VASH1-SVBP and VASH2-SVBP generate different detyrosination profiles on microtubules.

The detyrosination/tyrosination cycle of α-tubulin is critical for proper cell functioning. VASH1-SVBP and VASH2-SVBP are ubiquitous enzymes involved in microtubule detyrosination, whose mode of action is little known. Here, we show in reconstituted systems and cells that VASH1-SVBP and VASH2-SVBP drive the global and local detyrosination of microtubules, respectively. We solved the cryo-electron microscopy structure of VASH2-SVBP bound to microtubules, revealing a different microtubule-binding configuration of its central catalytic region compared to VASH1-SVBP. We show that the divergent mode of detyrosination between the two enzymes is correlated with the microtubule-binding properties of their disordered N- and C-terminal regions. Specifically, the N-terminal region is responsible for a significantly longer residence time of VASH2-SVBP on microtubules compared to VASH1-SVBP. We suggest that this VASH region is critical for microtubule detachment and diffusion of VASH-SVBP enzymes on lattices. Our results suggest a mechanism by which VASH1-SVBP and VASH2-SVBP could generate distinct microtubule subpopulations and confined areas of detyrosinated lattices to drive various microtubule-based cellular functions.

Cryptosporulation in Kurthia spp. forces a rethinking of asporogenesis in Firmicutes.

Endosporulation is a complex morphophysiological process resulting in a more resistant cellular structure that is produced within the mother cell and is called endospore. Endosporulation evolved in the common ancestor of Firmicutes, but it is lost in descendant lineages classified as asporogenic. While Kurthia spp. is considered to comprise only asporogenic species, we show here that strain 11kri321, which was isolated from an oligotrophic geothermal reservoir, produces phase-bright spore-like structures. Phylogenomics of strain 11kri321 and other Kurthia strains reveals little similarity to genetic determinants of sporulation known from endosporulating Bacilli. However, morphological hallmarks of endosporulation were observed in two of the four Kurthia strains tested, resulting in spore-like structures (cryptospores). In contrast to classic endospores, these cryptospores did not protect against heat or UV damage and successive sub-culturing led to the loss of the cryptosporulating phenotype. Our findings imply that a cryptosporulation phenotype may have been prevalent and subsequently lost by laboratory culturing in other Firmicutes currently considered as asporogenic. Cryptosporulation might thus represent an ancestral but unstable and adaptive developmental state in Firmicutes that is under selection under harsh environmental conditions.

Rational Design of a Novel Tubulin Inhibitor with a Unique Mechanism of Action.

In this study, we capitalized on our previously performed crystallographic fragment screen and developed the antitubulin small molecule Todalam with only two rounds of straightforward chemical synthesis. Todalam binds to a novel tubulin site, disrupts microtubule networks in cells, arrests cells in G2/M, induces cell death, and synergizes with vinblastine. The compound destabilizes microtubules by acting as a molecular plug that sterically inhibits the curved-to-straight conformational switch in the α-tubulin subunit, and by sequestering tubulin dimers into assembly incompetent oligomers. Our results describe for the first time the generation of a fully rationally designed small molecule tubulin inhibitor from a fragment, which displays a unique molecular mechanism of action. They thus demonstrate the usefulness of tubulin-binding fragments as valuable starting points for innovative antitubulin drug and chemical probe discovery campaigns.

Cryo-electron Microscopy Imaging of Alzheimer's Amyloid-beta 42 Oligomer Displayed on a Functionally and Structurally Relevant Scaffold.

Amyloid-ß peptide (Aß) oligomers are pathogenic species of amyloid aggregates in Alzheimer's disease. Like certain protein toxins, Aß oligomers permeabilize cellular membranes, presumably through a pore formation mechanism. Owing to their structural and stoichiometric heterogeneity, the structure of these pores remains to be characterized. We studied a functional Aß42-pore equivalent, created by fusing Aß42 to the oligomerizing, soluble domain of the α-hemolysin (αHL) toxin. Our data reveal Aß42-αHL oligomers to share major structural, functional, and biological properties with wild-type Aß42-pores. Single-particle cryo-EM analysis of Aß42-αHL oligomers (with an overall 3.3 Šresolution) reveals the Aß42-pore region to be intrinsically flexible. The Aß42-αHL oligomers will allow many of the features of the wild-type amyloid oligomers to be studied that cannot be otherwise, and may be a highly specific antigen for the development of immuno-base diagnostics and therapies.

Statistically correcting dynamical electron scattering improves the refinement of protein nanocrystals, including charge refinement of coordinated metals.

Electron diffraction allows protein structure determination when only nanosized crystals are available. Nevertheless, multiple elastic (or dynamical) scattering, which is prominent in electron diffraction, is a concern. Current methods for modeling dynamical scattering by multi-slice or Bloch wave approaches are not suitable for protein crystals because they are not designed to cope with large molecules. Here, dynamical scattering of nanocrystals of insulin, thermolysin and thaumatin was limited by collecting data from thin crystals. To accurately measure the weak diffraction signal from the few unit cells in the thin crystals, a low-noise hybrid pixel Timepix electron-counting detector was used. The remaining dynamical component was further reduced in refinement using a likelihood-based correction, which was introduced previously for analyzing electron diffraction data of small-molecule nanocrystals and was adapted here for protein crystals. The procedure is shown to notably improve the structural refinement, in one case allowing the location of solvent molecules. It also allowed refinement of the charge states of bound metal atoms, an important element in protein function, through B-factor analysis of the metal atoms and their ligands. These results clearly increase the value of macromolecular electron crystallography as a complementary structural biology technique.

Shape-Shifting Peptide Nanomaterials: Surface Asymmetry Enables pH-Dependent Formation and Interconversion of Collagen Tubes and Sheets.

The fabrication of dynamic, transformable biomaterials that respond to environmental cues represents a significant step forward in the development of synthetic materials that rival their highly functional, natural counterparts. Here, we describe the design and synthesis of crystalline supramolecular architectures from charge-complementary heteromeric pairs of collagen-mimetic peptides (CMPs). Under appropriate conditions, CMP pairs spontaneously assemble into either 1D ultraporous (pore diameter >100 nm) tubes or 2D bilayer nanosheets due to the structural asymmetry that arises from heteromeric self-association. Crystalline collagen tubes represent a heretofore unobserved morphology of this common biomaterial. In-depth structural characterization from a suite of biophysical methods, including TEM, AFM, high-resolution cryo-EM, and SAXS/WAXS measurements, reveals that the sheet and tube assemblies possess a similar underlying lattice structure. The experimental evidence suggests that the tubular structures are a consequence of the self-scrolling of incipient 2D layers of collagen triple helices and that the scrolling direction determines the formation of two distinct structural isoforms. Furthermore, we show that nanosheets and tubes can spontaneously interconvert through manipulation of the assembly pH and systematic adjustment of the CMP sequence. Altogether, we establish initial guidelines for the construction of dynamically responsive 1D and 2D assemblies that undergo a structurally programmed morphological transition.

A lipocalin mediates unidirectional heme biomineralization in malaria parasites.

During blood-stage development, malaria parasites are challenged with the detoxification of enormous amounts of heme released during the proteolytic catabolism of erythrocytic hemoglobin. They tackle this problem by sequestering heme into bioinert crystals known as hemozoin. The mechanisms underlying this biomineralization process remain enigmatic. Here, we demonstrate that both rodent and human malaria parasite species secrete and internalize a lipocalin-like protein, PV5, to control heme crystallization. Transcriptional deregulation of PV5 in the rodent parasite Plasmodium berghei results in inordinate elongation of hemozoin crystals, while conditional PV5 inactivation in the human malaria agent Plasmodium falciparum causes excessive multidirectional crystal branching. Although hemoglobin processing remains unaffected, PV5-deficient parasites generate less hemozoin. Electron diffraction analysis indicates that despite the distinct changes in crystal morphology, neither the crystalline order nor unit cell of hemozoin are affected by impaired PV5 function. Deregulation of PV5 expression renders P. berghei hypersensitive to the antimalarial drugs artesunate, chloroquine, and atovaquone, resulting in accelerated parasite clearance following drug treatment in vivo. Together, our findings demonstrate the Plasmodium-tailored role of a lipocalin family member in hemozoin formation and underscore the heme biomineralization pathway as an attractive target for therapeutic exploitation.

Seeded Heteroepitaxial Growth of Crystallizable Collagen Triple Helices: Engineering Multifunctional Two-Dimensional Core-Shell Nanostructures.

Engineering free-standing 2D nanomaterials with compositional, spatial, and functional control across size regimes from the nano- to mesoscale represents a significant challenge. Herein, we demonstrate a straightforward strategy for the thermodynamically controlled fabrication of multicomponent sectored nanosheets in which each sector can be chemically and spatially addressed independently and orthogonally. Collagen triple helices, comprising collagen-mimetic peptides (CMPs), are employed as molecularly programmable crystallizable units. Modulating their thermodynamic stability affords the controlled synthesis of 2D core-shell nanostructures via thermally driven heteroepitaxial growth. Structural information, gathered from SAXS and cryo-TEM, reveals that the distinct peptide domains maintain their intrinsic lattice structure and illuminates various mechanisms employed by CMP triple helices to alleviate the elastic strain associated with the interfacial lattice mismatch. Finally, we demonstrate that different sectors of the sheet surface can be selectively functionalized using bioorthogonal conjugation chemistry. Altogether, we establish a robust platform for constructing multifunctional 2D nanoarchitectures in which one can systematically program their compositional, spatial, and functional properties, which is a significant step toward their deployment into functional nanoscale devices.

The wild-type flagellar filament of the Firmicute Kurthia at 2.8 Å resolution in vivo.

Bacteria swim and swarm by rotating the micrometers long, helical filaments of their flagella. They change direction by reversing their flagellar rotation, which switches the handedness of the filament's supercoil. So far, all studied functional filaments are composed of a mixture of L- and R-state flagellin monomers. Here we show in a study of the wild type Firmicute Kurthia sp., that curved, functional filaments can adopt a conformation in vivo that is closely related to a uniform, all-L-state. This sheds additional light on transitions of the flagellar supercoil and uniquely reveals the atomic structure of a wild-type flagellar filament in vivo, including six residues showing clearly densities of O-linked glycosylation.

2D Crystal Engineering of Nanosheets Assembled from Helical Peptide Building Blocks.

The successful integration of 2D nanomaterials into functional devices hinges on developing fabrication methods that afford hierarchical control across length scales of the entire assembly. We demonstrate structural control over a class of crystalline 2D nanosheets assembled from collagen triple helices. By lengthening the triple helix unit through sequential additions of Pro-Hyp-Gly triads, we achieved sub-angstrom tuning over the 2D lattice. These subtle changes influence the overall nanosheet size, which can be adjusted across the mesoscale size regime. The internal structure was observed by cryo-TEM with direct electron detection, which provides real-space high-resolution images, in which individual triple helices comprising the lattice can be clearly discerned. These results establish a general strategy for tuning the structural hierarchy of 2D nanomaterials that employ rigid, cylindrical structural units.

Dimers of mitochondrial ATP synthase induce membrane curvature and self-assemble into rows.

Mitochondrial ATP synthases form dimers, which assemble into long ribbons at the rims of the inner membrane cristae. We reconstituted detergent-purified mitochondrial ATP synthase dimers from the green algae Polytomella sp. and the yeast Yarrowia lipolytica into liposomes and examined them by electron cryotomography. Tomographic volumes revealed that ATP synthase dimers from both species self-assemble into rows and bend the lipid bilayer locally. The dimer rows and the induced degree of membrane curvature closely resemble those in the inner membrane cristae. Monomers of mitochondrial ATP synthase reconstituted into liposomes do not bend membrane visibly and do not form rows. No specific lipids or proteins other than ATP synthase dimers are required for row formation and membrane remodelling. Long rows of ATP synthase dimers are a conserved feature of mitochondrial inner membranes. They are required for cristae formation and a main factor in mitochondrial morphogenesis.

Conserved in situ arrangement of complex I and III2 in mitochondrial respiratory chain supercomplexes of mammals, yeast, and plants.

We used electron cryo-tomography and subtomogram averaging to investigate the structure of complex I and its supramolecular assemblies in the inner mitochondrial membrane of mammals, fungi, and plants. Tomographic volumes containing complex I were averaged at ∼4 nm resolution. Principal component analysis indicated that ∼60% of complex I formed a supercomplex with dimeric complex III, while ∼40% were not associated with other respiratory chain complexes. The mutual arrangement of complex I and III2 was essentially conserved in all supercomplexes investigated. In addition, up to two copies of monomeric complex IV were associated with the complex I1III2 assembly in bovine heart and the yeast Yarrowia lipolytica, but their positions varied. No complex IV was detected in the respiratory supercomplex of the plant Asparagus officinalis Instead, an ∼4.5-nm globular protein density was observed on the matrix side of the complex I membrane arm, which we assign to γ-carbonic anhydrase. Our results demonstrate that respiratory chain supercomplexes in situ have a conserved core of complex I and III2, but otherwise their stoichiometry and structure varies. The conserved features of supercomplex assemblies indicate an important role in respiratory electron transfer.

Visualization of ATP synthase dimers in mitochondria by electron cryo-tomography.

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.