No abstract

Zinc metalloproteins are one of the most abundant and structurally diverse proteins in nature. In these proteins, the Zn(II) ion possesses a multifunctional role as it stabilizes the fold of small zinc fingers, catalyzes essential reactions in enzymes of all six classes, or assists in the formation of biological oligomers. Previously, a number of database surveys have been conducted on zinc proteins to gain broader insights into their rich coordination chemistry. However, many of these surveys suffer from severe flaws and misinterpretations or are otherwise limited. To provide a more comprehensive, up-to-date picture on zinc coordination environments in proteins, zinc containing protein structures deposited in the Protein Data Bank (PDB) were analyzed in detail. A statistical analysis in terms of zinc coordinating amino acids, metal-to-ligand bond lengths, coordination number, and structural classification was performed, revealing coordination spheres from classical tetrahedral cysteine/histidine binding sites to more complex binuclear sites with carboxylated lysine residues. According to the results, coordination spheres of hundreds of crystal structures in the PDB could be misinterpreted due to symmetry-related molecules or missing electron densities for ligands. The analysis also revealed increasing average metal-to-ligand bond length as a function of crystallographic resolution, which should be taken into account when interrogating metal ion binding sites. Moreover, one-third of the zinc ions present in crystal structures are artifacts, merely aiding crystal formation and packing with no biological significance. Our analysis provides solid evidence that a minimal stable zinc coordination sphere is made up by four ligands and adopts a tetrahedral coordination geometry.

Deacetylation of histones is carried out by a corepressor complex in which Sin3A is an essential scaffold protein. Two proteins in this complex, the Sin3A-associated proteins SAP30L and SAP30, have previously been suggested to function as linker molecules between various corepressors. In this report, we demonstrate new functions for human SAP30L and SAP30 by showing that they can associate directly with core histones as well as naked DNA. A zinc-coordinating structure is necessary for DNA binding, one consequence of which is bending of the DNA. We provide evidence that a sequence motif previously shown to be a nuclear localization signal is also a phosphatidylinositol (PI)-binding element and that binding of specific nuclear monophosphoinositides regulates DNA binding and chromatin association of SAP30L. PI binding also decreases the repression activity of SAP30L and affects its translocation from the nucleus to the cytoplasm. Our results suggest that SAP30L and SAP30 play active roles in recruitment of deacetylating enzymes to nucleosomes, and mediate key protein-protein and protein-DNA interactions involved in chromatin remodeling and transcription.

Rhizobium etli CFN42 is a symbiotic nitrogen-fixing bacterium of the common bean Phaseolus vulgaris. The symbiotic plasmid p42d of R. etli comprises a gene encoding a putative (strept)avidin-like protein, named rhizavidin. The amino acid sequence identity of rhizavidin in relation to other known avidin-like proteins is 20-30%. The amino acid residues involved in the (strept)avidin-biotin interaction are well conserved in rhizavidin. The structural and functional properties of rhizavidin were carefully studied, and we found that rhizavidin shares characteristics with bradavidin, streptavidin and avidin. However, we found that it is the first naturally occurring dimeric protein in the avidin protein family, in contrast with tetrameric (strept)avidin and bradavidin. Moreover, it possesses a proline residue after a flexible loop (GGSG) in a position close to Trp-110 in avidin, which is an important biotin-binding residue. [3H]Biotin dissociation and ITC (isothermal titration calorimetry) experiments showed dimeric rhizavidin to be a high-affinity biotin-binding protein. Its thermal stability was lower than that of avidin; although similar to streptavidin, it was insensitive to proteinase K. The immunological cross-reactivity of rhizavidin was tested with human serum samples obtained from cancer patients exposed to (strept)avidin. No significant cross-reactivity was observed. The biodistribution of the protein was studied by SPECT (single-photon emission computed tomography) imaging in rats. Similarly to avidin, rhizavidin was observed to accumulate rapidly, mainly in the liver. Evidently, rhizavidin could be used as a complement to (strept)avidin in (strept)avidin-biotin technology.

Twelve members of the family 11 xylanases, including both mesophilic and thermophilic proteins, were studied using molecular dynamics (MD). Simulations of xylanases were carried out in an explicit water environment at four different temperatures, 300, 400, 500 and 600 K. A difference in thermotolerance between mesophilic and thermophilic xylanases became clear: thermophilic xylanases endured heat in higher simulation temperatures better than mesophilic ones. The unfolding pathways seemed to be similar for all simulations regardless of the protein. The unfolding initiates at the N-terminal region or alternatively from the alpha-helix region and proceeds to the 'finger region'. Unfolding of these regions led to denaturated structures within the 4.5 ns simulation at 600 K. The results are in agreement with experimental mutant studies. The results show clearly that the stability of the protein is not evenly distributed over the whole structure. The MD analysis suggests regions in the protein structure which are more unstable and thus potential targets for mutation experiments to improve thermostability.