11 research outputs found
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Open and cut: allosteric motion and membrane fission by dynamin superfamily proteins.
Cells have evolved diverse protein-based machinery to reshape, cut, or fuse their membrane-delimited compartments. Dynamin superfamily proteins are principal components of this machinery and use their ability to hydrolyze GTP and to polymerize into helices and rings to achieve these goals. Nucleotide-binding, hydrolysis, and exchange reactions drive significant conformational changes across the dynamin family, and these changes alter the shape and stability of supramolecular dynamin oligomers, as well as the ability of dynamins to bind receptors and membranes. Mutations that interfere with the conformational repertoire of these enzymes, and hence with membrane fission, exist in several inherited human diseases. Here, we discuss insights from new x-ray crystal structures and cryo-EM reconstructions that have enabled us to infer some of the allosteric dynamics for these proteins. Together, these studies help us to understand how dynamins perform mechanical work, as well as how specific mutants of dynamin family proteins exhibit pathogenic properties
Doctor of Philosophy
dissertationThe morphology and function of mitochondria, the energy producing organelles of the eukaryotic cell, determine the fate of diverse cellular processes such as metabolic demand, embryonic development, and cell death. The cell uses a dedicated protein based machinery to divide mitochondria for distribution to daughter cells and to ensure faithful distribution of the mitochondrial genome. As a consequence, the impact of this division machinery applies to all processes that are influenced by the mitochondrial network. In mammals, this machinery is composed of the soluble protein Dynamin-Related Protein 1 and its membrane protein receptors Mitochondrial Dynamics 49kDa/51kDa (MiD49/51) and Mitochondrial Fission Factor (Mff). Drp1 forms assemblies that encircle the mitochondria. In addition, it binds and hydrolyzes the nucleotide guanosine triphosphate (GTP) which is thought to provide the necessary energy for mitochondrial membrane fission. Although the importance of these proteins to the mitochondrial fission process is established, the mechanisms by which the receptor proteins recruit and mediate Drp1 activity are unknown. In this dissertation, I present functional and structural analyses of the interaction between Drp1 and its receptor proteins. In Chapter 3, I was a part of a study that showed that each single receptor can recruit Drp1 to the membrane of the mitochondria and cause mitochondrial fission. In Chapter 4, I extended this finding and used cryogenic electron microscopy (cryo-EM) to determine structures of Drp1 bound to MiD49. These structures help us visualize Drp1 conformations in the recruited state on the mitochondrion and establish the role of nucleotide binding and hydrolysis on Drp1 activity. Specifically, we find that Drp1 assumes an extended conformational state upon nucleotide binding. This state enables Drp1 to bind to MiD49 and polymerize into filaments that are structurally suited to encircle mitochondrial tubules. Furthermore, the addition of GTP to this structure induces receptor dissociation and conversion to a ringlike state that is suited to constrict mitochondria. The dimensions of this ring-like state correspond to the Drp1 mediated constrictions observed in human cell cultures. Taken together, this work helps us understand the functional context for multiple receptors in mitochondrial fission and enables us to visualize the conformational dynamics of Drp1 required for its engagement with receptor proteins
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Structural basis of mitochondrial receptor binding and constriction by DRP1.
Mitochondrial inheritance, genome maintenance and metabolic adaptation depend on organelle fission by dynamin-related protein 1 (DRP1) and its mitochondrial receptors. DRP1 receptors include the paralogues mitochondrial dynamics proteins of 49 and 51 kDa (MID49 and MID51) and mitochondrial fission factor (MFF); however, the mechanisms by which these proteins recruit and regulate DRP1 are unknown. Here we present a cryo-electron microscopy structure of full-length human DRP1 co-assembled with MID49 and an analysis of structure- and disease-based mutations. We report that GTP induces a marked elongation and rotation of the GTPase domain, bundle-signalling element and connecting hinge loops of DRP1. In this conformation, a network of multivalent interactions promotes the polymerization of a linear DRP1 filament with MID49 or MID51. After co-assembly, GTP hydrolysis and exchange lead to MID receptor dissociation, filament shortening and curling of DRP1 oligomers into constricted and closed rings. Together, these views of full-length, receptor- and nucleotide-bound conformations reveal how DRP1 performs mechanical work through nucleotide-driven allostery
Recommended from our members
Open and cut: allosteric motion and membrane fission by dynamin superfamily proteins.
Cells have evolved diverse protein-based machinery to reshape, cut, or fuse their membrane-delimited compartments. Dynamin superfamily proteins are principal components of this machinery and use their ability to hydrolyze GTP and to polymerize into helices and rings to achieve these goals. Nucleotide-binding, hydrolysis, and exchange reactions drive significant conformational changes across the dynamin family, and these changes alter the shape and stability of supramolecular dynamin oligomers, as well as the ability of dynamins to bind receptors and membranes. Mutations that interfere with the conformational repertoire of these enzymes, and hence with membrane fission, exist in several inherited human diseases. Here, we discuss insights from new x-ray crystal structures and cryo-EM reconstructions that have enabled us to infer some of the allosteric dynamics for these proteins. Together, these studies help us to understand how dynamins perform mechanical work, as well as how specific mutants of dynamin family proteins exhibit pathogenic properties
Recommended from our members
Structural basis of mitochondrial receptor binding and constriction by DRP1.
Mitochondrial inheritance, genome maintenance and metabolic adaptation depend on organelle fission by dynamin-related protein 1 (DRP1) and its mitochondrial receptors. DRP1 receptors include the paralogues mitochondrial dynamics proteins of 49 and 51 kDa (MID49 and MID51) and mitochondrial fission factor (MFF); however, the mechanisms by which these proteins recruit and regulate DRP1 are unknown. Here we present a cryo-electron microscopy structure of full-length human DRP1 co-assembled with MID49 and an analysis of structure- and disease-based mutations. We report that GTP induces a marked elongation and rotation of the GTPase domain, bundle-signalling element and connecting hinge loops of DRP1. In this conformation, a network of multivalent interactions promotes the polymerization of a linear DRP1 filament with MID49 or MID51. After co-assembly, GTP hydrolysis and exchange lead to MID receptor dissociation, filament shortening and curling of DRP1 oligomers into constricted and closed rings. Together, these views of full-length, receptor- and nucleotide-bound conformations reveal how DRP1 performs mechanical work through nucleotide-driven allostery
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Vms1p is a release factor for the ribosome-associated quality control complex.
Eukaryotic cells employ the ribosome-associated quality control complex (RQC) to maintain homeostasis despite defects that cause ribosomes to stall. The RQC comprises the E3 ubiquitin ligase Ltn1p, the ATPase Cdc48p, Rqc1p, and Rqc2p. Upon ribosome stalling and splitting, the RQC assembles on the 60S species containing unreleased peptidyl-tRNA (60S:peptidyl-tRNA). Ltn1p and Rqc1p facilitate ubiquitination of the incomplete nascent chain, marking it for degradation. Rqc2p stabilizes Ltn1p on the 60S and recruits charged tRNAs to the 60S to catalyze elongation of the nascent protein with carboxy-terminal alanine and threonine extensions (CAT tails). By mobilizing the nascent chain, CAT tailing can expose lysine residues that are hidden in the exit tunnel, thereby supporting efficient ubiquitination. If the ubiquitin-proteasome system is overwhelmed or unavailable, CAT-tailed nascent chains can aggregate in the cytosol or within organelles like mitochondria. Here we identify Vms1p as a tRNA hydrolase that releases stalled polypeptides engaged by the RQC