Identification of Unknown Protein Function Using Metabolite Cocktail Screening

• Published in: Structure (London) 2012-10, Vol.20 (10), p.1715-1725
• Main Authors: Shumilin, Igor A., Cymborowski, Marcin, Chertihin, Olga, Jha, Kula N., Herr, John C., Lesley, Scott A., Joachimiak, Andrzej, Minor, Wladek
• Format: Article
• Language: English
• Subjects: ADP Ribose Transferases - chemistry; Animals; Bacillus subtilis - enzymology; Bacterial Proteins - chemistry; Carrier Proteins - chemistry; Catalytic Domain; Crystallography, X-Ray; Hydro-Lyases - chemistry; Hydrogen Bonding; Mice; Models, Molecular; Molecular Sequence Annotation; Phosphoproteins - chemistry; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Small Molecule Libraries - chemistry; Thermotoga maritima - enzymology
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2012.07.016

Proteins of unknown function comprise a significant fraction of sequenced genomes. Defining the roles of these proteins is vital to understanding cellular processes. Here, we describe a method to determine a protein function based on the identification of its natural ligand(s) by the crystallographic screening of the binding of a metabolite library, followed by a focused search in the metabolic space. The method was applied to two protein families with unknown function, PF01256 and YjeF_N. The PF01256 proteins, represented by YxkO from Bacillus subtilis and the C-terminal domain of Tm0922 from Thermotoga maritima, were shown to catalyze ADP/ATP-dependent NAD(P)H-hydrate dehydratation, a previously described orphan activity. The YjeF_N proteins, represented by mouse apolipoprotein A-I binding protein and the N-terminal domain of Tm0922, were found to interact with an adenosine diphosphoribose-related substrate and likely serve as ADP-ribosyltransferases. Crystallographic screening of metabolites serves as an efficient tool in functional analyses of uncharacterized proteins. ► Crystallographic screening of ligand binding can be used in protein function analysis ► Proteins from family PF01256 serve as ADP/ATP-dependent NAD(P)H-hydrate dehydratases ► Proteins of family YjeF_N interact with substrate structurally related to ADP-ribose Proteins of unknown function comprise a significant fraction of sequenced genomes. Here, Shumilin et al. describe a method to determine protein function based on identification of its natural ligand(s) by crystallographic screening of a metabolite library, followed by a focused search in the metabolic space.