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 translates the genetic information into proteins, 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 disuccinimidyl diacetic urea (DSAU). We analyzed 143 E. coli ribosomal structures, mapping a total of 10,771 intramolecular distances for 126 cross-link-pairs 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 cross-links, 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.

Molecular architecture of the human tRNA ligase complex

“…A notable reduction of cross-links between the C-terminal regions of DDX1 and FAM98B was observed in the five-subunit full complex ( Figure 2A ) as compared to the four-subunit core complex ( Figure 2—figure supplement 1B ), suggesting that ASW is positioned between DDX1 and FAM98B or modulates their conformations. It is important to note, however, that formation of cross-links between protein regions depends not only on their spatial proximity but also on their conformational flexibility and the availability of lysine residues within them ( Tüting et al, 2020 ).…”

Puf6 primes 60S pre-ribosome nuclear export at low temperature

“…5c, d ). We performed crosslinking-mass spectrometry (XL-MS) analyses of a reconstituted 60S:Puf6 complex using disuccinimidyl suberate (DSS) that can covalently link lysine residues that are up to 37 Å apart 39 , 40 . These analyses revealed protein:protein cross-links between Puf6 and the r-protein uL2 (Fig.…”

Leveraging crosslinking mass spectrometry in structural and cell biology

“…In these cases, crosslink satisfaction is assessed on the basis of an upper distance limit, which is often set to the crosslinker spacer length plus the maximum length of the appropriate side-chain rotamers and a small additional tolerance to account for molecular motions. As an example, the maximum distance for DSS and BS 3 , which have an 11.4 A ˚spacer, has often been set as high as 30 A ˚Ca-Ca (Lee et al, 2020;T€ uting et al, 2020;Yan et al, 2019). Testing this upper-bound assumption for DSS by examining lysine-lysine distances in molecular dynamics simulations and comparing them with experimentally observed crosslinks led to a good agreement based on average protein motions, supporting the incorporation of tolerances (Merkley et al, 2014).…”

“…In addition to their use in selecting between alternative models generated by other structural approaches, such scoring methods may also be used to provide experimental validation or to select between different structural models (Bullock et al, 2016;T€ uting et al, 2020). In multistate systems, quantitative crosslinking-MS may be used to disentangle the contribution of individual states, especially in response to perturbation, as reviewed in Chen and Rappsilber (2018).…”