Robin E. Stanley

Formation of the First Peptide Bond: The Structure of EF-P Bound to the 70S Ribosome

Protein Synthesis Initiation Complex The final step in the initiation phase of protein synthesis is the formation of the first peptide bond, which requires initiator transfer RNA (tRNA) to be bound at the ribosomal P site. Elongation factor P (EF-P) is a protein conserved in all eubacteria that stimulates this initial bond formation. Insight into how this is achieved comes from a structure of Thermus thermophilus 70S ribosome bound to EF-P, initiator tRNA, and a short piece of messenger RNA presented by Blaha et al. (p. 966). EF-P binds between the P and E sites and facilitates proper positioning of initiator tRNA in the P site. A similar mechanism is likely to apply to structurally homologous initiation factors in archea and eukarya. Elongation factor P binds to the ribosome so as to position the initiator transfer RNA for the first bond formation. Elongation factor P (EF-P) is an essential protein that stimulates the formation of the first peptide bond in protein synthesis. Here we report the crystal structure of EF-P bound to the Thermus thermophilus 70S ribosome along with the initiator transfer RNA N-formyl-methionyl-tRNAi (fMet-tRNAifMet) and a short piece of messenger RNA (mRNA) at a resolution of 3.5 angstroms. EF-P binds to a site located between the binding site for the peptidyl tRNA (P site) and the exiting tRNA (E site). It spans both ribosomal subunits with its amino-terminal domain positioned adjacent to the aminoacyl acceptor stem and its carboxyl-terminal domain positioned next to the anticodon stem-loop of the P site–bound initiator tRNA. Domain II of EF-P interacts with the ribosomal protein L1, which results in the largest movement of the L1 stalk that has been observed in the absence of ratcheting of the ribosomal subunits. EF-P facilitates the proper positioning of the fMet-tRNAifMet for the formation of the first peptide bond during translation initiation.

The structures of the anti-tuberculosis antibiotics viomycin and capreomycin bound to the 70S ribosome

Viomycin and capreomycin belong to the tuberactinomycin family of antibiotics, which are among the most effective antibiotics against multidrug-resistant tuberculosis. Here we present two crystal structures of the 70S ribosome in complex with three tRNAs and bound to either viomycin or capreomycin at 3.3- and 3.5-Å resolution, respectively. Both antibiotics bind to the same site on the ribosome, which lies at the interface between helix 44 of the small ribosomal subunit and helix 69 of the large ribosomal subunit. The structures of these complexes suggest that the tuberactinomycins inhibit translocation by stabilizing the tRNA in the A site in the pretranslocation state. In addition, these structures show that the tuberactinomycins bind adjacent to the binding sites for the paromomycin and hygromycin B antibiotics, which may enable the development of new derivatives of tuberactinomycins that are effective against drug-resistant strains.

A HORMA domain in Atg13 mediates PI 3-kinase recruitment in autophagy

Autophagy-related 13 (Atg13) is a key early-acting factor in autophagy and the major locus for nutrient-dependent regulation of autophagy by Tor. The 2.3-Å resolution crystal structure of the N-terminal domain of Atg13 reveals a previously unidentified HORMA (Hop1p, Rev1p and Mad2) domain similar to that of the spindle checkpoint protein Mad2. Mad2 has two different stable conformations, O-Mad2 and C-Mad2, and the Atg13 HORMA structure corresponds to the C-Mad2 state. The Atg13 HORMA domain is required for autophagy and for recruitment of the phosphatidylinositol (PI) 3-kinase subunit Atg14 but is not required for Atg1 interaction or Atg13 recruitment to the preautophagosomal structure. The Atg13 HORMA structure reveals a pair of conserved Arg residues that constitute a putative phosphate sensor. One of the Arg residues is in the region corresponding to the “safety belt” conformational switch of Mad2, suggesting conformational regulation of phosphate binding. These two Arg residues are essential for autophagy, suggesting that the Atg13 HORMA domain could function as a phosphoregulated conformational switch.

Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen–deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity. DOI: http://dx.doi.org/10.7554/eLife.05115.001

The beginning of the end: how scaffolds nucleate autophagosome biogenesis

Autophagy is a conserved mechanism that is essential for cell survival in starvation. Moreover, autophagy maintains cellular health by clearing unneeded or harmful materials from cells. Autophagy proceeds by the engulfment of bulk cytosol and organelles by a cup-shaped double-membrane sheet known as the phagophore. The phagophore closes on itself to form the autophagosome, which delivers its contents to the vacuole or lysosome for degradation. A multiprotein complex comprising the protein kinase autophagy-related protein 1 (Atg1) together with Atg13, Atg17, Atg29, and Atg31 (ULK1, ATG13, FIP200, and ATG101 in humans) has a pivotal role in the earliest steps of this process. This review summarizes recent structural and ultrastructural analysis of the earliest step in autophagosome biogenesis and discusses a model in which the Atg1 complex clusters high-curvature vesicles containing the integral membrane protein Atg9, thereby initiating the phagophore.

Assembly and dynamics of the autophagy-initiating Atg1 complex

Significance Autophagy is conserved and essential for cellular survival during starvation and stress, and is initiated by the autophagy-related 1 (Atg1) complex. In yeast, the three subunits Atg17, Atg29, and Atg31 assemble first, and their structure is known. There has been a debate over how the Atg1 and Atg13 subunits assemble when autophagy is triggered. Here we use mass spectrometry to show that the C-terminal domain of Atg1 is highly dynamic on its own. Atg1 forms a stable complex when it binds with high affinity to Atg13. The combined Atg1–Atg13 subcomplex then binds with moderate affinity to the preformed Atg17–Atg31–Atg29 scaffold. This highlights the binding of Atg1–Atg13 to Atg17–Atg31–Atg29 as a pivotal step in autophagy initiation. The autophagy-related 1 (Atg1) complex of Saccharomyces cerevisiae has a central role in the initiation of autophagy following starvation and TORC1 inactivation. The complex consists of the protein kinase Atg1, the TORC1 substrate Atg13, and the trimeric Atg17–Atg31–Atg29 scaffolding subcomplex. Autophagy is triggered when Atg1 and Atg13 assemble with the trimeric scaffold. Here we show by hydrogen–deuterium exchange coupled to mass spectrometry that the mutually interacting Atg1 early autophagy targeting/tethering domain and the Atg13 central domain are highly dynamic in isolation but together form a stable complex with ∼100-nM affinity. The Atg1–Atg13 complex in turn binds as a unit to the Atg17–Atg31–Atg29 scaffold with ∼10-μM affinity via Atg13. The resulting complex consists primarily of a dimer of pentamers in solution. These results lead to a model for autophagy initiation in which Atg1 and Atg13 are tightly associated with one another and assemble transiently into the pentameric Atg1 complex during starvation.

What the N-terminal domain of Atg13 looks like and what it does: A HORMA fold required for PtdIns 3-kinase recruitment

Atg13 is a subunit of the Atg1 complex that is involved in autophagy. The middle and C-terminal regions of Atg13 are intrinsically disordered and rich in regulatory phosphorylation sites. Thus far, there have been no structural data for any part of Atg13, and no function assigned to its N-terminal domain. We crystallized this domain, and found that it has a HORMA (Hop1, Rev7, Mad2) fold. We showed that the Atg13 HORMA domain is required for autophagy and for recruitment of the phosphatidylinositol (PtdIns) 3-kinase subunit Atg14, but is not required for Atg1 interaction or Atg13 recruitment to the PAS. The HORMA domain of Atg13 is similar to the closed conformation of the spindle checkpoint protein Mad2. A pair of conserved arginines was identified in the structure, and tested functionally in yeast. These residues are important for autophagy, as mutations abrogate autophagy and block Atg14 recruitment. The location of these Arg residues in the structure suggests that the Atg13 HORMA domain could act as a phosphorylation-dependent conformational switch.

Grc3 programs the essential endoribonuclease Las1 for specific RNA cleavage

Significance Ribonucleases are molecular scissors that catalyze the cleavage of RNA phosphodiester bonds and play essential roles in RNA processing and maturation. Precursor ribosomal RNA (rRNA) must be processed by several ribonucleases, including the endonuclease Las1, in a carefully orchestrated manner to generate the mature ribosomal subunits. Las1 is essential for cell viability, and mutations in the mammalian gene have been linked with human disease, underscoring the importance of this enzyme. Here, we show that, on its own, Las1 has weak activity; however, when associated with its binding partner, the polynucleotide kinase Grc3, Las1 is programmed to efficiently cleave pre-rRNA at the C2 site. Together, Grc3 and Las1 assemble into a higher-order complex exquisitely primed for cleavage and phosphorylation of RNA. Las1 is a recently discovered endoribonuclease that collaborates with Grc3–Rat1–Rai1 to process precursor ribosomal RNA (rRNA), yet its mechanism of action remains unknown. Disruption of the mammalian Las1 gene has been linked to congenital lethal motor neuron disease and X-linked intellectual disability disorders, thus highlighting the necessity to understand Las1 regulation and function. Here, we report that the essential Las1 endoribonuclease requires its binding partner, the polynucleotide kinase Grc3, for specific C2 cleavage. Our results establish that Grc3 drives Las1 endoribonuclease cleavage to its targeted C2 site both in vitro and in Saccharomyces cerevisiae. Moreover, we observed Las1-dependent activation of the Grc3 kinase activity exclusively toward single-stranded RNA. Together, Las1 and Grc3 assemble into a tetrameric complex that is required for competent rRNA processing. The tetrameric Grc3/Las1 cross talk draws unexpected parallels to endoribonucleases RNaseL and Ire1, and establishes Grc3/Las1 as a unique member of the RNaseL/Ire1 RNA splicing family. Together, our work provides mechanistic insight for the regulation of the Las1 endoribonuclease and identifies the tetrameric Grc3/Las1 complex as a unique example of a protein-guided programmable endoribonuclease.

Nuclease integrated kinase super assemblies (NiKs) and their role in RNA processing

Here we highlight the Grc3/Las1 complex, an essential RNA processing machine that is well conserved across eukaryotes and required for processing the pre-ribosomal RNA (pre-rRNA). Las1 is an endoribonuclease that cleaves the pre-rRNA while Grc3 is a polynucleotide kinase that phosphorylates the Las1-cleaved RNA product. Recently we showed that Grc3 and Las1 assemble into a higher-order complex composed of a dimer of Grc3/Las1 heterodimers that is required for nuclease and kinase activity. Unexpectedly, we found that the Grc3/Las1 complex draws numerous parallels with two other eukaryotic nucleases, Ire1 and RNase L. In this perspective we explore the similarities and differences between this family of nuclease integrated kinase super assemblies (NiKs) and their distinct roles in RNA cleavage.

Characterization of the molecular crosstalk within the essential Grc3/Las1 pre-rRNA processing complex

Grc3 is an essential well-conserved eukaryotic polynucleotide kinase (PNK) that cooperates with the endoribonuclease Las1 to process the preribosomal RNA (rRNA). Aside from being dependent upon Las1 for coordinated kinase and nuclease function, little is known about Grc3 substrate specificity and the molecular mechanisms governing kinase activity. Here we characterize the kinase activity of Grc3 and identify key similarities and differences between Grc3 and other polynucleotide kinase family members. In contrast to other PNK family members, Grc3 has distinct substrate preference for RNA substrates in vitro. By disrupting conserved residues found at the Grc3 kinase active site, we identified specific residues required to support Grc3-directed Las1-mediated pre-rRNA cleavage in vitro and in vivo. The crosstalk between Grc3 and Las1 ensures the direct coupling of cleavage and phosphorylation during pre-rRNA processing. Taken together, our studies provide key insight into the polynucleotide kinase activity of the essential enzyme Grc3 and its molecular crosstalk with the endoribonuclease Las1.