Marc Leibundgut

Crystal Structure of the Eukaryotic 60S Ribosomal Subunit in Complex with Initiation Factor 6

The 3.5 angstrom–resolution structure provides insights into the architecture of the eukaryotic ribosome and its regulation. Protein synthesis in all organisms is catalyzed by ribosomes. In comparison to their prokaryotic counterparts, eukaryotic ribosomes are considerably larger and are subject to more complex regulation. The large ribosomal subunit (60S) catalyzes peptide bond formation and contains the nascent polypeptide exit tunnel. We present the structure of the 60S ribosomal subunit from Tetrahymena thermophila in complex with eukaryotic initiation factor 6 (eIF6), cocrystallized with the antibiotic cycloheximide (a eukaryotic-specific inhibitor of protein synthesis), at a resolution of 3.5 angstroms. The structure illustrates the complex functional architecture of the eukaryotic 60S subunit, which comprises an intricate network of interactions between eukaryotic-specific ribosomal protein features and RNA expansion segments. It reveals the roles of eukaryotic ribosomal protein elements in the stabilization of the active site and the extent of eukaryotic-specific differences in other functional regions of the subunit. Furthermore, it elucidates the molecular basis of the interaction with eIF6 and provides a structural framework for further studies of ribosome-associated diseases and the role of the 60S subunit in the initiation of protein synthesis.

The Crystal Structure of a Mammalian Fatty Acid Synthase

Mammalian fatty acid synthase is a large multienzyme that catalyzes all steps of fatty acid synthesis. We have determined its crystal structure at 3.2 angstrom resolution covering five catalytic domains, whereas the flexibly tethered terminal acyl carrier protein and thioesterase domains remain unresolved. The structure reveals a complex architecture of alternating linkers and enzymatic domains. Substrate shuttling is facilitated by flexible tethering of the acyl carrier protein domain and by the limited contact between the condensing and modifying portions of the multienzyme, which are mainly connected by linkers rather than direct interaction. The structure identifies two additional nonenzymatic domains: (i) a pseudo-ketoreductase and (ii) a peripheral pseudo-methyltransferase that is probably a remnant of an ancestral methyltransferase domain maintained in some related polyketide synthases. The structural comparison of mammalian fatty acid synthase with modular polyketide synthases shows how their segmental construction allows the variation of domain composition to achieve diverse product synthesis.

Structure of the Eukaryotic Thiamine Pyrophosphate Riboswitch with Its Regulatory Ligand

Riboswitches are untranslated regions of messenger RNA, which adopt alternate structures depending on the binding of specific metabolites. Such conformational switching regulates the expression of proteins involved in the biosynthesis of riboswitch substrates. Here, we present the 2.9 angstrom–resolution crystal structure of the eukaryotic Arabidopsis thaliana thiamine pyrophosphate (TPP)–specific riboswitch in complex with its natural ligand. The riboswitch specifically recognizes the TPP via conserved residues located within two highly distorted parallel “sensor” helices. The structure provides the basis for understanding the reorganization of the riboswitch fold upon TPP binding and explains the mechanism of resistance to the antibiotic pyrithiamine.

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.

Architecture of a Fungal Fatty Acid Synthase at 5 Å Resolution

All steps of fatty acid synthesis in fungi are catalyzed by the fatty acid synthase, which forms a 2.6-megadalton α6β6 complex. We have determined the molecular architecture of this multienzyme by fitting the structures of homologous enzymes that catalyze the individual steps of the reaction pathway into a 5 angstrom x-ray crystallographic electron density map. The huge assembly contains two separated reaction chambers, each equipped with three sets of active sites separated by distances up to ∼130 angstroms, across which acyl carrier protein shuttles substrates during the reaction cycle. Regions of the electron density arising from well-defined structural features outside the catalytic domains separate the two reaction chambers and serve as a matrix in which domains carrying the various active sites are embedded. The structure rationalizes the compartmentalization of fatty acid synthesis, and the spatial arrangement of the active sites has specific implications for our understanding of the reaction cycle mechanism and of the architecture of multienzymes in general.

The multienzyme architecture of eukaryotic fatty acid synthases.

Eukaryotic fatty acid synthases (FASs) are huge multifunctional enzymes that carry out all enzymatic steps essential for fatty acid biosynthesis. Recent crystallographic studies provide new insights into the architecture of the two distinct eukaryotic FAS systems, the 2.6 MDa heterododecameric fungal and the 540 kDa dimeric animal FAS. In this review, we compare the fundamentally different organization of these two megasynthases and discuss the structural principles of enzyme integration and substrate shuttling in FAS multienzymes.

Atomic structures of the eukaryotic ribosome

Eukaryotic ribosomes are significantly larger and more complex than their prokaryotic counterparts. This parallels the increased complexity of the associated cellular machinery responsible for translation initiation, ribosome assembly, and the regulation of protein synthesis in eukaryotic cells. The recently determined crystal structures of the small (40S) and large (60S) ribosomal subunits and the 80S ribosome now provide an atomic description of this essential molecular machine and reveal its eukaryote-specific features. In this review, we discuss the common structural principles underlying the evolution of both ribosomal subunits. The recently obtained structural information provides a framework for further genetic, biochemical and structural studies of eukaryotic ribosomes. At the same time, it facilitates a direct comparison between prokaryotic and eukaryotic ribosomal features.

Selenocysteine tRNA-specific elongation factor SelB is a structural chimaera of elongation and initiation factors.

In all three kingdoms of life, SelB is a specialized translation elongation factor responsible for the cotranslational incorporation of selenocysteine into proteins by recoding of a UGA stop codon in the presence of a downstream mRNA hairpin loop. Here, we present the X‐ray structures of SelB from the archaeon Methanococcus maripaludis in the apo‐, GDP‐ and GppNHp‐bound form and use mutational analysis to investigate the role of individual amino acids in its aminoacyl‐binding pocket. All three SelB structures reveal an EF‐Tu:GTP‐like domain arrangement. Upon binding of the GTP analogue GppNHp, a conformational change of the Switch 2 region in the GTPase domain leads to the exposure of SelB residues involved in clamping the 5′ phosphate of the tRNA. A conserved extended loop in domain III of SelB may be responsible for specific interactions with tRNASec and act as a ruler for measuring the extra long acceptor arm. Domain IV of SelB adopts a β barrel fold and is flexibly tethered to domain III. The overall domain arrangement of SelB resembles a ‘chalice’ observed so far only for initiation factor IF2/eIF5B. In our model of SelB bound to the ribosome, domain IV points towards the 3′ mRNA entrance cleft ready to interact with the downstream secondary structure element.

Structure and function of eukaryotic fatty acid synthases

In all organisms, fatty acid synthesis is achieved in variations of a common cyclic reaction pathway by stepwise, iterative elongation of precursors with two-carbon extender units. In bacteria, all individual reaction steps are carried out by monofunctional dissociated enzymes, whereas in eukaryotes the fatty acid synthases (FASs) have evolved into large multifunctional enzymes that integrate the whole process of fatty acid synthesis. During the last few years, important advances in understanding the structural and functional organization of eukaryotic FASs have been made through a combination of biochemical, electron microscopic and X-ray crystallographic approaches. They have revealed the strikingly different architectures of the two distinct types of eukaryotic FASs, the fungal and the animal enzyme system. Fungal FAS is a 2·6 MDa α₆β₆ heterododecamer with a barrel shape enclosing two large chambers, each containing three sets of active sites separated by a central wheel-like structure. It represents a highly specialized micro-compartment strictly optimized for the production of saturated fatty acids. In contrast, the animal FAS is a 540 kDa X-shaped homodimer with two lateral reaction clefts characterized by a modular domain architecture and large extent of conformational flexibility that appears to contribute to catalytic efficiency.