Biology subjects

Agard, D. A.

Publications and source records attributed to Agard, D. A..

35 records · Page 2Linked to original sources

bioRxiv2021

Entropy Regularized Deconvolution of Cellular Cryo-Transmission Electron Tomograms

Cryo-electron tomography (cryo-ET) allows for the high resolution visualization of biological macromolecules. However, the technique is limited by a low signal-to-noise ratio (SNR) and variance in contrast at different frequencies, as well as reduced Z resolution. Here, we applied entropy regularized deconvolution (ER DC) to cryo-electron tomography data generated from transmission electron microscopy (TEM) and reconstructed using weighted back projection (WBP). We applied DC to several in situ cryo-ET data sets, and assess the results by Fourier analysis and subtomogram analysis (STA).

biophysics↗

bioRxiv2020

CM1-driven assembly and activation of Yeast γ-Tubulin Small Complex underlies microtubule nucleation

Microtubule (MT) nucleation is regulated by the {gamma}-tubulin ring complex ({gamma}TuRC), conserved from yeast to humans. In Saccharomyces cerevisiae, {gamma}TuRC is composed of seven identical {gamma}-tubulin small complex ({gamma}TuSC) sub-assemblies which associate helically to template microtubule growth. {gamma}TuRC assembly provides a key point of regulation for the MT cytoskeleton. Here we combine cross-linking mass spectrometry (XL-MS), X-ray crystallography and cryo-EM structures of both monomeric and dimeric {gamma}TuSCs, and open and closed helical {gamma}TuRC assemblies in complex with Spc110p to elucidate the mechanisms of {gamma}TuRC assembly. {gamma}TuRC assembly is substantially aided by the evolutionarily conserved CM1 motif in Spc110p spanning a pair of adjacent {gamma}TuSCs. By providing the highest resolution and most complete views of any {gamma}TuSC assembly, our structures allow phosphorylation sites to be mapped, surprisingly suggesting that they are mostly inhibitory. A comparison of our structures with the CM1 binding site in the human {gamma}TuRC structure at the interface between GCP2 and GCP6 allows for the interpretation of significant structural changes arising from CM1 helix binding to metazoan {gamma}TuRC.

biochemistry↗

bioRxiv2020

Practical considerations for using K3 cameras in CDS mode for high-resolution and high-throughput single particle cryo-EM

Detector technology plays a pivotal role in high-resolution and high-throughput cryo-EM structure determination. Compared with the first-generation, single-electron counting direct detection camera (Gatan K2), the latest K3 camera is faster, larger, and now offers a correlated-double sampling mode (CDS). Importantly this results in a higher DQE and improved throughput compared to its predecessor. In this study, we focused on optimizing camera data collection parameters for daily use within a cryo-EM facility and explored the balance between throughput and resolution. In total, eight data sets of murine heavy-chain apoferritin were collected at different dose rates and magnifications, using 9-hole image shift data collection strategies. The performance of the camera was characterized by the quality of the resultant 3D reconstructions. Our results demonstrated that the Gatan K3 operating in CDS mode outperformed nonCDS mode in terms of reconstruction resolution in all tested conditions with 8 electrons per pixel per second being the optimal dose rate. At low magnification (64kx) we were able to achieve reconstruction resolutions of 149% of the physical Nyquist limit (1.8 [A] with a 1.346 [A] physical pixel). Low magnification allows more particles to be collected per image, aiding analysis of heterogeneous samples requiring large data sets. At moderate magnification (105kx, 0.834[A] physical pixel size) we achieved a resolution of 1.65 [A] within 9 hours of data collection, a condition optimal for achieving high-resolution on well behaved samples. Our results also show that for an optimal sample like apoferritin, one can achieve better than 2.5 [A] resolution with 5 minutes of data collection. Together, our studies validate the most efficient ways of imaging protein complexes using the K3 direct detector and will greatly benefit the cryo-EM community.

biophysics↗

bioRxiv2020

GR chaperone cycle mechanism revealed by cryo-EM: inactivation of GR by GR:Hsp90:Hsp70:Hop client-loading complex

Maintaining a healthy proteome is fundamental for organism survival1,2. Integral to this are Hsp90 and Hsp70 molecular chaperones that together facilitate the folding, remodeling and maturation of Hsp90s many "client" proteins3-7. The glucocorticoid receptor (GR) is a model client strictly dependent upon Hsp90/Hsp70 for activity8-13. Chaperoning GR involves a cycle of inactivation by Hsp70, formation of an inactive GR:Hsp90:Hsp70:Hop "loading" complex, conversion to an active GR:Hsp90:p23 "maturation" complex, and subsequent GR release14. Unfortunately, a molecular understanding of this intricate chaperone cycle is lacking for any client. Here, we report the cryo-EM structure of the GR loading complex, in which Hsp70 loads GR onto Hsp90, revealing the molecular basis of direct Hsp90/Hsp70 coordination. The structure reveals two Hsp70s--one delivering GR and the other scaffolding Hop. Unexpectedly, the Hop cochaperone interacts with all components of the complex including GR, poising Hsp90 for subsequent ATP hydrolysis. GR is partially unfolded and recognized via an extended binding pocket composed of Hsp90, Hsp70 and Hop, revealing the mechanism of GR loading and inactivation. Together with the GR maturation complex (Noddings et al., 2020), we present the first complete molecular mechanism of chaperone-dependent client remodeling, establishing general principles of client recognition, inhibition, transfer and activation.

biochemistry↗

bioRxiv2020

Cryo-EM analysis of human mitochondrial Hsp90 in multiple tetrameric states

Hsp90 is a ubiquitous molecular chaperone that mediates the folding and maturation of hundreds of "client" proteins. Although Hsp90s generally function as homodimers, recent discoveries suggested that the mitochondrion specific Hsp90 (TRAP1) also forms functionally relevant tetramers. The structural mechanism of tetramer formation remains elusive. Here we used a combination of solution, biochemical and cryo-electron microscopy (cryo-EM) approaches to confirm that, independent of nucleotide state, a subpopulation of TRAP1 exists as tetramers. Unexpectedly, cryo-EM reveals multiple tetramer conformations having TRAP1 dimers arranged in orthogonal, parallel, or antiparallel configurations. The cryo-EM structure of one of the orthogonal tetrameric states was determined at 3.5 [A] resolution. Each of the two TRAP1 dimers is in a symmetric AMP{middle dot}PNP-bound closed state with the tetramer being stabilized through three distinct dimer-dimer interaction sites. In unique ways, each of the three TRAP1 domains contributes to tetramer formation. In addition to tetramerization via direct dimer-dimer contacts, our structure suggests that additional stabilization could come from domain swapping between the dimers. These results expand our understanding of TRAP1 biology beyond the conventional view of a functional dimer and provide a platform to further explore the function and regulation of tetrameric TRAP1 in mitochondria.

biophysics↗

bioRxiv2020

GR chaperone cycle mechanism revealed by cryo-EM: reactivation of GR by the GR:Hsp90:p23 client-maturation complex

Hsp90 is a conserved and essential molecular chaperone responsible for the folding and activation of hundreds of client proteins1,2. The glucocorticoid receptor (GR) is a model client that constantly depends on Hsp90 for activity3. Previously, we revealed GR ligand binding is inhibited by Hsp70 and restored by Hsp90, aided by the cochaperone p234. However, a molecular understanding of the chaperone-induced transformations that occur between the inactive Hsp70:Hsp90 client-loading complex and an activated Hsp90:p23 client-maturation complex is lacking for GR, or for any client. Here, we present a 2.56[A] cryo-EM structure of the GR-maturation complex (GR:Hsp90:p23), revealing that the GR ligand binding domain is, surprisingly, restored to a folded, ligand-bound conformation, while simultaneously threaded through the Hsp90 lumen. Also, unexpectedly, p23 directly stabilizes native GR using a previously uncharacterized C-terminal helix, resulting in enhanced ligand-binding. This is the highest resolution Hsp90 structure to date and the first atomic resolution structure of a client bound to Hsp90 in a native conformation, sharply contrasting with the unfolded kinase:Hsp90 structure5. Thus, aided by direct cochaperone:client interactions, Hsp90 dictates client-specific folding outcomes. Together with the GR-loading complex structure (Wang et al. 2020), we present the molecular mechanism of chaperone-mediated GR remodeling, establishing the first complete chaperone cycle for any client.

biophysics↗

bioRxiv2020

A molecular pore spans the double membrane of the coronavirus replication organelle

Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored micro-environment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and mRNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. Here, using cellular electron cryo-microscopy, we unveiled a molecular pore complex that spans both membranes of the double-membrane vesicle and would allow export of RNA to the cytosol. A hexameric assembly of a large viral transmembrane protein was found to form the core of the crown-shaped complex. This coronavirus-specific structure likely plays a critical role in coronavirus replication and thus constitutes a novel drug target

microbiology↗

bioRxiv2020

Hsp70 chaperone blocks α-synuclein oligomer formation via a novel engagement mechanism

Over-expression and aggregation of -synuclein (ASyn) are linked to the onset and pathology of Parkinsons disease and related synucleinopathies. Elevated levels of the stress induced chaperone, Hsp70, protects against ASyn misfolding and ASyn-driven neurodegeneration in cell and animal models, yet there is minimal mechanistic understanding of this important protective pathway. It is generally assumed that Hsp70 binds to ASyn using its canonical and promiscuous substrate-binding cleft to limit aggregation. Here we report that this activity is due to a novel and unexpected mode of Hsp70 action, involving neither ATP nor the typical substrate-binding cleft. We use novel ASyn oligomerization assays to show that Hsp70 directly blocks ASyn oligomerization, an early event in ASyn misfolding. Using truncations, mutations and inhibitors, we confirmed that Hsp70 interacts with ASyn via an as yet unidentified, non-canonical interaction site in the C-terminal domain. Finally, a biological role for a non-canonical interaction was observed in H4 neuroglioma cells. Together, these findings suggest that new chemical approaches will be required to target Hsp70-ASyn interaction in synucleinopathies. Such approaches are likely to be more specific than targeting Hsp70 canonical actions. Additionally, these results raise the question of whether other misfolded proteins might also engage via the same non-canonical mechanism.

neuroscience↗

bioRxiv2019

Microtubules form by progressively faster tubulin accretion, not by nucleation-elongation

Microtubules are dynamic polymers that play fundamental roles in all eukaryotes. Despite their importance, how new microtubules form is poorly understood. Textbooks have focused on variations of a nucleation-elongation mechanism in which monomers rapidly equilibrate with an unstable oligomer (nucleus) that limits the rate of polymer formation; once formed, the polymer then elongates efficiently from this nucleus by monomer addition. Such models faithfully describe actin assembly, but they fail to account for how more complex polymers like hollow microtubules assemble. Here we articulate a new model for microtubule formation that has three key features: i) microtubules initiate via rectangular, sheet-like structures which grow faster the larger they become; ii) the dominant pathway proceeds via accretion, stepwise addition of longitudinal or lateral layers; iii) a straightening penalty to account for the energetic cost of tubulins curved-to-straight conformational transition. This model can quantitatively fit experimental assembly data, providing new insights into biochemical determinants and assembly pathways for microtubule nucleation.

cell biology↗

bioRxiv2018

The Atomic Structure of the Microtubule-Nucleating γ-Tubulin Small Complex and its Implications for Regulation

The microtubule cytoskeleton is essential in mediating a number of critical cellular processes, affecting cell shape, transport, organelle organization, and chromosomal segregation during mitosis. Microtubule network dynamics are controlled by many factors including the efficiency and localization of the nucleation machinery. Microtubule nucleation is dependent on the universally conserved {gamma}-tubulin small complex ({gamma}TuSC), a 300 kDa heterotetramer composed of two copies of {gamma}-tubulin and one each of accessory proteins GCP2 and GCP3. In yeast, nucleation is mediated by a heptameric ring of {gamma}TuSC, which presents 13 {gamma}-tubulins to form a template for microtubule nucleation.\n\nWe have obtained single-particle structures of the {gamma}TuSC as a monomer and dimer at resolutions of 3.6-4.6[A], allowing us to build an atomic model for this important complex. By comparison with a crystal structure of isolated {gamma}-tubulin, it is clear that {gamma}-tubulin is activated upon assembly into the {gamma}TuSC, in a manner analogous to the bent to straight transition in {beta}-tubulin upon assembly into the microtubule lattice. Our structures allow us to map phosphorylation sites, revealing several at key interfaces, highly suggestive of their role in regulating spindle pole body attachment and assembly into rings. When combined with previous lower resolution structures of helical assemblies, we observe that additional conformational changes occur during ring assembly and activation.

biophysics↗

bioRxiv2018

Spc110 N-Terminal Domains Act Independently to Mediate Stable γ-Tubulin Small Complex Binding and γ-Tubulin Ring Complex Assembly

Microtubule (MT) nucleation in vivo is regulated by the {gamma}-tubulin ring complex ({gamma}TuRC), an approximately 2-megadalton complex conserved from yeast to humans. In Saccharomyces cerevisiae, {gamma}TuRC assembly is a key point of regulation over the MT cytoskeleton. Budding yeast {gamma}TuRC is composed of seven {gamma}-tubulin small complex ({gamma}TuSC) subassemblies which associate helically to form a template from which microtubules grow. This assembly process requires higher-order oligomers of the coiled-coil protein Spc110 to bind multiple {gamma}TuSCs, thereby stabilizing the otherwise low-affinity interface between {gamma}TuSCs. While Spc110 oligomerization is critical, its N-terminal domain (NTD) also plays a role that is poorly understood both functionally and structurally. In this work, we sought a mechanistic understanding of Spc110 NTD using a combination of structural and biochemical analyses. Through crosslinking-mass spectrometry (XL-MS), we determined that a segment of Spc110 coiled-coil is a major point of contact with {gamma}TuSC. We determined the structure of this coiled-coil segment by X-ray crystallography and used it in combination with our XL-MS dataset to generate an integrative structural model of the {gamma}TuSC-Spc110 complex. This structural model, in combination with biochemical analyses of Spc110 heterodimers lacking one NTD, suggests that the two NTDs within an Spc110 dimer act independently, one stabilizing association between Spc110 and {gamma}TuSC and the other stabilizing the interface between adjacent {gamma}TuSCs.

biochemistry↗

bioRxiv2018

Mechanistic Origins of Dynamic Instability in Filaments from the Phage Tubulin, PhuZ

A bacteriophage-encoded tubulin homologue, PhuZ, harnesses dynamic instability to position genomes of KZ-like bacteriophage at the midline of their Pseudomonas hosts, facilitating phage infectivity. While much has been learned about molecular origins of microtubule dynamics, how GTP binding and hydrolysis control dynamics in the divergent 3-stranded PhuZ filaments is not understood. Here we present cryo-EM reconstructions of the PhuZ filamentin a pre-hydrolysis (3.5[A]) and three post-hydrolysis states (4.2 [A], 7.3 [A] and 8.1 [A] resolutions), likely representing distinct depolymerization stages. Core polymerization-induced structural changes reveal similarities to {beta}-tubulin, suggesting broad conservation within the tubulin family. By contrast, GTP hydrolysis is sensed quite differently and is communicated by the divergent PhuZ C-terminus to the lateral interface, leading to PhuZ polymer destabilization. This provides a contrasting molecular description of how nucleotide state can be harnessed by the tubulin fold to regulate filament assembly, metastability and disassembly.

cell biology↗

bioRxiv2018

Insights into centriole biogenesis and evolution revealed by cryoTomography of doublet and triplet centrioles.

Centrioles are cylindrical assemblies comprised of 9 singlet, doublet, or triplet microtubules, essential for the formation of motile and sensory cilia. While the structure of the cilium is being defined at increasing resolution, centriolar structure remains poorly understood. Here, we used electron cryo-tomography to determine the structure of mammalian (triplet) and Drosophila (doublet) centrioles at 25 [A] resolution. Mammalian centrioles have two distinct domains: a 200 nm proximal core region connected by A-C linkers, and a distal domain where the C-tubule is incomplete and a pair of novel linkages stabilize the assembly producing a geometry more closely resembling the ciliary axoneme. Drosophila centrioles more closely resemble the mammalian core, but with their doublets linked through A tubules. The commonality of core region length, and the abrupt transition in mammalian centrioles, suggests a conserved length-setting mechanism. The unexpected linker diversity suggests how unique centriolar architectures arise in different tissues and organisms.

cell biology↗

bioRxiv2018

Modulation of mitochondrial Hsp90 (TRAP1) ATPase activity by calcium and magnesium

The Hsp90 protein family are ATP-dependent molecular chaperones that maintain protein homeostasis and regulate many essential cellular processes. Higher eukaryotic cells have organelle-specific Hsp90 paralogs that are adapted to each unique sub-cellular environment. The mitochondrial Hsp90, TRAP1, supports the folding and activity of electron transport components and is increasingly being appreciated as a critical player in mitochondrial signaling. It is well known that calcium plays an important regulatory role in mitochondria and can even accumulate to much higher concentrations than in the cytoplasm. Surprisingly, we find that calcium can replace the requirement for magnesium to support TRAP1 ATPase activity. Using anomalous x-ray diffraction, we reveal a novel calcium-binding site within the TRAP1 nucleotide-binding pocket located near the ATP -phosphate and completely distinct from the magnesium site adjacent to the {beta} and {gamma}-phosphates. In the presence of magnesium, ATP hydrolysis by TRAP1, as with other Hsp90s, is non-cooperative, whereas calcium binding results in cooperative ATP hydrolysis by the two protomers within the Hsp90 dimer. The structural data suggest a mechanism for the cooperative behavior. Owing to the cooperativity, at high ATP concentrations, ATPase activity is higher with calcium, whereas the converse is true at low ATP concentrations. Integrating these observations, we propose a model where the divalent cations choice can control switching between non-cooperative and cooperative TRAP1 ATPase mechanisms in response ATP concentrations. This may facilitate coordination between cellular energetics, mitochondrial signaling, and protein homeostasis via alterations in the TRAP1 ATP-driven cycle.

biochemistry↗

bioRxiv2018

A simple and robust procedure for preparing graphene-oxide cryo-EM grids

Graphene oxide (GO) sheets have been used successfully as a supporting substrate film in several recent cryogenic electron-microscopy (cryo-EM) studies of challenging biological macromolecules. However, difficulties in preparing GO-covered holey carbon EM grids have limited its widespread use. Here, we report a simple and robust method for covering holey carbon EM grids with GO sheets and demonstrate that these grids are suitable for high-resolution single particle cryo-EM. GO substrates adhere macromolecules, allowing cryo-EM grid preparation with lower specimen concentrations and providing partial protection from the air-water interface. Additionally, the signal from images of the GO lattice beneath the frozen-hydrated specimen can be discerned in many motion-corrected micrographs, providing a high-resolution fiducial for evaluating beam-induced motion correction.

biophysics↗

bioRxiv2017

The centrosomin CM2 domain is a multi-functional binding domain with distinct cell cycle roles

The centrosome serves as the main microtubule-organizing center in metazoan cells, yet despite its functional importance, little is known mechanistically about the structure and organizational principles that dictate protein organization in the centrosome. In particular, the protein-protein interactions that allow for the massive structural transition between the tightly organized interphase centrosome and the highly expanded matrix-like arrangement of the mitotic centrosome have been largely uncharacterized. Among the proteins that undergo a major transition is the Drosophila melanogaster protein centrosomin that contains a conserved carboxyl terminus motif, CM2. Recent crystal structures have shown this motif to be dimeric and capable of forming an intramolecular interaction with a central region of centrosomin. Here we use a combination of in-cell microscopy and in vitro oligomer assessment to show that dimerization is not necessary for CM2 recruitment to the centrosome and that CM2 alone undergoes a significant cell cycle dependent rearrangement. We use NMR binding assays to confirm this intramolecular interaction and show that residues involved in solution interactions are consistent with the published crystal structure and identify L1137 as critical for binding. Additionally, we show for the first time an in vitro interaction of CM2 with the Drosophila pericentrin-like-protein that exploits the same set of residues as the intramolecular interaction. Furthermore, NMR experiments reveal a calcium sensitive interaction between CM2 and calmodulin. Although unexpected because of sequence divergence, this suggests that centrosomin-mediated assemblies, like the mammalian pericentrin, may be calcium regulated. From these results we suggest a model where during interphase CM2 interacts with pericentrin-like-protein to form a layer of centrosomin around the centriole wall and that at the onset of mitosis this population acts as a nucleation site of intramolecular centrosomin interactions that support the expansion into the metaphase matrix.

biochemistry↗

bioRxiv2017

Structural Basis of Mitochondrial Receptor Binding and GTP Driven Conformational Constriction by Dynamin-Related Protein 1

Mitochondrial inheritance, genome maintenance, and metabolic adaptation all depend on organelle fission by Dynamin-Related Protein 1 (DRP1) and its mitochondrial receptors. DRP1 receptors include the paralogs Mitochondrial Dynamics 49 and 51 (MID49/MID51) and Mitochondrial Fission Factor (MFF), but the mechanisms by which these proteins recruit DRP1 and regulate its activities are unknown. Here we present a cryoEM structure of human, full-length DRP1 bound to MID49 and an analysis of structure- and disease-based mutations. We report that GTP binding allosterically induces a remarkable elongation and rotation of the G-domain, Bundle-Signaling Element (BSE) and connecting hinge loops of DRP1. In this nucleotide-bound conformation, a distributed network of multivalent interactions promotes DRP1 copolymerization into a linear filament with MID49, MID51 or both. Subsequent GTP hydrolysis and exchange within the filament leads to receptor dissociation, shortening through disassembly, and concomitant curling of DRP1 oligomers into closed rings. The dimensions of the closed DRP1 rings are consistent with DRP1-constricted mitochondrial tubules observed in human cells. These structures are the first views of full-length, receptor- and nucleotide-bound dynamin-family GTPases and--in comparison with nucleotide-free crystal structures--teach us how these molecular machines perform mechanical work through nucleotide-driven allostery.

biophysics↗