Blue copper proteins: A comparative analysis of their molecular interaction properties

Abstract

Blue copper proteins are type-I copper-containing redox proteins whose role is to shuttle electrons from an electron donor to an electron acceptor in bacteria and plants. A large amount of experimental data is available on blue copper proteins; however, their functional characterization is hindered by the complexity of redox processes in biological systems. We describe here the application of a semiquantitative method based on a comparative analysis of molecular interaction fields to gain insights into the recognition properties of blue copper proteins. Molecular electrostatic and hydrophobic potentials were computed and compared for a set of 33 experimentally-determined structures of proteins from seven blue copper subfamilies, and the results were quantified by means of similarity indices. The analysis provides a classification of the blue copper proteins and shows that~1! comparison of the molecular electrostatic potentials provides useful information complementary to that highlighted by sequence analysis;~2! similarities in recognition properties can be detected for proteins belonging to different subfamilies, such as amicyanins and pseudoazurins, that may be isofunctional proteins;~3! dissimilarities in interaction properties, consistent with experimentally different binding specificities, may be observed between proteins belonging to the same subfamily, such as cyanobacterial and eukaryotic plastocyanins;~4! proteins with low sequence identity, such as azurins and pseudoazurins, can have sufficient similarity to bind to similar electron donors and acceptors while having different binding specificity profiles.Keywords: blue copper proteins; electrostatic potential; electron transfer; hydrophobic potential; protein-protein interactions; redox proteins; similarity index Blue copper proteins, which are also known as cupredoxins, are small, soluble proteins~10-14 kDa! whose active site contains a type-I copper. As far as it is known, they exert their function by shuttling electrons from a protein acting as an electron donor to another acting as an electron acceptor in various biological systems, such as bacterial and plant photosynthesis~Baker, 1994; Sykes, 1994!.A large amount of structural and spectroscopic data is available for the blue copper proteins, which have been named and classified into subfamilies according to their spectroscopic properties~Ad-man, 1991!. High resolution X-ray and NMR structures are known for several members of each of the plastocyanin, azurin, pseudoazurin, and amicyanin subfamilies~Baker, 1994!, and for single members of three further subfamilies, the rusticyanins~Harvey et al., 1998!, the cucumber basic proteins~CBP!~Guss et al., 1996!, and the stellacyanins~Hart et al., 1996!. Despite often showing low~Ͻ20%! sequence identity, all these proteins possess an eight-stranded Greek key b-barrel or b-sandwich fold and have a highly conserved active site architecture~Baker, 1994; Sykes, 1994!. Important information concerning the functional role and the binding properties of th...

Apoptosis or growth arrest: Modulation of tumor suppressor p53's specificity by bacterial redox protein azurin

“…Wt azurin, which forms a stable complex at the N-terminal to mid-region of p53 (16) without affecting p53 oligomer formation, mostly directs p53 specificity toward activation of the bax gene, leading to apoptosis (9,10). The hydrophobic patch of azurin is known to be involved in protein-protein interaction with its in vitro electron transfer partners (12)(13)(14). A replacement of two hydrophobic amino acids by polar amino acids in this region changes the way the M44KM64E mutant azurin interacts with p53, suppressing its oligomer formation.…”

“…On entry into such cells, the WT azurin formed a complex with the tumor suppressor protein p53, stabilized it to raise its intracellular level, generated enhanced levels of reactive oxygen species, and induced apoptosis (9,10). Azurin has a hydrophobic patch exposed to the surface that is believed to be involved in protein-protein interaction with its in vitro electron transfer partners such as cytochrome c 551 (12)(13)(14). When two methionine residues in this hydrophobic patch were replaced by two polar amino acids (lysine and glutamic acid) to reduce the hydrophobicity of the patch, the resultant M44KM64E mutant azurin could still enter the J774 cells, as determined by immunoblotting of the subcellular fractions of the cells incubated for various times with the mutant azurin protein, but demonstrated very little cytotoxicity and induction of apoptosis in J774 cells (15).…”

Genomic View of Energy Metabolism in <i>Ralstonia eutropha</i> H16

“…In contrast to several related ␤ -proteobacteria (e.g. from the genus Alcaligenes ), R. eutropha H16 does not encode blue-copper type single-electron mediators like cupredoxin, azurin, or pseudoazurin [De Rienzo et al, 2000;Farver and Pecht, 1991]. More than 60 genes for putative cytochrome c proteins are present in the genome.…”

A peptide fragment of azurin induces a p53-mediated cell cycle arrest in human breast cancer cells

“…Azurin, a member of the cupredoxin family of redox proteins present in bacteria and green plants, is a type I copper-containing soluble protein (10-14 kDa) that transports electrons during denitrification and photosynthesis (1). We and others have shown that azurin preferentially enters cancer cells (2)(3)(4)(5) and induces a p53-mediated apoptosis in murine J774, human breast cancer, melanoma (2,3,5), and osteosarcoma cells but not in p53-negative osteosarcoma and normal liver cells (p53 wild-type; ref.…”