Research paper Identification of new protein complexes of Escherichia coli inorganic pyrophosphatase using pull-down assay E. Rodina a, * , N. Vorobieva a , S. Kurilova b , Ju. Mikulovich a , J. Vainonen c , E.-M. Aro c , T. Nazarova b a Chemistry Department, M.V. Lomonosov Moscow State University, Leninskie Gory 1, Moscow119992, Russian Federation b A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninskie Gory 1, Moscow 119992, Russian Federation c Department of Biochemistry and Food Chemistry, Molecular Plant Biology, University of Turku, FI-20520 Turku, Finland article info Article history: Received 25 November 2010 Accepted 10 May 2011 Available online 31 May 2011 Keywords: Protein complex Metabolic regulation Inorganic pyrophosphatase abstract Inorganic pyrophosphatase (PPase) is a conserved and essential enzyme catalyzing the hydrolysis of pyrophosphate PP i . Its activity is required to promote a lot of thermodynamically unfavorable reactions including biosynthesis of activated precursors of sugars and amino acids. Several protein partners of PPase were found so far in Escherichia coli by large-scale approaches. Functional role of these interactions was not studied. In this paper we report the identification of three protein partners of E. coli PPase not found earlier. Pull-down assay on the Ni 2þ -chelating column using 6His-tagged PPase as bait was used to isolate PPase complexes from stationary-phase cells. Of several isolated protein components, five were identified by MALDIeTOF mass-spectrometry: two chaperones (DnaK and GroEL) and three enzymes of carbohydrate and amino acid metabolism (FbaB, fructose-1,6-bisphosphate aldolase, class I; GadA, L- glutamate decarboxylase; and KduI, 5-keto-4-deoxyuronate isomerase). These three proteins were cloned, expressed and purified in 6His-tagged and/or tag-free forms. Their binary interactions with PPase were verified by independent approaches. Initial characterization of the complexes indicates that PPase may stabilize its protein partners against unfolding or degradation. Comparative analysis of the PPase protein partners allowed an insight into its possible involvement in the cell metabolic regulation. Ó 2011 Elsevier Masson SAS. All rights reserved. 1. Introduction From the systems biology point of view, any cell is a complex adaptive system where proteins and their intermolecular interac- tions are the key components. Understanding of its work as a whole requires complete mapping and characterization of physical and functional relations between individual players. This gargantuan task is additionally complicated by a dynamic character of a cell composition and interactions which are subject to constant varia- tion in response to outer signals or developmental stage. Although Escherichia coli is often regarded as a model system for understanding the workings of living cells, its interactome is far from complete characterization. Large-scale mapping of physically interacting proteins in E. coli were performed in two studies. But- land and coworkers [1] purified stable protein complexes on a global scale using the tandem affinity purification (TAP) or sequential peptide affinity (SPA) tags. Tagged bait proteins were expressed, purified, and protein components of the isolated complexes were identified using mass spectrometry. More than 5000 binary inter- actions were found, of which 716 were validated by reciprocal experiments. Another large-scale pull-down assay was performed by Arifuzzaman and coworkers [2] using a His-tagged E. coli ORF clone library. Interactions were found for more than 2500 bait proteins. For proper understanding of the cell machinery and its regulation, physical interactions of every player in the network have to be apprehended on the functional level. The present work is focused on the protein complexes of inor- ganic pyrophosphatase (PPase). It is a cytosolic enzyme that cata- lyzes hydrolytic cleavage of phosphoanhydride bond in pyrophosphate, MgP 2 O 7 2 [3]. PPase activity is essential for the cell viability since it supports a lot of endergonic metabolic processes where diphosphate is produced, including biosynthesis of proteins and nucleic acids [4]. This renders PPase a possible component of various multiprotein complexes. E. coli PPase is well characterized structurally and kinetically, but it is poorly understood in what way it may be involved in the regulatory networks. Neither is known if PPase has other functions in vitro aside from it being a scavenger Abbreviations: PPase, inorganic pyrophosphatase; FbaB, fructose-1,6-bisphosphate aldolase, class I; GadA, L-glutamate decarboxylase, a isoform; KduI, 5-keto-4- deoxyuronate isomerase. * Corresponding author. Tel.: þ7 495 939 5362; fax: þ7 495 939 3181. E-mail address: rodina@belozersky.msu.ru (E. Rodina). Contents lists available at ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi 0300-9084/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2011.05.023 Biochimie 93 (2011) 1576e1583