Deciphering structure and topology of conserved COG2042 orphan proteins

Background: The cluster of orthologous group COG2042 has members in all sequenced Eukaryota as well as in many Archaea. The cellular function of these proteins of ancient origin remains unknown. PSI- BLAST analysis does not indicate a possible link with even remotely- related proteins that have been functionally or structurally characterized. As a prototype among COG2042 orthologs, SSO0551 protein from the hyperthermophilic archaeon Sulfolobus solfataricus was purified to homogeneity for biophysical characterization. Results: The untagged protein is thermostable and behaves as a monomeric protein in gel filtration experiment. Several mass spectrometry- based strategies were combined to obtain a set of low resolution structural information. Kinetic data from limited proteolysis with various endoproteases are concordant in pointing out that region Glu(73)- Arg(78) is hyper- sensitive, and thus accessible and flexible. Lysine labeling with NHS- biotin and cross- linking with DTSSP revealed that the 35 amino acid RLI motif at the N terminus is solvent exposed. Cross- links between Lys(10)- Lys(14) and Lys(23)-Lys(25) indicate that these residues are spatially close and in adequate conformation to be crosslinked. These experimental data have been used to rank multiple three- dimensional models generated by a de novo procedure. Conclusion: Our data indicate that COG2042 proteins may share a novel fold. Combining biophysical, mass- spectrometry data and molecular model is a useful strategy to obtain structural information and to help in prioritizing targets in structural genomics programs.