Identification and Mapping of Self-Assembling Protein Domains Encoded by theEscherichia coliK-12 Genome by Use of λ Repressor Fusions

Abstract

Self-assembling proteins and protein fragments encoded by the Escherichia coli genome were identified from E. coli K-12 strain MG1655. Libraries of random DNA fragments cloned into a series of repressor fusion vectors were subjected to selection for immunity to infection by phage . Survivors were identified by sequencing the ends of the inserts, and the fused protein sequence was inferred from the known genomic sequence. Four hundred sixty-three nonredundant open reading frame-encoded interacting sequence tags (ISTs) were recovered from sequencing 2,089 candidates. These ISTs, which range from 16 to 794 amino acids in length, were clustered into families of overlapping fragments, identifying potential homotypic interactions encoded by 232 E. coli genes. Repressor fusions identified ISTs from genes in every protein-based functional category, but membrane proteins were underrepresented. The IST-containing genes were enriched for regulatory proteins and for proteins that form higher-order oligomers. Forty-eight (20.7%) homotypic proteins identified by ISTs are predicted to contain coiled coils. Although most of the IST-containing genes are identifiably related to proteins in other bacterial genomes, more than half of the ISTs do not have identifiable homologs in the Protein Data Bank, suggesting that they may include many novel structures. The data are available online at http://oligomers.tamu.edu/.For many proteins, quaternary structure is intimately coupled to function and stability. This coupling allows the regulation of many cellular processes to be controlled through specific assembly or disassembly of protein complexes as well as by conformational changes that alter how subunits contact one another.Proteins use a wide variety of quaternary structures to assemble multisubunit complexes. Genome-wide identification of protein interactions by use of genetic (21,36,46,57,58) or biochemical (15,19,41) screens has provided a wealth of insight into the diversity of structures used for self-assembly. In the annotation of predicted open reading frames (ORFs), assembly interactions are an important feature that provides insights into structure and function. In addition, the involvement of a gene product in a multimeric complex suggests strategies for the generation of assembly-based inhibitors for functional studies (18). The possibility that protein interactions represent a large and largely underexploited target for drug discovery has also been discussed (6,55) The study of the protein interactome has focused on heterotypic interactions, as these can provide links between proteins of unknown function and proteins of known function. However, homotypic interactions, which are found in both homomultimeric proteins and as subcomplexes of heteromultimeric proteins, may be the most common way to form protein complexes in nature (31). Although by definition self-interaction does not link a protein's function to that of another protein, homotypic interactions are important in the study of protein structure, function, and evol...

Novel Structure of the Conserved Gram-Negative Lipopolysaccharide Transport Protein A and Mutagenesis Analysis

“…LptA sequence alignments also revealed that residues 28-80, which delineate the N-terminal cleft in LptA, also share a sequence similarity with Imp, suggesting a structural connection between these segments of the two proteins, both of which are associated with LPS transport. 12 LptA was thought to possess an N-terminal signal sequence (M1-A27) 21,22 that is removed upon secretion to the periplasm. 14,23 To examine this processing, we performed mass spectrometry analysis of purified LptA-His 6 that suggested a molecular mass of 18,603 Da, corresponding to a processed recombinant LptA-His 6 protein in which the 27 N-terminal residues (M1-A27) were removed (predicted molecular mass of 18,645 Da) 24 prior to crystallization (data not shown).…”

Recombinational branch migration by the RadA/Sms paralog of RecA in Escherichia coli

“…Threading of RadA onto the RecA presynaptic crystal structure (our unpublished results) suggests that RadA possesses subunit interfaces similar to that of RecA that assemble the ATPase site. In a bacterial one-hybrid assay, RadA was shown to interact with itself ( Marino-Ramirez et al, 2004 ), indicating that it forms a multimeric complex. Our early experiments with His 6 -tagged RadA protein, a less active protein than the more native protein characterized here, exhibited multiple bound species in gel-shift experiments with poly(dT)(data not shown), consistent with oligomer formation.…”

AbmR (Rv1265) is a novel transcription factor of Mycobacterium tuberculosis that regulates host cell association and expression of the non‐coding small RNA Mcr11

“…Database of oligomerization domains from lambda experiments (Doodle), maintained by the Hu Lab at Texas A&M (Marino‐Ramirez et al ., ), was accessed on 09/24/14 and was searched for hits in the abmR ORF. The database indicated AbmR might oligomerize, so the ability of purified recombinant AbmR to self‐associate was tested in vitro .…”