Daniel Boehringer

The ribosome as a platform for co-translational processing, folding and targeting of newly synthesized proteins

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.

Structural probing of a protein phosphatase 2A network by chemical cross-linking and mass spectrometry.

Dynamic Assembly Structural characterization of protein complexes has yielded significant insight into biological function; however, most structural techniques require stable, homogenous samples. This presents a challenge in characterizing transient signaling complexes. Herzog et al. (p. 1348) used chemical cross-linking and mass spectroscopy (XL-MS) to characterize the modular and dynamic interaction network involving phosphatase 2A (PP2A), which interacts with tens of regulatory and adaptor proteins in diverse signaling pathways. They found 176 interprotein and 569 intraprotein distance restraints that delineated the topology of the network. The study establishes the importance of XL-MS in the suite of structural methods used to characterize dynamic assemblies. Spatial restraints revealed by chemical cross-linking and mass spectrometry elucidate the topology of a dynamic signaling network. 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.

GraFix: sample preparation for single-particle electron cryomicroscopy.

We developed a method, named GraFix, that considerably improves sample quality for structure determination by single-particle electron cryomicroscopy (cryo-EM). GraFix uses a glycerol gradient centrifugation step in which the complexes are centrifuged into an increasing concentration of a chemical fixation reagent to prevent aggregation and to stabilize individual macromolecules. The method can be used to prepare samples for negative-stain, cryo-negative-stain and, particularly, unstained cryo-EM.

Protein Composition and Electron Microscopy Structure of Affinity-Purified Human Spliceosomal B Complexes Isolated under Physiological Conditions

ABSTRACT The spliceosomal B complex is the substrate that undergoes catalytic activation leading to catalysis of pre-mRNA splicing. Previous characterization of this complex was performed in the presence of heparin, which dissociates less stably associated components. To obtain a more comprehensive inventory of the B complex proteome, we isolated this complex under low-stringency conditions using two independent methods. MS2 affinity-selected B complexes supported splicing when incubated in nuclear extract depleted of snRNPs. Mass spectrometry identified over 110 proteins in both independently purified B complex preparations, including ∼50 non-snRNP proteins not previously found in the spliceosomal A complex. Unexpectedly, the heteromeric hPrp19/CDC5 complex and 10 additional hPrp19/CDC5-related proteins were detected, indicating that they are recruited prior to spliceosome activation. Electron microscopy studies revealed that MS2 affinity-selected B complexes exhibit a rhombic shape with a maximum dimension of 420 Å and are structurally more homogeneous than B complexes treated with heparin. These data provide novel insights into the composition and structure of the spliceosome just prior to its catalytic activation and suggest a potential role in activation for proteins recruited at this stage. Furthermore, the spliceosomal complexes isolated here are well suited for complementation studies with purified proteins to dissect factor requirements for spliceosome activation and splicing catalysis.

The complete structure of the 55S mammalian mitochondrial ribosome

Resolving whole mitoribosomes Mitochondria probably evolved from a prokaryotic cell living within a proto-eukaryotic cell. Consequently, mitochondria have lost much of their genomic DNA, except for a few genes that require highly divergent mitoribosomes for protein translation. Greber et al. and Amunts et al. have used cryo–electron microscopy to uncover the structure of this complex (see the Perspective by Beckmann and Hermann) and reveal an unusual mRNA binding channel. The structure supplies clues for how aminoglycoside antibiotics might inhibit mitoribosomes and how mutations in mitoribosomes might cause human disease. Science, this issue p. 303, p. 288; see also A. Amunts et al., Science, 3 April, p. 95 The protein-synthesizing machinery of mammalian mitochondria differs substantially from bacterial and eukaryotic ribosomes. [Also see Perspective by Beckmann and Hermann] 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.

Architecture of the large subunit of the mammalian mitochondrial ribosome

Mitochondrial ribosomes synthesize a number of highly hydrophobic proteins encoded on the genome of mitochondria, the organelles in eukaryotic cells that are responsible for energy conversion by oxidative phosphorylation. The ribosomes in mammalian mitochondria have undergone massive structural changes throughout their evolution, including ribosomal RNA shortening and acquisition of mitochondria-specific ribosomal proteins. Here we present the three-dimensional structure of the 39S large subunit of the porcine mitochondrial ribosome determined by cryo-electron microscopy at 4.9 Å resolution. The structure, combined with data from chemical crosslinking and mass spectrometry experiments, reveals the unique features of the 39S subunit at near-atomic resolution and provides detailed insight into the architecture of the polypeptide exit site. This region of the mitochondrial ribosome has been considerably remodelled compared to its bacterial counterpart, providing a specialized platform for the synthesis and membrane insertion of the highly hydrophobic protein components of the respiratory chain.

Molecular mechanism and structure of Trigger Factor bound to the translating ribosome

Ribosome‐associated chaperone Trigger Factor (TF) initiates folding of newly synthesized proteins in bacteria. Here, we pinpoint by site‐specific crosslinking the sequence of molecular interactions of Escherichia coli TF and nascent chains during translation. Furthermore, we provide the first full‐length structure of TF associated with ribosome–nascent chain complexes by using cryo‐electron microscopy. In its active state, TF arches over the ribosomal exit tunnel accepting nascent chains in a protective void. The growing nascent chain initially follows a predefined path through the entire interior of TF in an unfolded conformation, and even after folding into a domain it remains accommodated inside the protective cavity of ribosome‐bound TF. The adaptability to accept nascent chains of different length and folding states may explain how TF is able to assist co‐translational folding of all kinds of nascent polypeptides during ongoing synthesis. Moreover, we suggest a model of how TFs chaperoning function can be coordinated with the co‐translational processing and membrane targeting of nascent polypeptides by other ribosome‐associated factors.

Architecture of Human Mtor Complex 1

From sensing leucine to metabolic control The mTORC1 protein kinase complex plays central roles in regulating cell growth and metabolism and is implicated in common human diseases such as diabetes and cancer. The level of the amino acid leucine tells an organism a lot about its physiological state, including how much food is available, how much insulin is going to be needed, and whether new muscle mass can be made (see the Perspective by Buel and Blenis). Wolfson et al. identified a biochemical sensor of leucine, Sestrin2, which connects the concentration of leucine to the control of organismal metabolism and growth. When leucine bound to Sestrin2, it was released from a complex with the mTORC1 regulatory factor GATOR2, activating the mTORC1 complex. Saxton et al. describe the crystal structure of Sestrin2 and show how it specifically detects leucine. Aylett et al. determined the structure of human mTORC1 by cryoelectron microscopy and the crystal structure of a regulatory subunit, Raptor. The results reveal the structural basis for the function and intricate regulation of this important enzyme, which is also a strategic drug target. Science, this issue p. 43, p. 48, p. 53; see also p. 25 Electron microscopy reveals the structure of a key regulator of growth and metabolism. [Also see Perspective by Buel and Blenis] Target of rapamycin (TOR), a conserved protein kinase and central controller of cell growth, functions in two structurally and functionally distinct complexes: TORC1 and TORC2. Dysregulation of mammalian TOR (mTOR) signaling is implicated in pathologies that include diabetes, cancer, and neurodegeneration. We resolved the architecture of human mTORC1 (mTOR with subunits Raptor and mLST8) bound to FK506 binding protein (FKBP)–rapamycin, by combining cryo–electron microscopy at 5.9 angstrom resolution with crystallographic studies of Chaetomium thermophilum Raptor at 4.3 angstrom resolution. The structure explains how FKBP-rapamycin and architectural elements of mTORC1 limit access to the recessed active site. Consistent with a role in substrate recognition and delivery, the conserved amino-terminal domain of Raptor is juxtaposed to the kinase active site.

Molecular Architecture of the 40S⋅eIF1⋅eIF3 Translation Initiation Complex

Summary Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core. These structures, together with electron microscopy reconstructions, cross-linking coupled to mass spectrometry, and integrative structure modeling, allowed us to position and orient all eIF3 components on the 40S⋅eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits. Yeast eIF3 engages 40S in a clamp-like manner, fully encircling 40S to position key initiation factors on opposite ends of the mRNA channel, providing a platform for the recruitment, assembly, and regulation of the translation initiation machinery. The structures of eIF3 components reported here also have implications for understanding the architecture of the mammalian 43S preinitiation complex and the complex of eIF3, 40S, and the hepatitis C internal ribosomal entry site RNA.

YidC and Oxa1 Form Dimeric Insertion Pores on the Translating Ribosome

The YidC/Oxa1/Alb3 family of membrane proteins facilitates the insertion and assembly of membrane proteins in bacteria, mitochondria, and chloroplasts. Here we present the structures of both Escherichia coli YidC and Saccharomyces cerevisiae Oxa1 bound to E. coli ribosome nascent chain complexes determined by cryo-electron microscopy. Dimers of YidC and Oxa1 are localized above the exit of the ribosomal tunnel. Crosslinking experiments show that the ribosome specifically stabilizes the dimeric state. Functionally important and conserved transmembrane helices of YidC and Oxa1 were localized at the dimer interface by cysteine crosslinking. Both Oxa1 and YidC dimers contact the ribosome at ribosomal protein L23 and conserved rRNA helices 59 and 24, similarly to what was observed for the nonhomologous SecYEG translocon. We suggest that dimers of the YidC and Oxa1 proteins form insertion pores and share a common overall architecture with the SecY monomer.