title: A proteomic approach to study Escherichia coli. Acetyl esterase interactors unveil a sequence motif involved in protein-protein interaction.
abstract: E. coli acetyl esterase (Aes) and beta-cysthationase (MalY) interact, probably with the same mechanism, with the N-terminus of transcriptional activator of maltose regulon (MalT). In order to investigate the basic mechanism of this interaction, we used both a proteomic and a bioinformatic approach. Affinity-based mass spectrometry experiments with purified Aes protein as bait allowed to fish twenty-three, apparently specific, interactors from crude extracts of E. coli cells grown in different conditions. The group of interactors appeared quite heterogeneous, comprising Aes itself, some molecular chaperons, metabolic enzymes, and several proteins of unknown function. Among the identified proteins, two are in some way related to the maltose metabolism and two are related to the lipopolysaccharide metabolism. By superposing the structures of the Alicyclobacillus acidocaldarius EST2, an Aes homolog, and MalY, a region of structural similarity was discovered that allowed detecting a short stretch of nine residues with sequence similarity among EST2, AES and MalY. Degenerated sequence consensuses derived from the alignment were used to analyse the E. coli proteome in the Swiss Prot database and permitted to retrieve sequences of Aes interactors already known or detected in this study. Most of these interactors (14 out of 25) contain the expected consensus. A site-directed mutant of Aes R179A made in the consensus sequence resulted in complete loss of interaction. Based on the analysis of the available three-dimensional structures and mutagenic and structural data inferred from literature, we predict a role of this motif in protein-protein interaction.
authors.affiliation: Institute of Protein Biochemistry, National Research Council, via P. Castellino 111, I-80131, Naples, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: D'Ambrosio
authors.forename: Luigi
authors.initials: L
authors.lastname: Mandrich
authors.forename: Mosè
authors.initials: M
authors.lastname: Rossi
authors.forename: Andrea
authors.initials: A
authors.lastname: Scaloni
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Manco
mesh_terms: D000115:Acetylesterase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020816:Amino Acid Motifs; D019295:Computational Biology; D003542:Cystathionine gamma-Lyase / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism; D008320:Maltose / Q000378:metabolism; D013058:Mass Spectrometry; D053858:Metabolic Networks and Pathways; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D040901:Proteomics; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D012097:Repressor Proteins; C079405:MalY protein, E coli; D008320:Maltose; D000115:Acetylesterase; D003542:Cystathionine gamma-Lyase