Crystal Structures of GCN2 Protein Kinase C-terminal Domains Suggest Regulatory Differences in Yeast and Mammals

In response to amino acid starvation, GCN2 phosphorylation of eIF2 leads to repression of general translation and initiation of gene reprogramming that facilitates adaptation to nutrient stress. GCN2 is a multidomain protein with key regulatory domains that directly monitor uncharged tRNAs which accumulate during nutrient limitation, leading to activation of this eIF2 kinase and translational control. A critical feature of regulation of this stress response kinase is its C-terminal domain (CTD). Here, we present high resolution crystal structures of murine and yeast CTDs, which guide a functional analysis of the mammalian GCN2. Despite low sequence identity, both yeast and mammalian CTDs share a core subunit structure and an unusual interdigitated dimeric form, albeit with significant differences. Disruption of the dimeric form of murine CTD led to loss of translational control by GCN2, suggesting that dimerization is critical for function as is true for yeast GCN2. However, although both CTDs bind single- and double-stranded RNA, murine GCN2 does not appear to stably associate with the ribosome, whereas yeast GCN2 does. This finding suggests that there are key regulatory differences between yeast and mammalian CTDs, which is consistent with structural differences.     

Endoplasmic Reticulum Targeting and Insertion of Tail-Anchored Membrane Proteins by the GET Pathway

Hundreds of eukaryotic membrane proteins are anchored to membranes by a single transmembrane domain at their carboxyl terminus. Many of these tail-anchored (TA) proteins are posttranslationally targeted to the endoplasmic reticulum (ER) membrane for insertion by the guided-entry of TA protein insertion (GET) pathway. In recent years, most of the components of this conserved pathway have been biochemically and structurally characterized. Get3 is the pathway-targeting factor that uses nucleotide-linked conformational changes to mediate the delivery of TA proteins between the GET pretargeting machinery in the cytosol and the transmembrane pathway components in the ER. Here we focus on the mechanism of the yeast GET pathway and make a speculative analogy between its membrane insertion step and the ATPase-driven cycle of ABC transporters.The mechanism of membrane protein insertion into the endoplasmic reticulum (ER) has been extensively studied for many years (Shao and Hegde 2011). From this work, the signal recognition particle (SRP)/Sec61 pathway has emerged as a textbook example of a cotranslational membrane insertion mechanism (Grudnik et al. 2009). The SRP binds a hydrophobic segment (either a cleavable amino-terminal signal sequence or a transmembrane domain) immediately after it emerges from the ribosomal exit tunnel. This results in a translational pause that persists until SRP engages its receptor in the ER and delivers the ribosome-nascent chain complex to the Sec61 channel. Last, the Sec61 channel enables protein translocation into the ER lumen along with partitioning of hydrophobic transmembrane domains into the lipid bilayer through the Sec61 lateral gate (Rapoport 2007).Approximately 5% of all eukaryotic membrane proteins have an ER targeting signal in a single carboxy-terminal transmembrane domain that emerges from the ribosome exit tunnel following completion of protein synthesis and is not recognized by the SRP (Stefanovic and Hegde 2007). Nonetheless, because hydrophobic peptides in the cytoplasm are prone to aggregation and subject to degradation by quality control systems (Hessa et al. 2011), these tail-anchored (TA) proteins still have to be specifically recognized, shielded from the aqueous environment, and guided to the ER membrane for insertion. In the past five years, the guided-entry of TA proteins (GET) pathway has come to prominence as the major machinery for performing these tasks and the enabler of many key cellular processes mediated by TA proteins including vesicle fusion, membrane protein insertion, and apoptosis. This research has rapidly yielded biochemical and structural insights (​and2)2) into many of the GET pathway components (Hegde and Keenan 2011; Chartron et al. 2012a; Denic 2012). In particular, Get3 is an ATPase that uses metabolic energy to bridge recognition of TA proteins by upstream pathway components with TA protein recruitment to the ER for membrane insertion. However, the precise mechanisms of nucleotide-dependent TA protein binding to Get3 and how the GET pathway inserts tail anchors into the membrane are still poorly understood. Here, we provide an overview of the budding yeast GET pathway with emphasis on mechanistic insights that have come from structural studies of its membrane-associated steps and make a speculative juxtaposition with the ABC transporter mechanism.

Table 1.

A catalog of GET pathway component structures

Crystal Structures of Leishmania mexicana Phosphoglycerate Mutase Suggest a One-Metal Mechanism and a New Enzyme Subclass

The structures of Leishmania mexicana cofactor-independent phosphoglycerate mutase (Lm iPGAM) crystallised with the substrate 3-phosphoglycerate at high and low cobalt concentrations have been solved at 2.00- and 1.90-Å resolutions. Both structures are very similar and the active site contains both 3-phosphoglycerate and 2-phosphoglycerate at equal occupancies (50%). Lm iPGAM co-crystallised with the product 2-phosphoglycerate yields the same structure. Two Co²⁺ are coordinated within the active site with different geometries and affinities. The cobalt at the M1 site has a distorted octahedral geometry and is present at 100% occupancy. The M2-site Co²⁺ binds with distorted tetrahedral geometry, with only partial occupancy, and coordinates with Ser75, the residue involved in phosphotransfer. When the M2 site is occupied, the side chain of Ser75 adopts a position that is unfavourable for catalysis, indicating that this site may not be occupied under physiological conditions and that catalysis may occur via a one-metal mechanism. The geometry of the M2 site suggests that it is possible for Ser75 to be activated for phosphotransfer by H-bonding to nearby residues rather than by metal coordination. The 16 active-site residues of Lm iPGAM are conserved in the Mn-dependent iPGAM from Bacillus stearothermophilus (33% overall sequence identity). However, Lm iPGAM has an inserted tyrosine (Tyr210) that causes the M2 site to diminish in size, consistent with its reduced metal affinity. Tyr210 is present in trypanosomatid and plant iPGAMs, but not in the enzymes from other organisms, indicating that there are two subclasses of iPGAMs.     

The Crystal Structures of TrkA and TrkB Suggest Key Regions for Achieving Selective Inhibition

The Trk family of neurotrophin receptors, which includes the three highly homologous proteins TrkA, TrkB and TrkC, is strongly associated with central and peripheral nervous system processes. Trk proteins are also of interest in oncology, since Trk activation has been observed in several cancer types. While Trk kinases are attractive oncology targets, selectivity might be more of an issue than for other kinases due to potential CNS side effects if several Trk kinases are simultaneously targeted. In order to address this issue, we present here the first structures of human TrkA and TrkB kinase domains and three complexes between TrkB and Trk inhibitors. These structures reveal different conformations of the kinase domain and suggest new regions of selectivity among the Trk family.     

Crystal Structures and Biochemical Analyses Suggest a Unique Mechanism and Role for Human Glycyl-tRNA Synthetase in Ap4A Homeostasis

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs for protein synthesis. However, the aminoacylation reaction can be diverted to produce diadenosine tetraphosphate (Ap4A), a universal pleiotropic signaling molecule needed for cell regulation pathways. The only known mechanism for Ap4A production by a tRNA synthetase is through the aminoacylation reaction intermediate aminoacyl-AMP, thus making Ap4A synthesis amino acid-dependent. Here, we demonstrate a new mechanism for Ap4A synthesis. Crystal structures and biochemical analyses show that human glycyl-tRNA synthetase (GlyRS) produces Ap4A by direct condensation of two ATPs, independent of glycine concentration. Interestingly, whereas the first ATP-binding pocket is conserved for all class II tRNA synthetases, the second ATP pocket is formed by an insertion domain that is unique to GlyRS, suggesting that GlyRS is the only tRNA synthetase catalyzing direct Ap4A synthesis. A special role for GlyRS in Ap4A homeostasis is proposed.Aminoacyl-tRNA synthetases (AARSs)⁴ are considered to be among the earliest proteins to have emerged during evolution. As a family of typically 20 members (one for each amino acid), AARSs catalyze the first step of protein synthesis by linking each amino acid onto the 3′-end of its cognate tRNA harboring the trinucleotide anticodon. Through evolution, the role of AARSs has also been broadened with expanded functions (reviewed in Refs. 1 and 2). These expanded functions often involve direct interaction partners. For example, human tyrosyl-tRNA synthetase interacts with chemokine receptor CXCR1 to induce cell migration (3); human glutaminyl-tRNA synthetase interacts with ASK1 to regulate apoptosis (4); human tryptophanyl-tRNA synthetase interacts with VE-cadherin to inhibit angiogenesis (5); human lysyl-tRNA synthetase interacts with the Gag protein of human immunodeficiency virus to facilitate viral assembly (6); and human glutamyl-prolyl-tRNA synthetase interacts with L13a and glyceraldehyde-3-phosphate dehydrogenase to form the GAIT complex for translational silencing to regulate inflammation (7). However, functional expansion also can be achieved indirectly via reaction products of AARSs. As examples, Lys-tRNA^Lys and Ala-tRNA^Ala are used to aminoacylate cytoplasmic membrane phosphatidylglycerol of Staphylococcus aureus and Pseudomonas aeruginosa, respectively, to enhance drug resistance in these microorganisms (8).In addition to tRNA aminoacylation, the majority of AARSs have the capacity to catalyze a side reaction to form diadenosine oligophosphates (ApnA) in the absence of cognate tRNA (9). These reactions of AARS are the most well known sources of ApnA in vivo (10). ApnA are made up of two adenosine moieties linked at the 5′-end of the ribose by a chain of two to six phosphates. In the 4 decades following the discovery of these molecules by Zamecnik et al. (10), ApnA have been linked to highly diverse physiological effects in prokaryotic and eukaryotic cells, including various types of mammalian cells and tissues, and to assorted functions associated with the nucleus, membrane receptors, and activities in the cytoplasm (reviewed in Refs. 11 and 12). The concentrations of ApnA molecules in vivo respond to numerous factors, including cell proliferation status, glucose level, heat shock, oxidative stress, and interferon stimulation. They have emerged as extracellular and intracellular signaling molecules (as pleiotropically acting “alarmones” (13) and second messengers (14)) implicated in the maintenance and regulation of vital cellular functions.The aminoacylation reaction proceeds in two steps. First, the amino acid is activated by condensation with ATP to form aminoacyl-AMP, the enzyme-bound intermediate. The aminoacyl moiety is then transferred to the 3′-end of the cognate tRNA. When tRNA is absent, the enzyme-bound aminoacyl-AMP can be attacked by the γ-phosphate of a second ATP molecule to form diadenosine tetraphosphate (Ap4A), the most common diadenosine oligophosphate produced by a tRNA synthetase (see Fig. 1A). The presence of tRNA in most cases inhibits Ap4A synthesis (11). Therefore, a subgroup of tRNA synthetases that requires tRNA as cofactor for synthesis of aminoacyl-AMP is not capable of producing Ap4A. This group includes tRNA synthetases that are specific for arginine, glutamine, and glutamic acid and an unusual class I lysyl-tRNA synthetase (LysRS).Open in a separate windowFIGURE 1.Amino acid-independent synthesis of Ap4A by human GlyRS. A, conventional mechanism for synthesis of Ap4A by a tRNA synthetase. The first step of the reaction involves the generation of an enzyme-bound aminoacyl-AMP (aa-AMP), which is then attacked either by cognate tRNA to form aminoacyl-tRNA or by ATP to form Ap4A. B, mechanism used by human GlyRS to produce Ap4A by direct condensation of two ATPs. C–E, synthesis of Ap4A by GlyRS, LysRS, and TyrRS, respectively, in the presence (●) and absence (○) of cognate amino acid. Amounts of Ap4A produced were quantitated from the TLC sheets and are plotted on the right. GlyRS activity was measured in triplicate, and the plotted values reflect the mean ± S.E.Although the amino acid recycles, the above mechanism requires the presence of the amino acid for the production of Ap4A via the aminoacyl-AMP intermediate. Using biochemical analyses and determinations of co-crystal structures, we demonstrate in this work that human glycyl-tRNA synthetase (GlyRS) produces Ap4A by direct condensation of two ATPs in the absence of glycine. Thus, the mechanism for GlyRS to synthesize Ap4A is decoupled from aminoacylation. Furthermore, GlyRS is likely to be the only synthetase that produces Ap4A by this mechanism. Our results raise the possibility that GlyRS plays a special role in Ap4A homeostasis.     

The Us9 Gene Product of Pseudorabies Virus, an Alphaherpesvirus, Is a Phosphorylated, Tail-Anchored Type II Membrane Protein

The Us9 gene is highly conserved among the alphaherpesviruses sequenced to date, yet its function remains unknown. In this report, we demonstrate that the pseudorabies virus (PRV) Us9 protein is present in infected cell lysates as several phosphorylated polypeptides ranging from 17 to 20 kDa. Synthesis is first detected at 6 h postinfection and is sensitive to the DNA synthesis inhibitor phosphonoacetic acid. Unlike the herpes simplex virus type 1 Us9 homolog, which was reported to be associated with nucleocapsids in the nuclei of infected cells (M. C. Frame, D. J. McGeoch, F. J. Rixon, A. C. Orr, and H. S. Marsden, Virology 150:321–332, 1986), PRV Us9 localizes to the secretory pathway (predominately to the Golgi apparatus) and not to the nucleus. By fusing the enhanced green fluorescent protein (EGFP) reporter molecule to the carboxy terminus of Us9, we demonstrated that Us9 not only is capable of targeting a Us9-EGFP fusion protein to the Golgi compartment but also is able to direct efficient incorporation of such chimeric molecules into infectious viral particles. Moreover, through protease digestion experiments with Us9-EGFP-containing viral particles, we demonstrated that the Us9 protein is inserted into the viral envelope as a type II, tail-anchored membrane protein.     

The Molecular Mechanism Underlying Recruitment and Insertion of Lipid-Anchored LC3 Protein into Membranes

Lipid modification of cytoplasmic proteins initiates membrane engagement that triggers diverse cellular processes. Despite the abundance of lipidated proteins in the human proteome, the key determinants underlying membrane recognition and insertion are poorly understood. Here, we define the course of spontaneous membrane insertion of LC3 protein modified with phosphatidylethanolamine using multiple coarse-grain simulations. The partitioning of the lipid anchor chains proceeds through a concerted process, with its two acyl chains inserting one after the other. Concurrently, a conformational rearrangement involving the α-helix III of LC3, especially in the three basic residues Lys⁶⁵, Arg⁶⁸, and Arg⁶⁹, ensures stable insertion of the phosphatidylethanolamine anchor into membranes. Mutational studies validate the crucial role of these residues, and further live-cell imaging analysis shows a substantial reduction in the formation of autophagic vesicles for the mutant proteins. Our study captures the process of water-favored LC3 protein recruitment to the membrane and thus opens, to our knowledge, new avenues to explore the cellular dynamics underlying vesicular trafficking.     

The F-BAR Domain of Rga7 Relies on a Cooperative Mechanism of Membrane Binding with a Partner Protein during Fission Yeast Cytokinesis

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Crystal Structures of Membrane Transporter MmpL3, an Anti-TB Drug Target

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Replication Intermediates of the Linear Mitochondrial DNA of Candida parapsilosis Suggest a Common Recombination Based Mechanism for Yeast Mitochondria

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations.     

Sequence Determinants for Regulated Degradation of Yeast 3-Hydroxy-3-Methylglutaryl-CoA Reductase, an Integral Endoplasmic Reticulum Membrane Protein

The degradation rate of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-R), a key enzyme of the mevalonate pathway, is regulated through a feedback mechanism by the mevalonate pathway. To discover the intrinsic determinants involved in the regulated degradation of the yeast HMG-R isozyme Hmg2p, we replaced small regions of the Hmg2p transmembrane domain with the corresponding regions from the other, stable yeast HMG-R isozyme Hmg1p. When the first 26 amino acids of Hmg2p were replaced with the same region from Hmg1p, Hmg2p was stabilized. The stability of this mutant was not due to mislocalization, but rather to an inability to be recognized for degradation. When amino acid residues 27–54 of Hmg2p were replaced with those from Hmg1p, the mutant was still degraded, but its degradation rate was poorly regulated. The degradation of this mutant was still dependent on the first 26 amino acid residues and on the function of the HRD genes. These mutants showed altered ubiquitination levels that were well correlated with their degradative phenotypes. Neither determinant was sufficient to impart regulated degradation to Hmg1p. These studies provide evidence that there are sequence determinants in Hmg2p necessary for degradation and optimal regulation, and that independent processes may be involved in Hmg2p degradation and its regulation.     

分裂泛素化酵母双杂交系统研究光合膜蛋白相互作用

光合类囊体膜主要由光系统Ⅱ、细胞色素b6f复合物、光系统Ⅰ以及ATP合酶4个超分子复合物组成.利用分裂泛素化酵母双杂交系统研究光合类囊体膜蛋白间的相互作用.将叶绿体psbA基因编码的D1蛋白作为诱饵蛋白,以叶绿体基因psbD编码的D2蛋白、petB编码的Cytb6蛋白作为靶蛋白,分别共转化酵母菌株后进行相互作用分析.实验结果表明,诱饵蛋白D1能与来源于同一复合物光系统Ⅱ的D2蛋白发生相互作用,而与来源于细胞色素b6f复合物的Cytb6蛋白没有互作.这一结果表明,分裂泛素化酵母双杂交系统可以用于检测光合膜蛋白间的相互作用,从而为研究光合膜蛋白生物发生的调控机理提供一个有效的工具.     

Mechanism and a Peptide Motif for Targeting Peripheral Proteins to the Yeast Inner Nuclear Membrane

Trm1 is a tRNA specific m₂²G methyltransferase shared by nuclei and mitochondria in Saccharomyces cerevisiae . In nuclei, Trm1 is peripherally associated with the inner nuclear membrane (INM). We investigated the mechanism delivering/tethering Trm1 to the INM. Analyses of mutations of the Ran pathway and nuclear pore components showed that Trm1 accesses the nucleoplasm via the classical nuclear import pathway. We identified a Trm1 cis-acting sequence sufficient to target passenger proteins to the INM. Detailed mutagenesis of this region uncovered specific amino acids necessary for authentic Trm1 to locate at the INM. The INM information is contained within a sequence of less than 20 amino acids, defining the first motif for addressing a peripheral protein to this important subnuclear location. The combined studies provide a multi-step process to direct Trm1 to the INM: (i) translation in the cytoplasm; (ii) Ran-dependent import into the nucleoplasm; and (iii) redistribution from the nucleoplasm to the INM via the INM motif. Furthermore, we demonstrate that the Trm1 mitochondrial targeting and nuclear localization signals are in competition with each other, as Trm1 becomes mitochondrial if prevented from entering the nucleus.     

A Soluble Protein Factor is Required in Vitro for Membrane Insertion of the Thylakoid Precursor Protein, pLHCP

The precursor to the light-harvesting chlorophyll a/b protein of photosystem II can insert into isolated thylakoid membranes if reaction mixtures also contain ATP and a soluble extract of chloroplasts. Optimization of this insertion process and the initial characterization of the soluble chloroplastic component are presented. With a fixed amount of precursor, maximum integration rates occurred during the first 30 minutes at pH 8.0 and 30°C when the soluble chloroplast extract was increased eight-fold over the stoichiometric amount. Under these conditions, insertion was routinely about 60% of that which occurred during import into intact chloroplasts. Integration also increased virtually linearly with increasing amounts of precursor. However, assays revealed that at least 40% of the in vitro-synthesized pLHCP was pelletable and inactive. The soluble chloroplastic component exhibited characteristics expected of a protein. It was inactivated by heat, protease, and N-ethylmaleimide, but was insensitive to ribonuclease. The soluble component migrated on a Sephacryl S-200 gel filtration column as a single peak with an M_r of approximately 65,000. The proteinaceous nature of this factor suggests a similarity to soluble factors required for protein transport/integration in other membrane systems.     

Consequences of Domain Insertion on the Stability and Folding Mechanism of a Protein

SlyD, the sensitive-to-lysis protein from Escherichia coli, consists of two domains. They are not arranged successively along the protein chain, but one domain, the “insert-in-flap” (IF) domain, is inserted internally as a guest into a surface loop of the host domain, which is a prolyl isomerase of the FK506 binding protein (FKBP) type. We used SlyD as a model to elucidate how such a domain insertion affects the stability and folding mechanism of the host and the guest domain. For these studies, the two-domain protein was compared with a single-domain variant SlyDΔIF, SlyD* without the chaperone domain (residues 1-69 and 130-165) in which the IF domain was removed and replaced by a short loop, as present in human FKBP12. Equilibrium unfolding and folding kinetics followed an apparent two-state mechanism in the absence and in the presence of the IF domain. The inserted domain decreased, however, the stability of the host domain in the transition region and decelerated its refolding reaction by about 10-fold. This originates from the interruption of the chain connectivity by the IF domain and its inherent instability. To monitor folding processes in this domain selectively, a Trp residue was introduced as fluorescent probe. Kinetic double-mixing experiments revealed that, in intact SlyD, the IF domain folds and unfolds about 1000-fold more rapidly than the FKBP domain, and that it is strongly stabilized when linked with the folded FKBP domain. The unfolding limbs of the kinetic chevrons of SlyD show a strong downward curvature. This deviation from linearity is not caused by a transition-state movement, as often assumed, but by the accumulation of a silent unfolding intermediate at high denaturant concentrations. In this kinetic intermediate, the FKBP domain is still folded, whereas the IF domain is already unfolded.     

酵母双杂交系统在膜蛋白相互作用研究中的应用

膜相关蛋白约占细胞总蛋白质中的1/3,它们大都参与了细胞的诸多生理、病理过程和药物反应机理。研究膜蛋白的相互作用对于揭示细胞的生命活动规律及寻找药物作用靶标都有重要的意义。由于膜蛋白本身的特性及其难以进入核内等原因,经典的酵母双杂交技术并不适用于检测膜蛋白间的相互作用。针对在活细胞中研究膜蛋白相互作用的需要,近年来国际上先后发展了一系列用于膜蛋白相互作用研究的酵母双杂交新系统,并取得了许多重要发现。