Guardian and chaperone - comparative Cu(II) coordination properties of CopD-derived motifs and the Met-rich loop of CopC.
While copper transport into bacterial cells remains poorly understood, the molecular basis of copper transfer across the periplasm is still largely unresolved. In this study, we investigated Cu(II) binding to peptide fragments derived from the periplasmic chaperone CopC and the inner membrane transporter CopD. The CopD protein contains two putative metal-binding domains, Ac-SPHLEHSIGHGDYTGAA-NH2 (SPH) and Ac-GHGAMDEGSRRFWHFATD-NH2 (GHG), characterized by three closely spaced and two distantly spaced histidine residues, respectively. In contrast, CopC features a Met-rich MX2MXHX2M motif (Ac-KLVMTAMPGMEHSPMAVKAAVSGGGDPKTMVIT-NH2, KLV), typically associated with Cu(I) binding. Using potentiometry, UV-Vis, CD, ESI-MS, ITC and DFT calculations, we show that all fragments form 1:1 Cu(II) complexes with distinct stability and coordination properties. At physiological pH, the SPH motif exhibits the highest Cu(II) affinity, while the GHG fragment shows reduced binding efficiency due to limited histidine cooperativity. The Met-rich CopC fragment binds Cu(II) significantly weaker and does not involve methionine residues in metal coordination, despite their abundance. This behaviour is consistent with the established preference of Met-rich motifs for Cu(I), while demonstrating that this region is not completely inert toward Cu(II). The formation of detectable yet labile Cu(II) complexes indicates limited but measurable Cu(II) binding by this sequence. Together with the strong Cu(II) binding observed for the SPH motif, these results support a model in which different sequence motifs contribute unequally to Cu(II) handling in the periplasm. Rather than demonstrating direct metal transfer, our data indicate that Met-rich regions provide comparatively labile Cu(II) binding, whereas His-rich sites offer more efficient Cu(II) capture under the studied conditions.