Capsella bursa-pastoris, commonly known as shepherd's purse, is a highly prevalent flowering plant found across the globe [1]. Despite being a widely recognized and challenging weed to manage due to its rapid growth in fields, pastures, and gardens, it has been used in traditional medicine since ancient times [2]. This plant, with its rich reservoir of valuable components including flavonoids, sterols, vitamins, and essential metal ions, plays a crucial role in supporting proper functioning of the body. Studies have shown its anti-haemorrhagic, antibacterial, anti-inflammatory and anti-cancer properties [3]. Antimicrobial properties were for a long time attributed mainly to sulforaphane, isothiocyanate compound, active against Gram-positive bacterium Bacillus anthracis and Vancomycin-Resistant Enterococci strains [2]. However, a few years ago, two novel antimicrobial peptides (AMPs) were isolated from the roots of the shepherd's purse, which show activity against Gram-negative bacteria and fungi: shepherin I (Shep I) and shepherin II (Shep II) [4].

In general, antimicrobial peptides can be found in almost all living organisms and are one of the most promising groups of compounds to fight antimicrobial resistance [5,6]. It is also well-documented that metal ions, such as Cu(II) and Zn(II), often exhibit antimicrobial properties. The exact mechanism of microbial copper-related death is not fully understood, but the most likely mechanisms include: (i) the physical interactions of copper ions and/or copper nanoparticles with the cell membrane, triggering plasma leakage and thus leading to its destruction and (ii) the generation of reactive oxygen species (ROS) by the reduction of copper through a Fenton-like reaction, leading to oxidative damage involving lipid peroxidation, protein oxidation and DNA damage. For fungi, the uptake of copper ions and the physical deterioration of the membrane leading to copper influx are considered to be the primary mechanisms [7]. In the case of zinc (which is one of the most important metal ions for all species, essential for the survival of both pathogenic microorganisms and their mammalian hosts), the crucial antimicrobial mechanism seems to be ‘zinc nutritional immunity’ - a process that describes the competition between the bacteria or fungi and the host for this metal, during which both the pathogen and host make huge efforts to control zinc availability [8]. Recently, new forms of zinc-based biocidal materials have been also reported, featuring distinct antimicrobial mechanisms, including zinc based nano-arrays, metal-organic framework (MOF) - based reactive oxygen species (ROS) release and zinc composites capable of self-generate ROS [9].

Antimicrobial peptides can be even more effective after having coordinated metal ions; in many cases, these ions are essential for their optimal activity via altering the physicochemical properties of the peptides, such as their local charge or structure [10].

Previously, we described Cu(II) and Zn(II) complexes of shepherin I and showed that presence of Zn(II) ions enhances antimicrobial activity. Overall, logical sequence of events is observed in which each step is influenced by the preceding one. First, Zn(II) binds to shepherin I, causing a structural change, which is a trigger for the formation of fibrils. Ultimately, these sequential structural changes culminate in a significant biological effect, where the Zn(II) – shepherin I fibrils exhibit remarkable antifungal properties. What makes this particularly intriguing is that both the formation of fibrils and the potent anticandidal activity are only evident in the Zn(II) – shepherin I complex, and not in the peptide itself, highlighting the connection between the structural rearrangement induced by metal binding and its impact on morphology and biological activity [11].

Shepherin II consists of 38 amino acid residues with a total of eight histidyl residues, which are perfect Cu(II) and Zn(II) anchors. A regular pattern of six GGH repeats may make the peptide act as a ‘metal-sponge’ (Fig. 1) [4]. Moreover, a characteristic fragment of Shep II is the ATCUN motif (amino terminal Cu(II) and Ni(II) binding site, also known as the ‘albumin-like’ binding site) [12], common in various naturally occurring peptides and proteins responsible for maintaining metal balance within the body. Examples of such proteins include human serum albumin (HSA), histatins or hepcidin- 25 [[13], [14], [15], [16], [17]]. The ATCUN motif comprises a free amine group at the N-terminus a His residue at the third position, and two amide groups in between; this arrangement enables the motif to bind Cu(II) and Ni(II) ions with exceptional affinity. Typically, within a pH range of 4.5 to 8, the ATCUN motif forms a 4 N square planar complex with Cu(II), featuring a donor set composed of groups [15,18]. In recent years there has been an increasing interest in the interaction of copper with the ATCUN motif, with particular emphasis on reaction kinetics [19,20]. The interaction between Cu(II) ions and peptides holds significant importance in comprehending how copper is distributed and transferred in bodily fluids, as well as how cells acquire copper. These matters have become increasingly significant due to recent findings indicating copper imbalances in various modern diseases, such as Alzheimer's disease [21,22]. The impact of these side chains on the rate of the reaction is still under ongoing investigation.

Previous studies show that shepherin II was active against various Gram-negative bacteria, with IC50 values within the 2.5–8 μg/mL range when tested against E. coli, P. putida, P. syringae, and Serratia sp. Additionally, shepherin II had marked antifungal activity against yeast-phase C. neoformans, S. cerevisiae and C. albicans [4].

In this work, we focus on shepherin II and its antimicrobial activity in complexes with Zn(II) and Cu(II), seeking a correlation between the coordination mode, thermodynamics, structure and antimicrobial potency of the Shep II complexes, and suggesting their possible mechanisms of action.