Structural characterization of the protein cce_0567 from Cyanothece 51142, a metalloprotein associated with nitrogen fixation in the DUF683 family

The genomes; of many cyanobacteria contain the sequence for a small protein with a common Domain of Unknown Function grouped into the DUF683 protein family. While the biological function of DUF683 is still not known, their genomic location within nitrogen fixation clusters suggests that DUF683 proteins may play a role in the process. The diurnal cyanobacterium Cyanothece sp. PCC 51142 contains a gene for a protein that falls into the DUF683 family, cce_0567 (78 aa, 9.0 kDa). In an effort to elucidate the biochemical role DUF683 proteins may play in nitrogen fixation, we have determined the first crystal structure for a protein in this family, cce_0567, to 1.84 A resolution. Cce_0567 crystallized in space group P2(1) with two protein molecules and one Ni2+ cation per asymmetric unit. The protein is composed of two (x-helices, residues P11 to G41 (alpha 1) and L49-E74 (alpha 2), with the second alpha-helix containing a short 3(10)-helix (Y46-N48). A four-residue linker (L42-D45) between the helices allows them to form an anti-parallel bundle and cross over each other towards their termini. In solution it is likely that two molecules of cce_0567 form a rod-like dimer by the stacking interactions of similar to 1/2 of the protein. Histidine-36 is highly conserved in all known DUF683 proteins and the N2 nitrogen of the H36 side chain of each molecule in the dimer is coordinated with Ni2+ in the crystal structure. The divalent cation Ni2+ was titrated into N-15-labeled cce_0567 and chemical shift perturbations were observed only in the H-1-N-15 HSQC spectra for residues at, or near, the site of Ni2+ binding observed in the crystal structure. There was no evidence for an increase in the size of cce_0567 upon binding Ni2+, even in large molar excess of Ni2+, indicating that a metal was not required for dimer formation. Circular dichroism spectroscopy indicated that cce_0567 was extremely robust, with a melting temperature of similar to 62 degrees C that was reversible. Published by Elsevier B.V.