The bis-histidine (bis-His) motif is an apparently simple system consisting of two adjacent histidine residues. Despite its seemingly simplicity, the bis-His motif exhibits a complex structural and functional framework, playing a pivotal role in metal coordination within biological systems. From a bioinorganic perspective, the uniqueness of histidine can be attributed to its imidazole ring, which contains two distinct nitrogen atoms, each with specific electronic and steric properties [1,2]. The nitrogen atom at position 1 of the imidazole ring is referred to as Nδ1, while the one at position 3 is called Nε2; for convenience, these will be referred to hereafter as Nπ and Nτ, respectively. The imidazole side chain of histidine provides nitrogen donors capable of interacting with metal ions, creating a versatile coordination environment that influences enzymatic activity, redox chemistry, and metal homeostasis. The bis-His motifs are widespread across various proteins and peptides, contributing significantly to their structural stability, catalytic mechanisms, and metal transport processes. Among these, histatin-5 (Hist-5) is one of the most abundant and biologically active variants of histatins. These family peptides are naturally secreted in human saliva and play a crucial role in oral defense mechanisms. Hist-5 contains multiple metal-binding motifs, including the ATCUN motif (Amino Terminal Cu(II), Ni(II) binding site) and a bis-His motif, both of which contribute to its interaction with copper ions (Cu) [3]. While the ATCUN motif predominantly binds Cu(II) in a square-planar 4 N coordination environment, the bis-His motif selectively binds Cu(I), stabilizing it in a near-linear geometry, sometimes involving additional ligands that contribute to modify the coordination structure. In full-length Hist-5, the coexistence of both ATCUN and bis-His motifs enables the peptide to bind and stabilize copper in both oxidation states, facilitating redox cycling and potentially playing a role in copper homeostasis and redox regulation, alongside its well-established antifungal activity [4].

Human copper transporter 1 (Ctr1) is a high-affinity membrane protein essential for cellular copper uptake and homeostasis. In Ctr1, the bis-His motif works alongside an ATCUN motif, which binds Cu(II) with high affinity. The proximity between these two motifs significantly influences copper reactivity, particularly in the presence of biological reductants like ascorbate [5]. The bis-His sequence enhances the reduction of Cu(II) to Cu(I), ensuring that copper is efficiently transported into the cell. The coordination environment of Cu(I) is also affected by the spacing between the ATCUN and bis-His motifs. When properly positioned, the bis-His motif helps maintain Cu(I) in a stable, protective state, preventing oxidative damage. However, when this spacing is altered, Cu(I) adopts a different 3 N(imidazole) distorted T-shaped geometry, which makes it more susceptible to oxidation, leading to the formation of reactive oxygen species (ROS) and peptide degradation [6].

The bis-His motif is also present in amyloidogenic proteins, which are recognized as hallmarks of neurodegenerative diseases. Alzheimer's disease (AD) is a progressive neurodegenerative disorder marked by cognitive decline and memory impairment. A defining characteristic of AD is protein aggregation, notably the accumulation of amyloid-beta (Aβ) peptides into extracellular plaques and tau protein into intracellular neurofibrillary tangles [7,8]. These pathological aggregates disrupt cellular function, contribute to synaptic loss, and trigger neuroinflammation, ultimately leading to neuronal degeneration [9]. Beyond the amyloid cascade hypothesis, growing evidence suggests that metal ion dyshomeostasis plays a pivotal role in AD pathology. Metal ions, including copper, zinc, and iron, are involved in key processes such as Aβ and tau metabolism, oxidative stress, and neuronal loss. Their abnormal accumulation or depletion can influence amyloid aggregation kinetics, altering the structural and pathological properties of Aβ and tau [[10], [11], [12]].

Tau protein is a microtubule-associated protein that plays a crucial role in stabilizing neuronal cytoskeleton structure. The R3 fragment, located within the microtubule-binding domain, contains a tandem His-His motif, which is involved in metal coordination, particularly with copper. This bis-His motif provides a highly favorable binding site for copper in both oxidation states. Potentiometric and spectroscopic studies reveal that Cu(II) coordination to R3 involves a (NIm, 2 N−, O) donor set, where one imidazole nitrogen, two deprotonated amide nitrogens, and possibly a water molecule participate in the equatorial coordination sphere. Additionally, the presence of two adjacent histidines in R3 enhances Cu(I) binding, forming a stable bis-His complex that facilitates redox cycling. The Cu(I)-R3 complex significantly enhances the catalytic oxidation of catecholic substrates such as dopamine and 4-methylcatechol, likely through an increased ability to activate molecular oxygen [13].

Aβ peptides are derived from the amyloid precursor protein (APP) through sequential cleavages by β- and γ-secretases. These peptides, primarily Aβ40 and Aβ42, exhibit varying aggregation propensities and can undergo further proteolytic processing and post-translational modifications, generating additional truncated, modified, or aggregated species that contribute to amyloid pathology [14]. Beyond full-length Aβ peptides, several N-terminally truncated variants, pyroglutamate-modified species, copper-bound Aβ complexes, and phosphorylated Aβ forms have been identified in CSF. Among these, AβpE3–40, AβpE11–42, Aβ4–40 Cu(II), and Aβ4–42 are particularly altered in Alzheimer's disease and mild cognitive impairment [15]. However, the precise role of these truncated and modified Aβ variants in AD progression remains unclear, requiring further investigation to develop targeted therapeutic strategies. A common feature of full-length amyloidogenic peptides is the absence of the ATCUN motif. Instead, they contain a single histidine at position 6 and two adjacent histidines at positions 13 and 14, forming the classical bis-His motif. In contrast, when the peptide is N-terminally truncated, it may incorporate the ATCUN motif, allowing two distinct coordination systems for copper ions. Given the structural diversity of the histidine residues involved, extensive investigations have been conducted to explore copper coordination, both in full-length Aβ1-x and its truncated model, Aβ4₋x [7,10,[16], [17], [18]]. In addition, the bis-His system is known to play a role in modulating peroxidase activity. In Aβ-heme complexes, His13 and His14 serve as key heme-binding ligands, with His13 preferentially coordinating the iron center when both residues are present [19]. The bis-His system appears to play a crucial role in modulating Aβ aggregation, even across species with distinct amyloid-beta (Aβ) sequences. Remarkably, studies have shown that the naked mole-rat (NMRat), differs from humans by a single amino acid substitution in its Aβ sequence (His13Arg). Both humans and NMRs exhibit lifespans 4- to 5-fold longer than expected for their body size, classifying them as long-lived species. This single substitution significantly reduces Aβ aggregation propensity in NMRats [20].

In this work, we investigated the coordination of copper ions to a truncated segment of the amyloidogenic Aβ peptide, (Aβ12–16; AcVHHQK-NH2), which includes the bis-His motif, while an acetylated valine prevents the formation of the ATCUN motif, effectively isolating the two histidine residues (His13 and 14). Our aim was to structurally assess the relevance of imidazole nitrogens in this bis-His motif for copper coordination. NMR spectroscopy was employed to fully characterize the metal complexes, utilizing systems in which histidine residues were selectively modified through methylation at the imidazole nitrogens. NMR experiments conducted on both the native peptide and its Nτ- and Nπ-methylhistidine (Fig. 1) derivatives elucidated the role of the bis-His motif in copper coordination, specifically clarifying the role played by the four imidazole nitrogens in the coordination system under investigation.