title: Identification of unknown protein function using metabolite cocktail screening.
abstract: 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.
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908, USA. ias2n@virginia.edu
authors.forename: Igor A
authors.initials: IA
authors.lastname: Shumilin
authors.forename: Marcin
authors.initials: M
authors.lastname: Cymborowski
authors.forename: Olga
authors.initials: O
authors.lastname: Chertihin
authors.forename: Kula N
authors.initials: KN
authors.lastname: Jha
authors.forename: John C
authors.initials: JC
authors.lastname: Herr
authors.forename: Scott A
authors.initials: SA
authors.lastname: Lesley
authors.forename: Andrzej
authors.initials: A
authors.lastname: Joachimiak
authors.forename: Wladek
authors.initials: W
authors.lastname: Minor
mesh_terms: D036002:ADP Ribose Transferases / Q000737:chemistry*; D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006836:Hydro-Lyases / Q000737:chemistry*; D006860:Hydrogen Bonding; D051379:Mice; D008958:Models, Molecular; D058977:Molecular Sequence Annotation; D010750:Phosphoproteins / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019998:Racemases and Epimerases; D054852:Small Molecule Libraries / Q000737:chemistry; D020124:Thermotoga maritima / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D010750:Phosphoproteins; D054852:Small Molecule Libraries; D036002:ADP Ribose Transferases; D006836:Hydro-Lyases; C529323:Naxe protein, mouse; D019998:Racemases and Epimerases