Gentili Silvia, Miglioli Francesca, Borghesani Valentina, Spagnoli Gloria, Bellotti Denise, Cavazzini Davide, Guerrini Remo, Remelli Maurizio, Maestri Giovanni, Ottonello Simone, Bolchi Angelo, Tegoni Matteo
Department of Chemistry, Life Sciences, and Environmental Sustainability, University of Parma, Parco Area delle Scienze 11A, 43124, Parma, Italy.
Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, 44100, Ferrara, Italy.
Chembiochem. 2025 Jul 18;26(14):e202500208. doi: 10.1002/cbic.202500208. Epub 2025 May 21.
Designing artificial metal binding sites within a protein is challenging since amino acid residues need to be placed in desired positions in the final construct and the use of non-natural amino acids is difficult. The alternative approach of directing the insertion of artificial metal coordination systems presents the difficulty of grafting such site in a single desired position. Spy protein is composed of a protein component (SpyCatcher) which binds spontaneously an oligopeptide (SpyTag) with formation of an isopeptide bond. A SpyTag peptide equipped with an ATCUN (amino terminal copper and nickel) binding site is designed to bind copper(II) with high femtomolar affinity both in the absence of SpyCatcher and in the reconstituted Spy construct. The Cu ATCUN site in the reconstituted Spy protein presents a catalytic activity in reactive oxygen species production, higher than that of the SpyTag peptide alone. This method offers a novel approach for constructing artificial metalloproteins by incorporating functional metal binding sites into a peptide, which can then be clicked onto its protein counterpart. The small size and modularity of this construct make it versatile for integration into other protein systems, eventually moving the complexity from a protein to a peptide and highlighting its potential for protein design.
在蛋白质中设计人工金属结合位点具有挑战性，因为需要将氨基酸残基置于最终构建体的期望位置，且使用非天然氨基酸存在困难。引导人工金属配位系统插入的另一种方法存在将此类位点嫁接到单个期望位置的难题。Spy蛋白由一个蛋白质组分（SpyCatcher）组成，该组分可自发结合一个寡肽（SpyTag）并形成异肽键。设计了一个配备有ATCUN（氨基末端铜和镍）结合位点的SpyTag肽，使其在不存在SpyCatcher以及在重组的Spy构建体中均能以高飞摩尔亲和力结合铜（II）。重组Spy蛋白中的铜ATCUN位点在活性氧生成方面呈现出催化活性，高于单独的SpyTag肽。该方法通过将功能性金属结合位点整合到肽中，为构建人工金属蛋白提供了一种新方法，然后该肽可与对应的蛋白质进行连接。这种构建体的小尺寸和模块化使其能够灵活地整合到其他蛋白质系统中，最终将复杂性从蛋白质转移到肽上，并突出了其在蛋白质设计方面的潜力。
