Functional role of PGAM5 multimeric assemblies and their polymerization into filaments.

PGAM5 is a mitochondrial protein phosphatase whose genetic ablation in mice results in mitochondria-related disorders, including neurodegeneration. Functions of PGAM5 include regulation of mitophagy, cell death, metabolism and aging. However, mechanisms regulating PGAM5 activation and signaling are poorly understood. Using electron cryo-microscopy, we show that PGAM5 forms dodecamers in solution. We also present a crystal structure of PGAM5 that reveals the determinants of dodecamer formation. Furthermore, we observe PGAM5 dodecamer assembly into filaments both in vitro and in cells. We find that PGAM5 oligomerization into a dodecamer is not only essential for catalytic activation, but this form also plays a structural role on mitochondrial membranes, which is independent of phosphatase activity. Together, these findings suggest that modulation of the oligomerization of PGAM5 may be a regulatory switch of potential therapeutic interest

Magnifying the multifaceted role of the ER membrane protein complex in membrane protein biogenesis

Membrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player in this process. Yet, we have limited understanding of how EMC enables insertion and integrity of diverse clients¬, from tail-anchored to polytopic transmembrane proteins. Here, yeast and human EMC cryo-EM structures reveal conserved intricate assemblies and human-specific features associated with pathologies. Structure-based functional studies distinguish between two separable EMC activities, as an insertase regulating tail-anchored protein levels and a broader role in polytopic membrane protein biogenesis. These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities which confer client-specific regulation, and a non-insertase EMC function mediated by the EMC lumenal domain. Our studies illuminate the structural and mechanistic basis of EMC’s multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes

eIF2B-catalyzed nucleotide exchange and phosphoregulation by the integrated stress response

The integrated stress response (ISR) tunes the rate of protein synthesis. Control is exerted by phosphorylation of the general translation initiation factor eIF2. eIF2 is a guanosine triphosphatase that becomes activated by eIF2B, a two-fold symmetric and heterodecameric complex that functions as eIF2's dedicated nucleotide exchange factor. Phosphorylation converts eIF2 from a substrate into an inhibitor of eIF2B. We report cryo-electron microscopy structures of eIF2 bound to eIF2B in the dephosphorylated state. The structures reveal that the eIF2B decamer is a static platform upon which one or two flexible eIF2 trimers bind and align with eIF2B's bipartite catalytic centers to catalyze nucleotide exchange. Phosphorylation refolds eIF2alpha, allowing it to contact eIF2B at a different interface and, we surmise, thereby sequestering it into a nonproductive complex

Structure of the nucleotide exchange factor eIF2B reveals mechanism of memory-enhancing molecule

Regulation by the integrated stress response (ISR) converges on the phosphorylation of translation initiation factor eIF2 in response to a variety of stresses. Phosphorylation converts eIF2 from a substrate to a competitive inhibitor of its dedicated guanine nucleotide exchange factor, eIF2B, thereby inhibiting translation. ISRIB, a drug-like eIF2B activator, reverses the effects of eIF2 phosphorylation, and in rodents it enhances cognition and corrects cognitive deficits after brain injury. To determine its mechanism of action, we solved an atomic-resolution structure of ISRIB bound in a deep cleft within decameric human eIF2B by cryo-electron microscopy. Formation of fully active, decameric eIF2B holoenzyme depended on the assembly of two identical tetrameric subcomplexes, and ISRIB promoted this step by cross-bridging a central symmetry interface. 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

eIF2B-catalyzed nucleotide exchange and phosphoregulation by the integrated stress response

The integrated stress response (ISR) tunes the rate of protein synthesis. Control is exerted by phosphorylation of the general translation initiation factor eIF2. eIF2 is a guanosine triphosphatase that becomes activated by eIF2B, a two-fold symmetric and heterodecameric complex that functions as eIF2's dedicated nucleotide exchange factor. Phosphorylation converts eIF2 from a substrate into an inhibitor of eIF2B. We report cryo-electron microscopy structures of eIF2 bound to eIF2B in the dephosphorylated state. The structures reveal that the eIF2B decamer is a static platform upon which one or two flexible eIF2 trimers bind and align with eIF2B's bipartite catalytic centers to catalyze nucleotide exchange. Phosphorylation refolds eIF2α, allowing it to contact eIF2B at a different interface and, we surmise, thereby sequestering it into a nonproductive complex

Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients.

Membrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player in this process. Yet, we have limited understanding of how EMC enables insertion and integrity of diverse clients, from tail-anchored to polytopic transmembrane proteins. Here, yeast and human EMC cryo-EM structures reveal conserved intricate assemblies and human-specific features associated with pathologies. Structure-based functional studies distinguish between two separable EMC activities, as an insertase regulating tail-anchored protein levels and a broader role in polytopic membrane protein biogenesis. These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities which confer client-specific regulation, and a non-insertase EMC function mediated by the EMC lumenal domain. Our studies illuminate the structural and mechanistic basis of EMC's multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes

Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding.

Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of α-synuclein, a dynamic protein linked to Parkinson's disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of α-synuclein missense mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to α-synuclein biology and pathobiology. We found that the conformation of α-synuclein previously shown to drive yeast toxicity-an extended, membrane-bound helix-is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress α-synuclein toxicity. Moreover, we find that sequence determinants of α-synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that α-synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework