Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice and is ...inhibited by anticancer agents like Taxol. We provide insight into the mechanism of dynamic instability, based on high-resolution cryo-EM structures (4.7–5.6 Å) of dynamic microtubules and microtubules stabilized by GMPCPP or Taxol. We infer that hydrolysis leads to a compaction around the E-site nucleotide at longitudinal interfaces, as well as movement of the α-tubulin intermediate domain and H7 helix. Displacement of the C-terminal helices in both α- and β-tubulin subunits suggests an effect on interactions with binding partners that contact this region. Taxol inhibits most of these conformational changes, allosterically inducing a GMPCPP-like state. Lateral interactions are similar in all conditions we examined, suggesting that microtubule lattice stability is primarily modulated at longitudinal interfaces.
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•Microtubule structures at 5 Å illustrate the effect of hydrolysis and drug binding•The longitudinal interface by the E site nucleotide compacts upon GTP hydrolysis•The intermediate domain and helix H7 of α-tubulin move, generating strain in the dimer•Taxol inhibits these changes, allosterically inducing a GTP-like state
GTP hydrolysis leads to a compaction around the E site nucleotide at longitudinal interfaces and displacement of regions involved in microtubule partner interactions. These data suggest that microtubule lattice stability is primarily modulated at longitudinal interfaces.
Near-atomic model of microtubule-tau interactions Kellogg, Elizabeth H; Hejab, Nisreen M A; Poepsel, Simon ...
Science (American Association for the Advancement of Science),
06/2018, Letnik:
360, Številka:
6394
Journal Article
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Tau is a developmentally regulated axonal protein that stabilizes and bundles microtubules (MTs). Its hyperphosphorylation is thought to cause detachment from MTs and subsequent aggregation into ...fibrils implicated in Alzheimer's disease. It is unclear which tau residues are crucial for tau-MT interactions, where tau binds on MTs, and how it stabilizes them. We used cryo-electron microscopy to visualize different tau constructs on MTs and computational approaches to generate atomic models of tau-tubulin interactions. The conserved tubulin-binding repeats within tau adopt similar extended structures along the crest of the protofilament, stabilizing the interface between tubulin dimers. Our structures explain the effect of phosphorylation on MT affinity and lead to a model of tau repeats binding in tandem along protofilaments, tethering together tubulin dimers and stabilizing polymerization interfaces.
Cryo-electron microscopy (cryo-EM) has enjoyed explosive recent growth due to revolutionary advances in hardware and software, resulting in a steady stream of long-awaited, high-resolution structures ...with unprecedented atomic detail. With this comes an increased number of microscopes, cryo-EM facilities, and scientists eager to leverage the ability to determine protein structures without crystallization. However, numerous pitfalls and considerations beset the path toward high-resolution structures and are not necessarily obvious from literature surveys. Here, we detail the most common misconceptions when initiating a cryo-EM project and common technical hurdles, as well as their solutions, and we conclude with a vision for the future of this exciting field.
CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism
. CAST elements contain multiple conserved proteins: a CRISPR ...effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA-TnsB) and a target-site-associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstituted the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined its structure using single-particle cryo-electon microscopy. The architecture of this transpososome reveals modular association between the components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Of note, we observe TnsC-TnsB interactions at the C-terminal face of TnsC, which contribute to the stimulation of ATPase activity. Although the TnsC oligomeric assembly deviates slightly from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs markedly in the transpososome. As a consequence, TnsC makes new protein-DNA interactions throughout the transpososome that are important for transposition activity. Finally, we identify two distinct transpososome populations that differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This ShCAST transpososome structure enhances our understanding of CAST transposition systems and suggests ways to improve CAST transposition for precision genome-editing applications.
We present a large-scale approach to investigate the functional consequences of sequence variation in a protein. The approach entails the display of hundreds of thousands of protein variants, ...moderate selection for activity and high-throughput DNA sequencing to quantify the performance of each variant. Using this strategy, we tracked the performance of >600,000 variants of a human WW domain after three and six rounds of selection by phage display for binding to its peptide ligand. Binding properties of these variants defined a high-resolution map of mutational preference across the WW domain; each position had unique features that could not be captured by a few representative mutations. Our approach could be applied to many in vitro or in vivo protein assays, providing a general means for understanding how protein function relates to sequence.
Deciphering the relationship between a gene and its genomic context is fundamental to understanding and engineering biological systems. Machine learning has shown promise in learning latent ...relationships underlying the sequence-structure-function paradigm from massive protein sequence datasets. However, to date, limited attempts have been made in extending this continuum to include higher order genomic context information. Evolutionary processes dictate the specificity of genomic contexts in which a gene is found across phylogenetic distances, and these emergent genomic patterns can be leveraged to uncover functional relationships between gene products. Here, we train a genomic language model (gLM) on millions of metagenomic scaffolds to learn the latent functional and regulatory relationships between genes. gLM learns contextualized protein embeddings that capture the genomic context as well as the protein sequence itself, and encode biologically meaningful and functionally relevant information (e.g. enzymatic function, taxonomy). Our analysis of the attention patterns demonstrates that gLM is learning co-regulated functional modules (i.e. operons). Our findings illustrate that gLM's unsupervised deep learning of the metagenomic corpus is an effective and promising approach to encode functional semantics and regulatory syntax of genes in their genomic contexts and uncover complex relationships between genes in a genomic region.
•Technical progress leads to high-resolution cryo-EM structures of microtubules (MTs).•Atomic details of MT-binding proteins and antimitotic drugs bound to MTs.•Characterization of the MT seam: a ...possible target of MT stability regulation.•Plasticity of structural modules in the binding of MTs with their cellular partners.•Cryo-EM of MTs shows high potential for drug development and improvement.
As non-crystallizable polymers, microtubules have been the target of cryo-electron microscopy (cryo-EM) studies since the technique was first established. Over the years, image processing strategies have been developed that take care of the unique, pseudo-helical symmetry of the microtubule. With recent progress in data quality and data processing, cryo-EM reconstructions are now reaching resolutions that allow the generation of atomic models of microtubules and the factors that bind them. These include cellular partners that contribute to microtubule cellular functions, or small ligands that interfere with those functions in the treatment of cancer. The stage is set to generate a family portrait for all identified microtubule interacting proteins and to use cryo-EM as a drug development tool in the targeting of tubulin.
A number of microtubule (MT)-stabilizing agents (MSAs) have demonstrated or predicted potential as anticancer agents, but a detailed structural basis for their mechanism of action is still lacking. ...We have obtained high-resolution (3.9–4.2Å) cryo-electron microscopy (cryo-EM) reconstructions of MTs stabilized by the taxane-site binders Taxol and zampanolide, and by peloruside, which targets a distinct, non-taxoid pocket on β-tubulin. We find that each molecule has unique distinct structural effects on the MT lattice structure. Peloruside acts primarily at lateral contacts and has an effect on the “seam” of heterologous interactions, enforcing a conformation more similar to that of homologous (i.e., non-seam) contacts by which it regularizes the MT lattice. In contrast, binding of either Taxol or zampanolide induces MT heterogeneity. In doubly bound MTs, peloruside overrides the heterogeneity induced by Taxol binding. Our structural analysis illustrates distinct mechanisms of these drugs for stabilizing the MT lattice and is of relevance to the possible use of combinations of MSAs to regulate MT activity and improve therapeutic potential.
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•High-resolution cryo-EM reconstructions of diverse MSAs•Taxane-binding results in MT structural heterogeneity.•Peloruside binding regularizes the MT lattice.•Peloruside has an overriding effect when combined with Taxol.
Microtubules are polymers of αβ-tubulin heterodimers essential for all eukaryotes. Despite sequence conservation, there are significant structural differences between microtubules assembled in vitro ...from mammalian or budding yeast tubulin. Yeast MTs were not observed to undergo compaction at the interdimer interface as seen for mammalian microtubules upon GTP hydrolysis. Lack of compaction might reflect slower GTP hydrolysis or a different degree of allosteric coupling in the lattice. The microtubule plus end-tracking protein Bim1 binds yeast microtubules both between αβ-tubulin heterodimers, as seen for other organisms, and within tubulin dimers, but binds mammalian tubulin only at interdimer contacts. At the concentrations used in cryo-electron microscopy, Bim1 causes the compaction of yeast microtubules and induces their rapid disassembly. Our studies demonstrate structural differences between yeast and mammalian microtubules that likely underlie their differing polymerization dynamics. These differences may reflect adaptations to the demands of different cell size or range of physiological growth temperatures.
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