A coordinated proteomic approach for identifying proteins that interact with the E. coli ribosomal protein S12

Abstract

The bacterial ribosomal protein S12 contains a universally conserved D88 residue on a loop region thought to be critically involved in translation due to its proximal location to the A site of the 30S subunit. While D88 mutants are lethal this residue has been found to be post-translationally modified to β-methylthioaspartic acid, a post-translational modification (PTM) identified in S12 orthologs from several phylogenetically distinct bacteria. In a previous report focused on characterizing this PTM, our results provided evidence that this conserved loop region might be involved in forming multiple proteins-protein interactions ( Strader , M. B. ; Costantino , N. ; Elkins , C. A. ; Chen , C. Y. ; Patel , I. ; Makusky , A. J. ; Choy , J. S. ; Court , D. L. ; Markey , S. P. ; Kowalak , J. A. A proteomic and transcriptomic approach reveals new insight into betamethylthiolation of Escherichia coli ribosomal protein S12. Mol. Cell. Proteomics 2011 , 10 , M110 005199 ). To follow-up on this study, the D88 containing loop was probed to identify candidate binders employing a two-step complementary affinity purification strategy. The first involved an endogenously expressed S12 protein containing a C-terminal tag for capturing S12 binding partners. The second strategy utilized a synthetic biotinylated peptide representing the D88 conserved loop region for capturing S12 loop interaction partners. Captured proteins from both approaches were detected by utilizing SDS-PAGE and one-dimensional liquid chromatography-tandem mass spectrometry. The results presented in this report revealed proteins that form direct interactions with the 30S subunit and elucidated which are likely to interact with S12. In addition, we provide evidence that two proteins involved in regulating ribosome and/or mRNA transcript levels under stress conditions, RNase R and Hfq, form direct interactions with the S12 conserved loop, suggesting that it is likely part of a protein binding interface.

Figure 1

The region surrounding D88 in S12, as determined in the structure from Thermus thermophilus 30S subunit (47) (Protein Data Back accession no. 1FJG). This region consists of 2 highly conserved loops (Loopl and Loop2) that are directed toward a site on the 30S subunit involved in translation. β-methylthio-D88 (highlighted in red) is located on the second conserved Loop 2 (highlighted in yellow) that connects two anti-parallel β strands This figure was created using the PyMol Molecular Graphics System (DeLano Scientific, Palo Alto, California, US).

Figure 2

SPA tagged S12 (SPA-S12) pull-downs in whole cell lysates enriched for proteins representing a heterogeneous population of isolated 30S subunits, 70S ribosomes and/or translationally active polysomes of ribosomal small subunit associated complexes. Pull-downs performed in polysome depleted extract resulted in the enrichment of ribosomal small subunit associated complexes (A) Polypeptides from eluates representing SPA-S12 purified from whole cell lysates (lane 2) and negative control (lane 3) (B) Polypeptides from eluates representing SPA-S12 purified from polysome depleted extract (lane 2) and negative control (lane 3). Pull-downs were separated by SDS-PAGE (10% polyacrylamide gels) prior to in-gel tryptic digestion and ID LC tandem mass spectrometry. Molecular weight standards are represented in lane 1. The image representing (B) have been displayed in a previous report (18).

Figure 3

Schematic diagram representing peptide pull-down strategy. Synthetic S12 and decoy peptide pull-downs were incubated in PDE minimal media (¹⁴N or ¹⁵N). The combined eluates were subjected to PAGE/in gel digestions and then analyzed by 1D-LC/MS/MS. To reduce the variability associated with background protein binding, cross experiments were performed in which the S12 peptide was incubated in the lysate from ¹⁵N labeled cells in one pull-down (the scrambled peptide would be incubated in ¹⁴N labeled lysate) and ¹⁴N labeled lysate was in the next pull-down. After 1D-LC/MS/MS analysis, extracted ion chromatograms and peptide abundance ratios were determined for isotopologous peptide pairs. Heavy and light versions of the same peptide were distinguishable with equal efficiency; the ion current ratio therefore, reflected the ratio of the proteins in two populations.

Figure 4

Venn diagram representing total protein identifications from each SPA-S12 experimental condition. The whole cell lysate pull-downs resulted in capturing proteins associated with a heterogeneous population of isolated 30S subunits, 70S ribosomes and/or translationally active polysomes. Proteins unique to mid-log and stationary phase PDE pull-downs represented approximately 2 fold and 20 fold fewer identifications, respectively, than polysomal samples.