Identification of unknown protein function using metabolite cocktail screening

Structure. 2012 Oct 10;20(10):1715-25. doi: 10.1016/j.str.2012.07.016. Epub 2012 Aug 30.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 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
  • Racemases and Epimerases
  • Small Molecule Libraries / chemistry
  • Thermotoga maritima / enzymology

Substances

  • Bacterial Proteins
  • Carrier Proteins
  • Phosphoproteins
  • Small Molecule Libraries
  • ADP Ribose Transferases
  • Hydro-Lyases
  • Naxe protein, mouse
  • Racemases and Epimerases

Associated data

  • PDB/3RNO
  • PDB/3RO7
  • PDB/3ROE
  • PDB/3ROG
  • PDB/3ROX
  • PDB/3ROZ
  • PDB/3RPH
  • PDB/3RPZ
  • PDB/3RQ2
  • PDB/3RQ5
  • PDB/3RQ6
  • PDB/3RQ8
  • PDB/3RQH
  • PDB/3RQQ
  • PDB/3RQX
  • PDB/3RRB
  • PDB/3RRE
  • PDB/3RRF
  • PDB/3RRJ
  • PDB/3RS9
  • PDB/3RSF
  • PDB/3RSG
  • PDB/3RSQ
  • PDB/3RSS
  • PDB/3RT7
  • PDB/3RT9
  • PDB/3RTA
  • PDB/3RTB
  • PDB/3RTC
  • PDB/3RTD
  • PDB/3RTE
  • PDB/3RTG
  • PDB/3RU2
  • PDB/3RU3