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The identification of proximate amino acids by chemical cross-linking and mass spectrometry (XL-MS) facilitates the structural analysis of homogeneous protein complexes. We gained distance restraints on a modular interaction network of protein complexes affinity-purified from human cells by applying an adapted XL-MS protocol. Systematic analysis of human protein phosphatase 2A (PP2A) complexes identified 176 interprotein and 570 intraprotein cross-links that link specific trimeric PP2A complexes to a multitude of adaptor proteins that control their cellular functions. Spatial restraints guided molecular modeling of the binding interface between immunoglobulin binding protein 1 (IGBP1) and PP2A and revealed the topology of TCP1 ring complex (TRiC) chaperonin interacting with the PP2A regulatory subunit 2ABG. This study establishes XL-MS as an integral part of hybrid structural biology approaches for the analysis of endogenous protein complexes.

Mammalian mitochondrial ribosomes (mitoribosomes) synthesize mitochondrially encoded membrane proteins that are critical for mitochondrial function. Here we present the complete atomic structure of the porcine 55S mitoribosome at 3.8 angstrom resolution by cryo-electron microscopy and chemical cross-linking/mass spectrometry. The structure of the 28S subunit in the complex was resolved at 3.6 angstrom resolution by focused alignment, which allowed building of a detailed atomic structure including all of its 15 mitoribosomal-specific proteins. The structure reveals the intersubunit contacts in the 55S mitoribosome, the molecular architecture of the mitoribosomal messenger RNA (mRNA) binding channel and its interaction with transfer RNAs, and provides insight into the highly specialized mechanism of mRNA recruitment to the 28S subunit. Furthermore, the structure contributes to a mechanistic understanding of aminoglycoside ototoxicity.

The early events in the life of newly synthesized proteins in the cellular environment are remarkably complex. Concurrently with their synthesis by the ribosome, nascent polypeptides are subjected to enzymatic processing, chaperone-assisted folding or targeting to translocation pores at membranes. The ribosome itself has a key role in these different tasks and governs the interplay between the various factors involved. Indeed, the ribosome serves as a platform for the spatially and temporally regulated association of enzymes, targeting factors and chaperones that act upon the nascent polypeptides emerging from the exit tunnel. Furthermore, the ribosome provides opportunities to coordinate the protein-synthesis activity of its peptidyl transferase center with the protein targeting and folding processes. Here we review the early co-translational events involving the ribosome that guide cytosolic proteins to their native state.