sphere, while the SpyCatcher protein potentially contributes to the second coordination sphere. Starting from these results, this solid SC-ST metal binding construct could be employed studying its reactivity with different metals, as well as different metal binding groups with both natural and unnatural amino acids, introduced in the ST sequence via solid-phase peptide synthesis. Project ART2HYDROGEN “Artificial enzymes for the photocatalytic production of hydrogen in photosynthetic bacteria” funded under the National Recovery and Resilience Plan (NRRP), Mission 2 Component 2 Investment 3.5 - Call for tender No. 4 of March 23, 2022 of Italian Ministry of Ecologic Transition funded by the European Union – NextGenerationEU. Project code RSH2A_000009, Concession Decree 445 of December 29, 2022 adopted by the Italian Ministry of Environment and Energy Security.