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Any living cell relies on translation to fuel its metabolism and perform essential functions. Eukaryotic initiation factor 2 (eIF2) is a heterotrimeric GTPase that lies at the core of protein synthesis, being responsible to load the ribosome with methionyl tRNA initiator (Met-tRNAi) at each round of translation. The functional cycle of eIF2 depends on additional factors including eIF2B and eIF5. These proteins have antagonist roles and compete for mutually exclusive interfaces of eIF2. As part of the integrated stress response, eIF2 is phosphorylated and traps all available eIF2B in an inhibitory interaction that results in the global shutdown of protein synthesis. Here, we gather insights into the molecular mechanism of eIF2 and eIF2B, and their interaction with eIF5 and the Met-tRNAi. We present the cryo-EM structures of phosphorylated and unphosphorylated yeast eIF2 in complex with eIF2B, revealing one of their two binding modes. Through structural modelling and computational analysis, we identify conserved elements within eIF2 that rearrange upon binding to two different pockets of eIF2B. Their role is addressed by complementary genetic and biochemical approaches. Consequently, we propose that phosphorylation constrains eIF2 to adopt a conformation that stably binds the regulatory site of eIF2B, preventing the dissociation of the complex. Finally, we advance a structural model of eIF2 recycling consisting of consecutive coupled interactions of eIF2 with eIF5, eIF2B and Met-tRNAi.

A key step in eukaryotic translation initiation is the recycling of the eukaryotic initiation factor, eIF2 from a GDP to GTP-bound translationally competent form. This nucleotide exchange reaction is mediated by eukaryotic translation initiation factor 2B (eIF2B). A range of starvation or stress conditions can inhibit translation by inducing the phosphorylation of the alpha subunit of eIF2 converting eIF2 from a substrate to an inhibitor of eIF2B. In addition, eIF2B localises to a large cytoplasmic body termed the '2B body', which appears important for translation activity and regulation. In yeasts, eIF2B is targeted to inhibit translation by various quorum-sensing alcohols. Toxicity of alcohols, such as butanol, is a major concern for industrial scale butanol production; therefore, the identification and characterisation of butanol resistant strains may well prove important in the search for commercial alternatives to fossil fuel products. In this study, specific eIF2B mutants are shown to have an altered response to alcohols and starvation and exhibit distinctive alterations in the dynamics and morphology of the 2B body. Purification, then proteomic and functional analysis of eIF2B suggests that alcohols act directly to inhibit guanine nucleotide exchange activity and that eIF2B may be associated with fatty acid and pyrimidine metabolic enzymes. Overall these data highlight the possibility that the eIF2B and the 2B body may serve as a hub connecting metabolic signalling with the control of translation initiation.

Axonal protein synthesis is rapidly regulated by extrinsic cues during neural wiring but the full landscape of proteomic changes and their translational control mechanisms remain unknown. The ability to investigate the nascent proteome on subcellular compartments has been hampered by the low sensitivity of existing methodology on quantity-limited samples combined with the difficulty of obtaining sufficient amounts of pure material. By combining pulsed Stable Isotope Labelling by Amino acids in Cell culture (pSILAC) with Single-Pot Solid-Phase-enhanced Sample Preparation (SP3), I have established an approach to characterize the nascent proteome from quantity-limited somaless retinal axons (~2μg) on an unparalleled rapid time-scale (5 min). The results show that a surprisingly large number of proteins (>350) is translated constitutively in axons, many of which are linked to neurological disease. Axons stimulated by different cues (Netrin-1, BDNF, Sema3A) each show a signature set of up/down newly synthesised protein (NSP) changes (>100) within 5 min. Remarkably, conversion of Netrin-1-induced responses from repulsion to attraction triggers opposite translational regulation for 73% of a common subset corresponding to >100 NSPs. Further, I show that pharmacological increase in cAMP, known to induce chemoattractive response, also leads to rapid and wide-scale remodelling of the nascent axonal proteome (~100 NSP changes). I find that the cAMP-elicited NSP changes underlie the attractive turning but are distinct from those induced by the physiological chemoattractant Netrin-1, suggesting that the same type of chemotropic response can be mediated by different protein synthesis-dependent mechanisms. Finally, I show that Sema3A, but not Slit1, triggers a physiological and non-canonical PERK-eIF2α-eIF2B signalling pathway required in neural wiring to elicit the rapid (< 15 min) local translation control of a specific subset of NSPs. Collectively my findings lead to the general conclusion that guidance molecules rapidly induce cue-specific remodelling of the nascent axonal proteome via distinct regulatory mechanisms.

The Integrated Stress Response (ISR) is a conserved eukaryotic translational and transcriptional program implicated in mammalian metabolism, memory and immunity. Although mainly considered to be a protective mechanism, prolonged and severe ISR can result in cell death. The ISR is activated by diverse stress pathways converged on phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2) that inhibits the guanine nucleotide exchange activity of its partner eIF2B and attenuates overall rates of protein synthesis. Numerous mutations in eIF2B are linked to a fatal neurodegenerative disease of vanishing white matter. A new chemical inhibitor of the ISR (ISRIB), a bis-O-arylglycolamide, can reverse the attenuation of mRNA translation by phosphorylated eIF2 protecting mice from prion-induced neurodegeneration and traumatic brain injury. The work presented in this dissertation describes identification of mammalian eIF2B as a cellular target of ISRIB by implementing biochemical, biophysical, structural and chemogenetic methods. The herein reported cryo-electron microscopy-based structure of eIF2B uncovers a novel allosteric site on the translation factor capturing the ISRIB-binding pocket at the interface between its β and δ regulatory subunits. The extensive CRISPR/ Cas9-based screen for ISRIB-resistant and analogue-sensitive phenotypes revealed residues on the eIF2B dimer interface important for ISRIB binding. Based on the results reported in this dissertation along with the similar findings of others the potential molecular basis of ISRIB action, and its implication for the regulation of eIF2B's activity is broadly discussed. The identification of the ISRIB binding pocket away from the known interaction sites between eIF2B and eIF2 is also put into the context of a possible molecular mechanism of eIF2B's guanine exchange inhibition by phosphorylated eIF2. The work described in this dissertation provides new insight into the translational regulation and points to the importance of fine-tuning the activity of translation factors by small chemical molecules.

In eukaryotic translation initiation, eIF2B, a 295 kDa multisubunit (from α to ε) complex,is the guanine nucleotide exchange factor (GEF) of eIF2, a GTP binding protein, and hasmultiple roles in regulating the level of active eIF2-GTP-Met-tRNAi ternary complexes inthe cytoplasm. Mutations in eIF2B subunits affect global protein synthesis and, in human,are responsible to cause a genetically inherited lethal childhood brain disease calledLeukoencephalopathy with Vanishing White Matter (VWM). Although the genetic aspectseIF2B have been widely studied over decades, detailed structural knowledge only becameavailable in recent years but is still limited. This study aims to gain structural insights intoyeast eIF2B by a range of electron microscopy techniques to improve our understandingtowards its GEF activity with eIF2 and regulatory response. By performing size-exclusion chromatography and multi-angle static light scattering (SECMALS), it was found that eIF2B is a stable dimer of pentamers (~600 kDa). Negativestaining (25.8 Å) and cryo-EM (12.1 Å) eIF2B decamer models that showed 2-foldrotational symmetry were generated by single particle reconstruction. Homology modelingof yeast eIF2B subunits revealed an eIF2B(αβδ)2 hexameric core and two separate arm-likeeIF2Bγε catalytic domains with potential flexibility. To constrain subunit position in thearm structure, Ni-NTA-Nanogold labeling against the multihistidine tag of eIF2Bγ wasperformed. In addition, genetic approaches were applied to eliminate synthesis of eIF2Bα(34 kDa) and eIF2B(βγδε)2 octamer complexes (532 kDa) were purified by SEC-MALSand analysed by negative staining single particle reconstruction. It was speculated thatdeletion of eIF2Bα might have triggered significant conformational rearrangement that ledto high uniformity in the 2D class averages. A hypothetical model was thus proposed forthe octamer where the two arm-like domains clamp together to form a compact structure.

Fusel alcohols signal nitrogen scarcity to elicit a range of responses in the yeast Saccharomyces cerevisiae. These alcohols activate pseudohyphal growth and cause rapid inhibition of translation initiation. Previous work from our lab has highlighted that the translation initiation factor eIF2B is a target for this regulation. eIF2B is the guanine nucleotide exchange factor required for recycling eIF2•GDP to eIF2•GTP. The GTP bound form of eIF2 can interact with the Methionyl initiator tRNA to form the ternary complex. Fusel alcohols target eIF2B leading to reduced ternary complex; however the mechanism by which alcohols cause this effect is currently unknown. This study aims to characterize the effects of fusel alcohols on eIF2B and identify post-translational modifications, which may be responsible for translation inhibition. Following purification of eIF2B, a number of novel phosphorylation sites have been identified using mass spectrometry. In particular, phosphorylated serine has been identified at position 131 within yeast eIF2Bδ. Phosphoantibody analysis suggests that the phosphorylation status of this residue differs following fusel alcohol treatment. Mutagenesis experiments are consistent with phosphorylation of this residue being essential for the translational inhibition seen following fusel alcohol exposure. Therefore, phosphorylation of this residue may prime eIF2B for regulation and provide a switch to sensitize the process of translation to particular conditions.

The ability to sense and respond to environmental cues is crucial for the survival of all organisms. This response is often manifested by exerting control at different levels of gene expression, i.e. transcription, translation and post translation levels. Global control of protein synthesis is frequently exercised at the initial step of translation initiation and is generally achieved by changes in the phosphorylation state of initiation factors or the regulators that interact with them. The formation of ternary complex (TC) is considered first step of translation initiation and depends on the recycling of inactive eIF2-GDP to active eIF2-GTP form. This nucleotide exchange reaction is catalyzed by the eukaryotic initiation factor-2B (eIF2B). eIF2B is composed of a regulatory sub-complex of alphaβdelta subunits and a catalytic sub-complex of the γε subunits. The guanine nucleotide exchange activity of eIF2B is regulated by phosphorylation of eIF2alpha and additionally in mammalian cells, by direct phosphorylation of eIF2B at multiple sites in ε subunit, where most of the catalytic activity of eIF2B resides. Recent unpublished studies in the Pavitt laboratory identified novel phosphorylation sites by Mass Spectrometry in γ and ε subunits of eIF2B catalytic sub-complex. In order to study the functional significance of these phospho-sites for translation initiation, Site Directed Mutagenesis (SDM) was performed to generate Ser to Ala mutants. All mutations are viable and have no significant growth defect on rich or minimal media; however the significance of these sites in yeast growth became apparent by growing yeast in different stress conditions (e.g. Rapamycin, Torin1, amino acid starvation and 1-butanol). Effects on the phosphorylation pattern at these sites were monitored by using custom generated phospho-specific antibodies. All phosphorylation events appear independent of the eIF2alpha kinase (Gcn2p in yeast). The phosphorylation of ε-S528 depends on the presence of ε-S525. This study finds that addition of rapamycin, Torin1, amino acid starvation and butanol, which each inhibits global translation initiation, alters the phosphorylation pattern at ε-S435, ε-S525 and ε-S528 sites. Linking growth to phosphorylation, it appears that phosphorylation at ε-S435 and ε-S525 is directly proportional to growth. Phosphorylation of ε-S435 is necessary for effect of eIF2alpha-Ser51 phosphorylation on protein synthesis while phosphorylation of ε-S528 seems to be a target of various mechanisms. This study also suggests that eIF2Bε may be a key player of the cell cycle progression and phosphorylation changes can serve as marker for the regulation of eIF2B activity. The kinases responsible for phosphorylation at these sites are not yet known in yeast. Further investigation is required to find the functional significance of alterations in phosphorylation pattern to definitively establish eIF2Bε phosphorylation as a mechanism for regulating eIF2B activity in yeast. Models are presented to account for the results obtained that show how phosphorylation of eIF2Bε at these sites may contribute to the control of protein synthesis.

Eukaryotic translation initiation is a complex and highly regulated process involving the ribosome, mRNA and proteins called eukaryotic initiation factors (eIFs). The overall aim of translation initiation is to position the ribosome at the initiation codon of the mRNA. eIF2, in its GTP-bound conformation, binds the initiator tRNA (Met-tRNAiMet) and delivers it to the 40S ribosomal subunit. When the anticodon of the tRNA is bound to the initiation codon, the GTP on eIF2 is hydrolysed to GDP. The guanine nucleotide exchange factor (GEF) eIF2B regenerates eIF2-GTP. eIF2 and eIF2B are multisubunit/multidomain protein complexes. Because information regarding the interface between each complex is limited, particularly the interface on the eIF2γ subunit, which binds the guanine-nucleotides and Met-tRNAiMet, interactions between the minimal GEF domain of eIF2Bε, εGEF, and eIF2 were mapped using mutagenesis and an in vitro cysteine cross-linking approach, with the cross-linker Mts-Atf-Biotin. Site-directed mutagenesis (SDM) was used to mutate five N-terminal and five C-terminal surface-exposed εGEF residues to cysteines. The mutant alleles were analysed in Saccharomyces cerevisiae and it was found that the gcd6-R574C allele was lethal and the gcd6-T572C was Gcd-. Further gcd6-R574 mutant alleles were also found to be lethal in yeast but expressed in vivo.εGEF-R574C has dramatically reduced GEF activity in vitro and binding assays showed that this mutant has significantly reduced affinity for eIF2. The εGEF-T572C and εGEF-S576C mutants also have severe and minor eIF2-binding defects respectively, while the C-terminal εGEF-Cys mutants have slightly reduced affinity for eIF2. The N-terminal εGEF-Cys mutants cross-link specifically to eIF2γ, while the C-terminal εGEF-Cys mutants interact predominantly with eIF2β. From the data obtained in this study, we propose a new model for eIF2B-mediated guanine-nucleotide exchange that reduces the importance of eIF2β and suggests εGEF resembles other GEFs in binding primarily to its G protein partner eIF2γ.

Coping with cellular stressors, manifesting as either intrinsic cues or environmental insults, is key to preserving cellular and organismal health. One strategy is to activate the Integrated Stress Response (ISR), a conserved eukaryotic signaling pathway that reprograms translation towards damage mitigation and recovery, or apoptosis when stress is irremediable. Although an inherently cytoprotective process, dysregulation of the ISR underlies a number of neurological disorders, and detailing its mechanism of action and precisely understanding modulators of the pathway is of critical importance. The ISR integrates diverse stresses through a set of stress-sensing kinases. Upon activation, they converge on a common task: the phosphorylation of a single serine in the translation initiation factor eIF2, the protein responsible for bringing the first amino acid to a translating ribosome. Phosphorylated eIF2 (eIF2-p), instead of acting as a substrate, inhibits its dedicated nucleotide exchange factor eIF2B, which is a two-fold symmetric heterodecameric complex. Inhibition of eIF2B by eIF2-p impairs the cycling of eIF2 from its inactive GDP-bound state to its active GTP-bound state, leading to diminished translation of most (and selective translation of a few) select mRNAs, the latter containing regulatory upstream open reading frames. Thus eIF2B serves as the central node regulating the critical first step of protein synthesis.In Chapter 1 I will be describing how through a suite of in vitro and in vivo tools, we uncovered the mechanism by which eIF2B activity is modulated. We discovered that, contrary to prior hypotheses, eIF2B activity is regulated through its conformation. eIF2-p binding to eIF2B induces a conformational change in eIF2B that disfavors substrate (eIF2) engagement by pulling the two halves of the complex apart and disrupting the eIF2 binding site. eIF2-p therefore activates the ISR by shifting eIF2B from its enzymatically active ‘A-State’ towards a conformationally inhibited ‘I-State’ with diminished substrate binding and enzymatic activity. This conformational inhibition of eIF2B can be overcome by the small-molecule Integrated Stress Response Inhibitor (ISRIB) that our lab discovered. ISRIB binds across the symmetry interface of eIF2B and staples the complex into its active conformation, allosterically competing off the inhibitor eIF2-p. We find orthogonal support for the A/I-State model of ISR activation through viral effector proteins and point mutations in eIF2B. In Chapter 2 I will describe how the NSs protein of Sandfly Fever Sicilian virus (SFSV) allows the virus to evade activation of the ISR. Mechanistically, NSs tightly binds to eIF2B and directly competes with inhibitory eIF2-p binding, restoring eIF2B’s conformation to its active A-State. Having the opposite effect (and detailed in Chapter 3), a single histidine to aspartate point mutation at eIF2B’s dimerization interface mimics the effects of eIF2-p binding by promoting an I-State like conformation. Through charge repulsion, this point mutation results in eIF2-p independent modulation of eIF2B’s conformation and concomitant activation of the ISR in vitro and in cells. Collectively, these data establish that eIF2B is a highly dynamic complex through which protein translation and cellular functioning can be modulated by small molecules (ISRIB), endogenous binding partners (eIF2 and eIF2-P), evolved viral proteins, and point mutations. My work on ISR regulation came to an abrupt halt when SARS-CoV-2 took over the world. During this involuntary pause, all non-Covid work stopped at UCSF. In an effort to address the global threat, I assembled and led an interdisciplinary team to develop agents that powerfully neutralize the virus (detailed in Chapter 4). In particular, we obtained single-domain antibodies (nanobodies) by screening a yeast surface-displayed library of synthetic nanobodies. We identified nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction. One exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a humanized trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains powerful binding and neutralization properties against the Delta-SARS-CoV-2. In hamster models of infection, mNb6-tri delivered via nasal-spray reduced the severity of infection in animals exposed to SARS-CoV-2, and served as a potent post-exposure prophylaxis in animals co-housed with infected individuals. Further, in initial safety trials in >200 human subjects after administration via a nasal spray, no safety concerns were observed. Most importantly mNb6-tri is fully resistant to aerosolization, lyophilization, and heat, and it can be readily and economically produced in yeast at industrial scale. These properties may allow these or similar agents to serve as a widely deployable, patient-friendly prophylactic and/or early infection therapeutic that can be self-administered directly to the initial sites of infection. Such a therapeutic is particularly needed in the developing world where vaccination levels remain low and access to expensive therapeutics is significantly limited.

Cells maintain their proteins in a functional and balanced state by regulating protein synthesis, folding, trafficking and degradation. A central regulatory target in this process is the nucleotide exchange factor eIF2B. Under favorable conditions eIF2B acts as a biological catalyst, efficiently unloading GDP from translation initiation factor 2 (eIF2), a GTPase that is required for protein synthesis. Under conditions of stress such as viral infection or starvation, a conserved signaling pathway known as the integrated stress response (ISR) couples stress detection to the phosphorylation of eIF2. This phosphorylation event renders eIF2 a potent inhibitor of eIF2B to restrict protein synthesis. Relieved of a heavy translational burden, cells are afforded more time and resources to cope with stress.Recently, a small molecule called ISRIB (integrated stress response inhibitor) was found to activate eIF2B. When systemically administered to mice, ISRIB enhances cognition, confers neuroprotection, and reduces inflammation. These cytoprotective effects highlight the importance of eIF2B in human health and the potential that this pathway offers for therapeutic intervention. To better understand eIF2B-mediated translational control and the physiological effects of ISRIB, we solved a 2.8Å cryo-EM structure of ISRIB-bound human eIF2B. The structure identified ISRIB’s binding site at the symmetric core of the eIF2B heterodecamer. Structural and biochemical analyses revealed that the fully active eIF2B heterodecamer depends on the assembly of two identical tetrameric subcomplexes, and that ISRIB promotes this step by cross-bridging the symmetry interface.Additional cryoEM structures of eIF2 bound to eIF2B in the dephosphorylated state revealed the eIF2B decamer to be a static platform upon which one or two flexible eIF2 trimers bind and align with eIF2B’s bipartite catalytic centers to catalyze guanine nucleotide exchange. Phosphorylation refolds eIF2 alpha, allowing it to contact eIF2B at a different interface that is only present in the full decamer, and thereby sequesters eIF2B into a non-productive complex. Thus, regulation of eIF2B assembly emerges as a rheostat for eIF2B activity that tunes translation during the ISR and that can be further modulated by ISRIB.

Translation begins when initiation factor-2 (eIF2) delivers methionyl initiator tRNA (Met-tRNAi) to the ribosome. The exchange of GDP bound to eIF2 for GTP is a prerequisite to binding Met-tRNAi and is mediated by a second initiation factor, eIF2B. Regulation of mRNA translation is achieved through phosphorylation of eIF2 α at Ser51 which converts eIF2 from a substrate into a competitive inhibitor of eIF2B. Using the latest cryo-electron microscopy (cryoEM) technologies in both collection and data processing we were able to obtain three high resolution structures to interrogate the structural basis of the integrated stress response.

Eukaryotic translation initiation factor 2 (eIF2) is a heterotrimeric G-protein that plays a critical role in protein synthesis regulation. eIF2-GTP binds Met-tRNAi to form the eIF2-GTP:Met-tRNAi ternary complex (TC), that is recruited to the 40S ribosomal subunit. Following GTP hydrolysis, eIF2-GDP is recycled back to TC by its guanine nucleotide exchange factor (GEF), eIF2B. Phosphorylation of the eIF2α subunit in response to various cellular stresses converts eIF2 into a competitive inhibitor of eIF2B, triggering the integrated stress response. Dysregulation of eIF2B activity is associated with a number of pathologies, including neurodegenerative diseases, metabolic disorders, and cancer. However, despite decades of research, the underlying molecular mechanisms remain unknown. This is due in large part to the absence of a structural understanding of the eIF2B assembly and of the eIF2B:eIF2 interaction. Common methods, such as yeast genetics, have been unable to unambiguously determine these mechanisms. Meanwhile, expanded interest in the integrated stress response has uncovered a diverse array of pathologies for which therapeutic modulation of the eIF2B:eIF2 interaction may ameliorate or overcome disease states. In this dissertation, a combination of structural and biochemical techniques is employed to elucidate the mechanisms of eIF2B action and its regulation by eIF2α phosphorylation. The aim is to provide a direct, unambiguous, structural understanding of eIF2B assembly and of eIF2B’s interactions with phosphorylated and unphosphorylated eIF2α. The work described here was among the first to challenge the widely held notion of a pentameric eIF2B assembly, as eventually confirmed by the recent publication of eIF2B’s crystal structure. The work further aims to overturn another long-standing assumption regarding the nature of inhibition of eIF2B activity: that competitive inhibition is mediated by a “direct effect” of the negatively charged phosphate group on the eIF2α:eIF2B interaction. Instead, we present evidence for an “indirect effect,” whereby phosphorylation disrupts a novel intramolecular interface within eIF2α, exposing an eIF2α surface that binds eIF2B and is responsible for inhibition of eIF2B. In the end, we combine a structural model of the eIF2B:eIF2 complex with our novel mechanism of inhibition, placing them within the larger thermodynamic context of eIF2-GDP recycling by eIF2B.