A prokaryotic superoxide dismutase paralog lacking two Cu ligands: From largely unstructured in solution to ordered in the crystal

Abstract

Little is known about prokaryotic homologs of Cu,Zn superoxide dismutase (SOD), an enzyme highly conserved among eukaryotic species. In 138 Archaea and Bacteria genomes, 57 of these putative homologs were found, 11 of which lack at least one of the metal ligands. Both the solution and the crystal structures of the SOD-like protein from Bacillus subtilis, lacking two Cu ligands and found to be enzymatically inactive, were determined. In solution, the protein is monomeric. The available nuclear Overhauser effects, together with chemical-shift index values, allowed us to define and to recognize the typical Cu,Zn SOD Greek ␤-barrel but with largely unstructured loops (which, therefore, sample a wide range of conformations). On the contrary, in the crystal structure (obtained in the presence of slight excess of Zn), the protein is well structured and organized in covalent dimers held by a symmetric bridge consisting of a Zn ion bound to an Asp-His dyad in a tetrahedral geometry. Couples of dimers held by hydrophobic interactions and H bonds are further organized in long chains. The order͞disorder transition is discussed in terms of metal binding and physical state.conformational mobility ͉ NMR structure determination ͉ Zn-mediated protein dimers ͉ x-ray crystallography ͉ superoxide dismutase T he availability of the complete sequence of an increasing number of genomes allows comparative analyses aimed at the identification of genes and of corresponding physiological functions conserved along evolution. Cu,Zn superoxide dismutase (SOD) is a ubiquitous family of enzymes that efficiently catalyze the dismutation of superoxide anions into oxygen and hydrogen peroxide (1, 2). The protein takes a classical Greek key ␤-barrel fold and adopts a dimeric quaternary structure (3). It binds one Zn and one Cu ion per subunit, the latter ion being the site of the enzymatic reaction, whose rate limiting step is the diffusion of superoxide through the channel that leads to the Cu site (4). SOD has been considered for a long time to be peculiar of eukaryotic organisms, in which its evolutionary rate has been studied (5). From the first description of a SOD activity in Escherichia coli (6), other putative homologous of the eukaryotic protein have been then found in the periplasmic space of other bacterial species (7) The x-ray structures have been solved for the proteins from Photobacterium leiognathi (8), E. coli (9), Salmonella typhimurium (10), and Actinobacillus pleuropneumoniae (11), all of them maintaining the typical metal sites of Cu,Zn eukaryotic SODs and normal SOD activity. With the aim of providing a more detailed picture of prokaryotic SODs, we browsed the available complete genomes of Archea and Bacteria to search for sequences with some degree of homology with human SOD (HSOD), which is found in 48 of the 138 prokaryotes with complete genome. In some of them, not all of the metal ligands are present. The SOD-like protein from Bacillus subtilis (BsSOD) lacks two binding-Cu residues (His-48 and His-63, human protein numbe...

Structural and dynamic aspects related to oligomerization of apo SOD1 and its mutants

“…Similar behavior was observed in the SOD-like protein from Bacillus subtilis (27), where its NMR properties indicate a conformational mobility for most of the protein, characterized by defined secondary-structure elements and a dynamic tertiary structure, at variance with the X-ray crystal structure of the same protein, which shows a well-ordered tertiary structure.…”

Crystallization of soluble proteins in vapor diffusion for x-ray crystallography

“…The case of Bacillus subtilis superoxide dismutase While facing the difficult task to crystallize a superoxide dismutase-like protein from Bacillus subtilis (BsSOD) we ran some NMR experiments, which revealed that BsSOD was only partially structured in solution 61,107 . The NMR data demonstrated the existence of secondary structure elements that fluctuate over several different conformations in solution.…”

“…We then set up a screen by using Zn(II) concentration as a variable and eventually we were able to grow well diffracting crystals of the protein. Step Problem Possible reason Solution located at the surface of the protein 61 . The coordination bonds promote a transition from a disordered structure to a completely folded structure of BsSOD.…”

“…6| Detect aggregate formation in dilute solution by LS [54][55][56][57][58][59][60] or by NMR/NMR dispersion 61,62 . In case of aggregate content of over 15-20% of the protein present in solution (monodispersity lower than 85-80%), reconsider protein purification and buffer composition 63 .…”

Predicting Metal-Binding Sites of Protein Residues

“…Metal ions in proteins are responsible for multiple tasks. They help stabilizing protein structure [4], induce conformational changes [5][6][7], and assist protein functions (e.g. electron transfer, nucleophilic catalysis).…”