Molecular mechanisms of copper homeostasis

Copper is an essential trace element which plays a pivotal role in cell physiology as it constitutes a core part of important cuproenzymes. Novel components of copper homeostasis in humans have been identified recently which have been characterised at the molecular level. These include copper-transporting P-type ATPases, Menkes and Wilson proteins, and copper chaperones. These findings have paved the way towards better understanding of the role of copper deficiency or copper toxicity in physiological and pathological conditions

Functional analysis of the N-terminal CXXC metal-binding motifs in the human Menkes copper-transporting P-type ATPase expressed in cultured mammalian cells

The Menkes protein (MNK) is a copper-transporting P-type ATPase, which has six highly conserved metal-binding sites, GMTCXXC, at the N terminus. The metal-binding sites may be involved in MNK trafficking and/or copper- translocating activity. In this study, we report the detailed functional analysis in mammalian cells of recombinant human MNK and its mutants with various metal-binding sites altered by site-directed mutagenesis. The results of the study, both in vitro and in vivo, provide evidence that the metal- binding sites of MNK are not essential for the ATP-dependent copper- translocating activity of MNK. Moreover, metal-binding site mutations, which resulted in a loss of ability of MNK to traffick to the plasma membrane, produced a copper hyperaccumulating phenotype. Using an in vitro vesicle assay, we demonstrated that the apparent K(m) and V(max) values for the wild type MNK and its mutants were not significantly different. The results of this study suggest that copper-translocating activity of MNK and its copper- induced relocalization to the plasma membrane represent a well coordinated copper homeostasis system. It is proposed that mutations in MNK which alter either its catalytic activity or/and ability to traffick can be the cause of Menkes disease

Erratum: Functional analysis of the N-terminal CXXC metal-binding motifs in the human Menkes copper-transporting P-type ATPase expressed in cultured mammalian cells (Journal of Biological Chemistry (1999) 274 (22008-22012))

Erratum: Functional analysis of the N-terminal CXXC metal-binding motifs in the human Menkes copper-transporting P-type ATPase expressed in cultured mammalian cells (Journal of Biological Chemistry (1999) 274 (22008-22012)

The structural basis for membrane binding and pore formation by lymphocyte perforin (3NSJ)

Natural killer cells and cytotoxic T lymphocytes accomplish the critically important function of killing virus-infected and neoplastic cells. They do this by releasing the pore-forming protein perforin and granzyme proteases from cytoplasmic granules into the cleft formed between the abutting killer and target cell membranes. Perforin, a 67-kilodalton multidomain protein, oligomerizes to form pores that deliver the pro-apoptopic granzymes into the cytosol of the target cell. The importance of perforin is highlighted by the fatal consequences of congenital perforin deficiency, with more than 50 different perforin mutations linked to familial haemophagocytic lymphohistiocytosis (type 2 FHL). Here we elucidate the mechanism of perforin pore formation by determining the X-ray crystal structure of monomeric murine perforin, together with a cryo-electron microscopy reconstruction of the entire perforin pore. Perforin is a thin ‘key-shaped’ molecule, comprising an amino-terminal membrane attack complex perforin-like (MACPF)/cholesterol dependent cytolysin (CDC) domain followed by an epidermal growth factor (EGF) domain that, together with the extreme carboxy-terminal sequence, forms a central shelf-like structure. A C-terminal C2 domain mediates initial, Ca2+-dependent membrane binding. Most unexpectedly, however, cryo-electron microscopy reveals that the orientation of the perforin MACPF domain in the pore is inside-out relative to the subunit arrangement in CDCs. These data reveal remarkable flexibility in the mechanism of action of the conserved MACPF/CDC fold and provide new insights into how related immune defence molecules such as complement proteins assemble into pores.</p