Structural Analysis of 70S Ribosomes by Cross-Linking/Mass Spectrometry Reveals Conformational Plasticity

Abstract

The ribosome is not only a highly complex molecular machine that executes translation according to the central dogma of molecular biology, but also an exceptional specimen for testing and optimizing cross-linking/mass spectrometry (XL-MS) workflows. Due to its high abundance, ribosomal proteins are frequently identified in proteome-wide XL-MS studies of cells or cell extracts. Here, we performed in-depth cross-linking of the E. coli ribosome using the amine-reactive cross-linker diacetyl dibutyric urea (DSAU). We analyzed 143 E. coli ribosomal structures, mapping a total of 10,771 intramolecular distances for 126 cross-linkpairs and 3,405 intermolecular distances for 97 protein pairs. Remarkably, 44% of intermolecular cross-links covered regions that have not been resolved in any high-resolution E. coli ribosome structure and point to a plasticity of cross-linked regions. We systematically characterized all cross-links and discovered flexible regions, conformational changes, and stoichiometric variations in bound ribosomal proteins, and ultimately remodeled 2,057 residues (15,794 atoms) in total. Our working model explains more than 95% of all crosslinks, resulting in an optimized E. coli ribosome structure based on the cross-linking data obtained. Our study might serve as benchmark for conducting biochemical experiments on newly modeled protein regions, guided by XL-MS. Data are available via ProteomeXchange with identifier PXD018935.

Cross-linking/Mass Spectrometry Combined with Ion Mobility on a timsTOF Pro Instrument for Structural Proteomics

“…The average Cα-Cα distances bridged by the three cross-linkers range between 18.9 Å for DSAU, 20.9 Å for DSPU, and 21.2 Å for DSBU (SI Figure S2), which is in perfect agreement with the different spacer lengths of the three cross-linkers (Scheme 1). For all three crosslinkers, the maximum Cα-Cα distance bridged is ~ 35 (40) Å, which harmonizes with our previous studies 3,6,21 . All unique cross-linking sites of BSA are summarized in SI Tables.…”

“…In total, 868 cross-linking sites were identified for the E. coli ribosome (Table 2, Figure 4A). The results with DSBU agree well with the data acquired recently in our group for the E. coli ribosome using the shorter DSAU linker 21 : 55% of unique cross-linking sites were found in both studies. The four replicates show an overall overlap of 34% with 295 unique cross-linking sites being identified in all four experiments (Figure 4B).…”

“…DSBU has been successfully used in the past for conformational studies of proteins as well as for mapping system-wide protein interaction networks 3, 6, 14, 20 . Here, we employ the three cross-linkers DSBU, DSPU (disuccinimidyl dipropionic urea), and DSAU (disuccinimidyl diacetic urea) 21 . All three cross-linkers are amine-reactive N -hydroxysuccinimide (NHS) esters and contain a central, MS-labile urea group with C4 to C2 spacers at each side of the urea moiety (Scheme 1).…”