Four Classes of BCB Domain-Containing Proteins

Close inspection of currently available sequences of proteins carrying BCB domains clearly indicated that they can be classified into four major classes, which are described below. This classification is based on their ability to bind copper and the specific features of their domain organization. Members of the first three classes harbor single or multiple type 1 blue copper-binding sites, while members of the fourth class do not appear to bind copper. Domain organizations of the precursors of aU currently known protein families that contain a BCB domain are shown in Fig. 1. 

The first class is cupredoxins—single-domain blue copper proteins composed of only one BCB domain. These proteins include plastocyanin, azurin, pseudoazurin, amicyanin, auracyanins, rusticyanin, halocyanin, and sulfocyanin (see Section IV). Plantacyanin of the phytocyanin family (Section V), subunit II of the cytochrome c oxidase, and the recently characterized nitrosocyanin also fall into this class. The last two are single BCB domain polypeptides closely related structurally to cupredoxins, but harboring, respectively, a binuclear copper site known as CuA and a novel type of copper-binding site called red (see Sections IX and X). 

The second class consists of multidomain blue copper proteins composed of exclusively two or more BCB domains and includes nitrite reductase (Section IV, E), multicopper blue oxidases such as laccase, ascorbate oxidase, ceruloplasmin, and hephaestin (Section VII), and some sequences found in extreme halophilic archaea (see Section V, E). 

The third class consists of proteins that are composed of one or more BCB domains fused to a sequence domain(s) characteristic of evolutionarily unrelated protein families. Such a mosaic domain organization has been found in the phytocyanin protein family, stellacyanins, uclacyanins, and the recently characterized dicyanins (Section V) in blood coagulation factor VIII (Section VIII) and in nitrous oxide reductase (Section IX). 

The functions of the above three classes of proteins are directly related to the protein-bound copper ions. In those cases where functions have been unequivocally established, they are either electron storage and transfer or redox catalysis. Representative proteins from each of the above-mentioned classes have been purified directly from their natural sources and extensively characterized both structurally and biophysically. In addition, there are well-established protocols for their expression and purification from different heterologous systems. [Factor VIII is a special case for which blue copper binding has not been experimentally demonstrated, although at least two such sites can be identified in its amino acid sequence (Section VIII).] 

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The rest of the known sequences of all four classes of BCB domain-containing proteins feature signal peptides in their precursors, indicating that they are translocated across the bacterial cytoplasmic membrane or are translated on the endoplasmic reticulum-bound ribosomes and sent to the secretory pathway in eukaryotes. Thus they are located in the bacterial periplasm, secreted into the extracellular milieu, or anchored to the cell surface. 

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