Using N-Terminal Coordination of Cu(II) and Ni(II) to Isolate the Coordination Environment of Cu(I) and Cu(II) Bound to His13 and His14 in Amyloid-ß(4-16).

The amyloid-ß (Aß) peptide is a cleavage product of the amyloid precursor protein and has been implicated as a central player in Alzheimer's disease. The N-terminal end of Aß is variable, and different proportions of these variable-length Aß peptides are present in healthy individuals and those with the disease. The N-terminally truncated form of Aß starting at position 4 (Aß4-x) has a His residue as the third amino acid (His6 using the formal Aß numbering). The N-terminal sequence Xaa-Xaa-His is known as an amino terminal copper and nickel binding motif (ATCUN), which avidly binds Cu(II). This motif is not present in the commonly studied Aß1-x peptides. In addition to the ATCUN site, Aß4-x contains an additional metal binding site located at the tandem His residues (bis-His at His13 and 14) which is also found in other isoforms of Aß. Using the ATCUN and bis-His motifs, the Aß4-x peptide is capable of binding multiple metal ions simultaneously. We confirm that Cu(II) bound to this particular ATCUN site is redox silent, but the second Cu(II) site is redox active and can be readily reduced with ascorbate. We have employed surrogate metal ions to block copper coordination at the ATCUN or the tandem His site in order to isolate spectral features of the copper coordination environment for structural characterization using extended X-ray absorption fine structure (EXAFS) spectroscopy. This approach reveals that each copper coordination environment is independent in the Cu2Aß4-x state. The identification of two functionally different copper binding environments within the Aß4-x sequence may have important implications for this peptide in vivo.

PCuAC domains from methane-oxidizing bacteria use a histidine brace to bind copper.

Copper is critically important for methanotrophic bacteria because their primary metabolic enzyme, particulate methane monooxygenase (pMMO), is copper-dependent. In addition to pMMO, many other copper proteins are encoded in the genomes of methanotrophs, including proteins that contain periplasmic copper-Achaperone (PCuAC) domains. Using bioinformatics analyses, we identified three distinct classes of PCuAC domain-containing proteins in methanotrophs, termed PmoF1, PmoF2, and PmoF3. PCuAC domains from other types of bacteria bind a single Cu(I) ion via an HXnMX21/22HXM motif, which is also present in PmoF3, but PmoF1 and PmoF2 lack this motif entirely. Instead, the PCuAC domains of PmoF1 and PmoF2 bind only Cu(II), and PmoF1 binds additional Cu(II) ions in a His-rich extension to its PCuAC domain. Crystal structures of the PmoF1 and PmoF2 PCuAC domains reveal that Cu(II) is coordinated by an N-terminal histidine brace HX10H motif. This binding site is distinct from those of previously characterized PCuAC domains but resembles copper centers in CopC proteins and lytic polysaccharide monooxygenase (LPMO) enzymes. Bioinformatics analysis of the entire PCuAC family reveals previously unappreciated diversity, including sequences that contain both the HXnMX21/22HXM and HX10H motifs, and sequences that lack either set of copper-binding ligands. These findings provide the first characterization of an additional class of copper proteins from methanotrophs, further expand the PCuAC family, and afford new insight into the biological significance of histidine brace-mediated copper coordination.