Interpretation of anomalously long crosslinks in ribosome crosslinking reveals the ribosome interaction in stationary phase E. coli

Abstract

Crosslinking mass spectrometry (XL-MS) of bacterial ribosomes revealed the dynamic intra- and intermolecular interactions within the ribosome structure. It has been also extended to capture the interactions of ribosome binding proteins during translation. Generally, XL-MS often identified the crosslinks within a cross-linkable distance (<40 Å) using amine-reactive crosslinkers. The crosslinks larger than cross-linkable distance (>40 Å) are always difficult to interpret and remain unnoticed. Here, we focused on stationary phase bacterial ribosome crosslinking that yields ultra-long crosslinks in an E. coli cell lysate. We explain these ultra-long crosslinks with the combination of sucrose density gradient centrifugation, chemical crosslinking, high-resolution mass spectrometry, and electron microscopy analysis. Multiple ultra-long crosslinks were observed in E. coli ribosomes for example ribosomal protein L19 (K63, K94) crosslinks with L21 (K71, K81) at two locations that are about 100 Å apart. Structural mapping of such ultra-long crosslinks in 70S ribosomes suggested that these crosslinks are not potentially formed within one 70S particle and could be a result of dimer and trimer formation as evidenced by negative staining electron microscopy. Ribosome dimerization captured by chemical crosslinking reaction could be an indication of ribosome-ribosome interactions in the stationary phase.

Fig. 1. Ribosome XL-MS workflow. The stationary phase E. coli ribosomes are crosslinked with DEST. Crosslinked proteins are digested with trypsin followed by crosslinked peptide enrichment using SCX chromatography and high-resolution mass spectrometry data acquisition with four different methods of precursor fragmentation.

Fig. 2. (a) Sucrose density gradient fractionation of non-crosslinked (control) and the crosslinked ribosome. Both crosslinked and non-crosslinked ribosomes were purified with a 10–50% sucrose gradient. Dimer and trimer peaks were the result of crosslinking. (b) 70S monomeric ribosome particles, dimer, and trimer peaks were analyzed using negative staining electron microscopy.

Fig. 3. Sucrose density gradient of crosslinked and non-crosslinked (control) ribosomes with high (10.5 mM, orange curve) and low magnesium ions (1 mM). Crosslinked ribosomes (XL) do not completely dissociate in a low magnesium sucrose gradient (blue curve). In the control sample, 70S ribosomes dissociate into 30S and 50S subunit particles in low magnesium as shown by the black curve.

Fig. 4. (a) Ultra-long crosslink between ribosomal proteins L19 and L21. (b) The crosslinked peptide is confidently identified by four different fragmentation methods. CID and HCD spectra give a series of b and y ions. The EThcD spectrum confirms the partially fragmented crosslinked peptide mass pairs and additional confirmation by the presence of immonium ions in the hcd spectrum.

Fig. 5. The ultra-long crosslinks (>40 Å) are mapped in the 70S ribosome crystal structure (PDB 4YBB) using the PyXlinkViewer plugin in PyMOL v2.4.1 – Schrodinger, LLC.

Fig. 6. Proposed ribosome dimer interaction models (1, 2, and 3) constructed using the PDB 4YBB crystal structure in PyMOL v2.4.1 – Schrodinger, LLC. The locations of ribosomal proteins/residues and the crosslink distance are approximate based on the crystal structure.