Copper/histidine sites in designed peptides and artificial proteins

Abstract

Copper bound to histidine binding sites is commonly found in a great number of biologically relevant metallo-peptides and metalloenzymes that operate essential functions for living organisms. Copper ion, however, may produce harmful effects when its homeostasis is not correctly regulated or when it binds to biomolecules such as neuropeptides. The coordination of copper(II) ions at His-His dyads of beta-amyloid or tau protein are exam-ples of metal sites which possess reactivities that were associated to the onset of neurodegenerative diseas-es. Among these reactivities, possibly the most important are the tendency to accelerate peptide aggregation and the propensity toward the production of ROS or other species relevant in oxidative stress. The work presented here initially aimed at elucidating the structural and thermodynamic stability of cop-per(II)/histidine adducts with short peptides that are models of tau protein. Oligopeptides containing His and tandem His-His motifs (named R1 and R3, respectively) were thoroughly studied in terms of Cu(II) and Cu(I) binding. Possibly the most important result of these investigations is that only the Cu/His-His site of R3 is ca-pable to bind copper with high affinity in both oxidation states. This result was crucial to explain the pseudo-catecholase activity of Cu(II)/R3 adducts in aerobic conditions. Many neuropeptides present His-His sites and, at short distance along the sequence, a third His site. Although there is a consensus in considering the His-His dyad as the principal copper(II) binding site, the effect of the additional His residue on the properties of the Cu(II)/His-His sites has yet to be elucidated. Therefore, taking inspiration from the R3 sequence of tau protein, in the second part of this work we de novo designed four peptides bearing two adjacent histidine residues and a third isolated His, the latter located at different posi-tions with respect to the His-His dyad. These peptides were synthesised, and the binding of copper(II) explored. The study of the pseudocatecholase activity of these Cu(II)/peptide adducts showed that the oxidase behavior remains centered at the His-His motif also in the presence of a third His site. The third part of this work describes the rational design of a novel artificial metalloprotein based on the Spy-Catcher/SpyTag (SC/ST) stystem, which is a construct that originally did not contain any metal binding site. The SC/ST construct consists of two moieties, namely the SpyCatcher protein and the SpyTag peptide, that spontaneously form a covalent adduct in solution. An ATCUN site was introduced on the SpyTag peptide, which was then used as ‘Trojan horse’ to take copper(II) on the SpyCatcher protein. As a result, we obtained an artificial metalloprotein bearing a copper/histidine site that was not originally present in the starting construct of which we have investigated the stability, the spectroscopic behaviour and the phosphoesterase activity.