The Structure of Sulfoindocarbocyanine 3 Terminally Attached to dsDNA via a Long, Flexible Tether

AbstractFluorescence resonance energy transfer (FRET) is an important source of long-range distance information in macromolecules. However, extracting maximum information requires knowledge of fluorophore, donor and acceptor, positions on the macromolecule. We previously determined the structure of the indocarbocyanine fluorophores Cy3 and Cy5 attached to DNA via three-carbon atom tethers, showing that they stacked onto the end of the helix in a manner similar to an additional basepair. Our recent FRET study has suggested that when they are attached via a longer 13-atom tether, these fluorophores are repositioned relative to the terminal basepair by a rotation of ∼30°, while remaining stacked. In this study, we have used NMR to extend our structural understanding to the commonly used fluorophore sulfoindocarbocyanine-3 (sCy3) attached to the 5′-terminus of the double-helical DNA via a 13-atom flexible tether (L13). We find that L13-sCy3 remains predominantly stacked onto the end of the duplex, but adopts a significantly different conformation, from that of either Cy3 or Cy5 attached by 3-atom tethers, with the long axes of the fluorophore and the terminal basepair approximately parallel. This result is in close agreement with our FRET data, supporting the contention that FRET data can be used to provide orientational information

MZT Proteins Form Multi-Faceted Structural Modules in the γ-Tubulin Ring Complex

Microtubule organization depends on the g-tubulin ring complex (g-TuRC), a 2.3-MDa nucleation factor comprising an asymmetric assembly of g-tubulin and GCP2-GCP6. However, it is currently unclear how the g-TuRC-associated microproteins MZT1 and MZT2 contribute to the structure and regulation of the holocomplex. Here, we report cryo-EM structures of MZT1 and MZT2 in the context of the native human g-TuRC. MZT1 forms two subcomplexes with the N-terminal a-helical domains of GCP3 or GCP6 (GCP-NHDs) within the g-TuRC ‘‘lumenal bridge.’’ We determine the X-ray structure of recombinant MZT1/GCP6-NHD and find it is similar to that within the native g-TuRC. We identify two additional MZT/GCP-NHD-like subcomplexes, one of which is located on the outer face of the g-TuRC and comprises MZT2 and GCP2-NHD in complex with a centrosomin motif 1 (CM1)-containing peptide. Our data reveal how MZT1 and MZT2 establish multi-faceted, structurally mimetic ‘‘modules’’ that can expand structural and regulatory interfaces in the g-TuRC.ISSN:2666-3864ISSN:2211-124

The cryoEM structure of the S<i>accharomyces cerevisiae</i> ribosome maturation factor Rea1

The biogenesis of 60S ribosomal subunits is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly factors. The ~5000 amino-acid AAA+ ATPase Rea1 (or Midasin) generates force to mechanically remove assembly factors from pre-60S particles, which promotes their export to the cytosol. Here we present three Rea1 cryoEM structures. We visualise the Rea1 engine, a hexameric ring of AAA+ domains, and identify an α-helical bundle of AAA2 as a major ATPase activity regulator. The α-helical bundle interferes with nucleotide-induced conformational changes that create a docking site for the substrate binding MIDAS domain on the AAA +ring. Furthermore, we reveal the architecture of the Rea1 linker, which is involved in force generation and extends from the AAA+ ring. The data presented here provide insights into the mechanism of one of the most complex ribosome maturation factors

Asymmetric molecular architecture of the human <i>γ</i>-tubulin ring complex

SUMMARYThe γ-tubulin ring complex (γ-TuRC) is an essential regulator of centrosomal and acentrosomal microtubule formation 1–4. Metazoan γ-TuRCs isolate as ∼2 MDa complexes containing the conserved proteins γ-tubulin, GCP2 and GCP3, as well as the expanded subunits GCP4, GCP5, and GCP6 3,5,6. However, in current structural models, γ-TuRCs assemble solely from subcomplexes of γ-tubulin, GCP2 and GCP3 7. The role of the metazoan-specific subunits in γ-TuRC assembly and architecture are not currently known, due to a lack of high resolution structural data for the native complex. Here, we present a cryo-EM structure of the native human γ-TuRC at 3.8Å resolution. Our reconstruction reveals an asymmetric, single helical-turn and cone-shaped structure built from at least 34 polypeptides. Pseudo-atomic models indicate that GCP4, GCP5 and GCP6 form distinct Y-shaped assemblies that structurally mimic GCP2/GCP3 subcomplexes and are distal to the γ-TuRC “seam”. Evolutionary expansion in metazoan-specific subunits diversifies the γ-TuRC by introducing large (&gt;100,000 Å2) surfaces that could interact with different regulatory factors. We also identify an unanticipated structural bridge that includes an actin-like protein and spans the γ-TuRC lumen. Despite its asymmetric composition and architecture, the human γ-TuRC arranges γ-tubulins into a helical geometry poised to nucleate microtubules. The observed compositional complexity of the γ-TuRC could self-regulate its assembly into a cone-shaped structure to control microtubule formation across diverse contexts, e.g. within biological condensates 8 or alongside existing filaments 9.</jats:p

Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function

The formation of cellular microtubule networks is regulated by the γ-tubulin ring complex (γ-TuRC). This ∼2.3 MD assembly of >31 proteins includes γ-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a “lumenal bridge” (LB). The challenge of reconstituting the γ-TuRC has limited dissections of its assembly and function. Here, we report a biochemical reconstitution of the human γ-TuRC (γ-TuRC-GFP) as a ∼35 S complex that nucleates microtubules in vitro. In addition, we generate a subcomplex, γ-TuRCΔLB-GFP, which lacks MZT1 and actin. We show that γ-TuRCΔLB-GFP nucleates microtubules in a guanine nucleotide–dependent manner and with similar efficiency as the holocomplex. Electron microscopy reveals that γ-TuRC-GFP resembles the native γ-TuRC architecture, while γ-TuRCΔLB-GFP adopts a partial cone shape presenting only 8–10 γ-tubulin subunits and lacks a well-ordered lumenal bridge. Our results show that the γ-TuRC can be reconstituted using a limited set of proteins and suggest that the LB facilitates the self-assembly of regulatory interfaces around a microtubulenucleating “core” in the holocomplex.ISSN:0021-9525ISSN:1540-814