Biology subjects

Kotecha, A.

Publications and source records attributed to Kotecha, A..

28 records · Page 2Linked to original sources

bioRxiv2021

Assembly of recombinant tau into filaments identical to those of Alzheimer's disease and chronic traumatic encephalopathy

Abundant filamentous inclusions of tau are characteristic of more than 20 neurodegenerative diseases that are collectively termed tauopathies. Electron cryo-microscopy (cryo-EM) structures of tau amyloid filaments from human brain revealed that distinct tau folds characterise many different diseases. A lack of laboratory-based model systems to generate these structures has hampered efforts to uncover the molecular mechanisms that underlie tauopathies. Here, we report in vitro assembly conditions with recombinant tau that replicate the structures of filaments from both Alzheimers disease (AD) and chronic traumatic encephalopathy (CTE), as determined by cryo-EM. Our results suggest that post-translational modifications of tau modulate filament assembly, and that previously observed additional densities in AD and CTE filaments may arise from the presence of inorganic salts, like phosphates and sodium chloride. In vitro assembly of tau into disease-relevant filaments will facilitate studies to determine their roles in different diseases, as well as the development of compounds that specifically bind to these structures or prevent their formation.

neuroscience↗

bioRxiv2021

Age-dependent formation of TMEM106B amyloid filaments in human brain

Many age-dependent neurodegenerative diseases, like Alzheimers and Parkinsons, are characterised by abundant inclusions of amyloid filaments. Filamentous inclusions of the proteins tau, amyloid-{beta} (A{beta}), -synuclein and TDP-43 are the most common. Here, we used electron cryo-microscopy (cryo-EM) structure determination to show that residues 120-254 of the lysosomal type II transmembrane protein 106B (TMEM106B) also form amyloid filaments in the human brain. We solved cryo-EM structures of TMEM106B filaments from the brains of 22 individuals with neurodegenerative conditions, including sporadic and inherited tauopathies, A{beta}-amyloidoses, synucleinopathies and TDP-43opathies, as well as from the brains of two neurologically normal individuals. We observed three different TMEM106B folds, with no clear relationship between folds and diseases. The presence of TMEM106B filaments correlated with that of a 29 kDa sarkosyl-insoluble fragment of the protein on Western blots. The presence of TMEM106B filaments in the brains of older, but not younger, neurologically normal individuals indicates that they form in an age-dependent manner.

neuroscience↗

bioRxiv2021

Cryo-EM Structures of Amyloid-β 42 Filaments from Human Brain

Filament assembly of amyloid-{beta} peptides ending at residue 42 (A{beta}42) is a central event in Alzheimers disease. We report the cryo-EM structures of A{beta}42 filaments from brain. Two structurally related S-shaped protofilament folds give rise to two types of filaments. Type I filaments were found mostly in the brains of individuals with sporadic Alzheimers disease and Type II filaments in individuals with familial Alzheimers disease and other conditions. The structures of A{beta}42 filaments from brain differ from those of filaments assembled in vitro. By contrast, in AppNL-F knock-in mice, A{beta}42 deposits were made of Type II filaments. Knowledge of A{beta}42 filament structures from human brain may lead to the development of inhibitors of assembly and improved imaging agents.

neuroscience↗

bioRxiv2021

Structure-based Classification of Tauopathies

Ordered assembly of the tau protein into filaments characterizes multiple neurodegenerative diseases, which are called tauopathies. We previously reported that by electron cryo-microscopy (cryo-EM), tau filament structures from Alzheimers disease (1,2), chronic traumatic encephalopathy (CTE) (3), Picks disease (4) and corticobasal degeneration (CBD) (5) are distinct. Here we show that the structures of tau filaments from typical and atypical progressive supranuclear palsy (PSP), the most common tauopathy after Alzheimers disease, define a previously unknown, three-layered fold. Moreover, the tau filament structures from globular glial tauopathy (GGT, Types I and II) are similar to those from PSP. The tau filament fold of argyrophilic grain disease (AGD) differs from the above and resembles the four-layered CBD fold. The majority of tau filaments from aging-related tau astrogliopathy (ARTAG) also have the AGD fold. Surprisingly, tau protofilament structures from inherited cases with mutations +3/+16 in intron 10 of MAPT, the microtubule-associated protein tau gene, are identical to those from AGD, suggesting that a relative overproduction of four-repeat tau can give rise to the AGD fold. Finally, tau filament structures from cases of familial British dementia (FBD) and familial Danish dementia (FDD) are the same as those from Alzheimers disease and primary age-related tauopathy (PART). These structures provide the basis for a classification of tauopathies that also allows identification of new entities, as we show here for a case diagnosed as PSP, but with abundant spherical 4R tau inclusions in limbic and other brain areas. The structures of the tau fold of this new disease (Limbic-predominant Neuronal inclusion body 4R Tauopathy, LNT) were intermediate between those of GGT and PSP.

neuroscience↗

bioRxiv2021

Evolving cryo-EM structural approaches for GPCR drug discovery

G protein-coupled receptors (GPCRs) are the largest class of cell surface drug targets. Advances in biochemical approaches for the stabilisation of GPCR:transducer complexes together with improvements in the technology and application of cryo-EM has recently opened up new possibilities for structure-assisted drug design of GPCR agonists. Nonetheless, limitations in the commercial application of some of these approaches, including the use of nanobody 35 (Nb35) for stabilisation of GPCR:Gs complexes, and the high cost of 300kV imaging have restricted broad application of cryo-EM in drug discovery. Here, using the PF 06882961-bound GLP-1R as exemplar, we validated formation of stable complexes with a modified Gs protein in the absence of Nb35 that had equivalent resolution in the drug binding pocket to complexes solved in the presence of Nb35, while the G protein displayed increased conformational dynamics. In parallel, we assessed the performance of 200kV versus 300kV image acquisition using a Falcon 4 or K3 direct electron detector. We show that with 300kV Krios, both bottom mounted Falcon 4 and energy filtered (25eV slit) Bio-Quantum K3 produced similar resolution. Moreover, the 200kV Glacios with bottom mounted Falcon 4 yielded a 3.2 [A] map with clear density for bound drug and multiple structurally ordered waters. Our work paves the way for broader commercial application of cryo-EM for GPCR drug discovery.

biochemistry↗

bioRxiv2020

CryoET structures of immature HIV Gag reveal a complete six-helix bundle and stabilizing small molecules distinct from IP6

Gag is the major HIV-1 structural polyprotein precursor. The Gag SP1 domain with the last residues of CA have been hypothesized to form a six-helix bundle necessary for particle assembly, but this bundle has not been fully resolved. Here, we determined the structures of complete CA-SP1 six-helix bundle connecting to the NC domain, from both in vitro Gag assemblies and viral-like particles (VLPs) carrying a T8I mutation in SP1, to near-atomic resolutions using cryoET and subtomogram averaging. The structures revealed novel densities, however distinct from IP6, inside the six-helix bundle of Gag assemblies, stabilizing the immature lattice. Interestingly, the T8I mutation impaired proteolytic cleavage of Gag at both SP1 boundaries. Our findings signify the involvement of small molecules in immature Gag assembly and provide the structural basis for development of small molecule inhibitors that stabilize SP1 helix, thus interfere with PR-mediated virus maturation.

microbiology↗

bioRxiv2020

Assembly intermediates of orthoreovirus captured in the cell

Traditionally molecular assembly pathways for viruses have been inferred from high resolution structures of stable intermediates purified in vitro, and from low resolution images of cell sections as well as from genetic approaches including conditionally lethal mutants. Here, we directly visualise a previously unsuspected single shelled icosahedral intermediate for a mammalian orthoreovirus, in addition to the expected virions, in cryo-preserved infected cells by cryo-electron tomography of cellular lamellae1,2. Particle classification and averaging yielded structures at resolutions as high as 5.6 [A], sufficient to identify secondary structural elements and place known molecular structures, allowing us to produce an atomic model of the intermediate, comprising 120 copies of protein {lambda}1 and 120 copies of {sigma}2. This {lambda}1 shell is in a collapsed form compared to the mature virions, with the molecules pushed inwards at the icosahedral 5-folds by ~100 [A]. This grossly indented shell, although produced by a mammalian reovirus, is reminiscent of the first assembly intermediate of prokaryotic dsRNA viruses belonging to a different virus family3, adding weight to the supposition that these diverse viruses share a common ancestor, and suggesting mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds enormous promise for the dissection of the replication cycle of many viruses.

microbiology↗

bioRxiv2020

A high resolution view of an adolescent flavivirus

Mosquito-transmitted flaviviruses, such as Dengue virus (DENV) or Zika virus (ZIKV), are responsible for significant economic damage and human misery. In infected cells, flaviviruses first assemble into an immature form within the endoplasmatic reticulum (ER), and then undergo further processing by furin protease in the trans-Golgi. Despite substantial efforts, previous cryogenic electron microscopy (cryo-EM) studies of immature flaviviruses were restricted to low to medium resolutions, limiting our understanding of maturation. To better grasp the process of maturation, we have carried out cryo-EM reconstructions of immature Spondweni virus (SPOV), an emerging human flavivirus belonging to the same serogroup as ZIKV (~75% amino acid identity). By combining localized reconstruction and focused refinement, we were able to improve the resolution to 3.8 [A], yielding unprecedented insight into the immature form. The structure elucidates how, at neutral pH, polar interactions conceal the furin recognition site within trimeric envelope (E) protein spikes. Furthermore, we identify how a strictly conserved pH sensor anchors the precursor membrane (prM) protein to immature E. We reconstructed mature forms of SPONV and DENV to 2.6[A] and 3.1[A], respectively. Comparison with immature virus shows a conserved binding pocket for a lipid headgroup, which forms as a consequence of the rearrangement of amphipathic stem-helices of E. We propose a structural role for the pocket and suggest it stabilizes mature E. Taken together, our data suggest a compelling rationale for low-pH triggered conformational rearrangement in the Golgi, which occurs during flavivirus maturation.

microbiology↗

bioRxiv2020

Single-particle cryo-EM at atomic resolution

The three-dimensional positions of atoms in protein molecules define their structure and provide mechanistic insights into the roles they perform in complex biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more knowledge about protein function may be inferred. With breakthroughs in electron detection and image processing technology, electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years1,2. However, obtaining cryo-EM reconstructions with sufficient resolution to visualise individual atoms in proteins has thus far been elusive. Here, we show that using a new electron source, energy filter and camera, we obtained a 1.7 [A] resolution cryo-EM reconstruction for a prototypical human membrane protein, the {beta}3 GABAA receptor homopentamer3. Such maps allow a detailed understanding of small molecule coordination, visualisation of solvent molecules and alternative conformations for multiple amino acids, as well as unambiguous building of ordered acidic side chains and glycans. Applied to mouse apo-ferritin, our strategy led to a 1.2 [A] resolution reconstruction that, for the first time, offers a genuine atomic resolution view of a protein molecule using single particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualised in difference maps, allowing a direct analysis of hydrogen bonding networks. Combination of the technological advances described here with further approaches to accelerate data acquisition and improve sample quality provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.

biophysics↗

bioRxiv2018

Heteromeric GABAA receptor structures in positively-modulated active states

Type-A {gamma}-aminobutyric acid (GABAA) receptors are pentameric ligand-gated ion channels (pLGICs), typically consisting of /{beta}/{gamma} subunit combinations. They are the principal mediators of inhibitory neurotransmission throughout the central nervous system and targets of major clinical drugs, such as benzodiazepines (BZDs) used to treat epilepsy, insomnia, anxiety, panic disorder and muscle spasm. However, the structures of heteromeric receptors and the molecular basis of BZD operation remain unknown. Here we report the cryo-EM structure of a human 1{beta}3{gamma}2 GABAAR in complex with GABA and a nanobody that acts as a novel positive allosteric modulator (PAM). The receptor subunits assume a unified quaternary activated conformation around an open pore. We also present crystal structures of engineered 5 and 5{gamma}2 GABAAR constructs, revealing the interfacial site for allosteric modulation by BZDs, including the binding modes and the conformational impact of the potent anxiolytic and partial PAM, bretazenil, and the BZD antagonist, flumazenil. These findings provide the foundation for understanding the mechanistic basis of GABAAR activation.

biophysics↗