Nadav Elad

Topologies of a Substrate Protein Bound to the Chaperonin GroEL

Summary The chaperonin GroEL assists polypeptide folding through sequential steps of binding nonnative protein in the central cavity of an open ring, via hydrophobic surfaces of its apical domains, followed by encapsulation in a hydrophilic cavity. To examine the binding state, we have classified a large data set of GroEL binary complexes with nonnative malate dehydrogenase (MDH), imaged by cryo-electron microscopy, to sort them into homogeneous subsets. The resulting electron density maps show MDH associated in several characteristic binding topologies either deep inside the cavity or at its inlet, contacting three to four consecutive GroEL apical domains. Consistent with visualization of bound polypeptide distributed over many parts of the central cavity, disulfide crosslinking could be carried out between a cysteine in a bound substrate protein and cysteines substituted anywhere inside GroEL. Finally, substrate binding induced adjustments in GroEL itself, observed mainly as clustering together of apical domains around sites of substrate binding.

Structural analysis of the nuclear pore complex by integrated approaches.

In eukaryotic cells, the nucleus is surrounded by a double membrane system, the nuclear envelope (NE), in which the outer membrane is continuous with the endoplasmic reticulum (ER). Nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes to form aqueous translocation channels that allow the free diffusion of small molecules and ions, as well as receptor-mediated transport of large macromolecules. Being the sole gateways for import and export to and from the nucleus, NPCs regulate the nucleocytoplasmic transport of macromolecules in a highly selective manner to maintain cellular functions. The large size and complexity of these multimolecular assemblies, which are composed of approximately 30 different proteins (termed nucleoporins), present a major challenge for structural biologists. Here, we discuss the latest structural findings related to the functional organization of the NPC.

Detection and separation of heterogeneity in molecular complexes by statistical analysis of their two-dimensional projections

Progress in molecular structure determination by cryo electron microscopy and single particle analysis has led to improvements in the resolution achievable. However, in many cases the limiting factor is structural heterogeneity of the sample. To address this problem, we have developed a method based on statistical analysis of the two-dimensional images to detect and sort localised structural variations caused, for example, by variable occupancy of a ligand. Images are sorted by two consecutive stages of multivariate statistical analysis (MSA) to dissect out the two main sources of variation, namely out of plane orientation and local structural changes. Heterogeneity caused by local changes is detected by MSA that reveals significant peaks in the higher order eigenimages. The eigenimages revealing local peaks are used for automated classification. Evaluation of differences between classes allows discrimination of molecular images with and without ligand. This method is very rapid, independent of any initial three-dimensional model, and can detect even minor subpopulations in an image ensemble. A strategy for using this technique was developed on model data sets. Here, we demonstrate the successful application of this method to both model and real EM data on chaperonin-substrate and ribosome-ligand complexes.

Profilin 1 is required for abscission during late cytokinesis of chondrocytes

Profilins are key factors for dynamic rearrangements of the actin cytoskeleton. However, the functions of profilins in differentiated mammalian cells are uncertain because profilin deficiency is early embryonic lethal for higher eukaryotes. To examine profilin function in chondrocytes, we disrupted the profilin 1 gene in cartilage (Col2pfn1). Homozygous Col2pfn1 mice develop progressive chondrodysplasia caused by disorganization of the growth plate and defective chondrocyte cytokinesis, indicated by the appearance of binucleated cells. Surprisingly, Col2pfn1 chondrocytes assemble and contract actomyosin rings normally during cell division; however, they display defects during late cytokinesis as they frequently fail to complete abscission due to their inability to develop strong traction forces. This reduced force generation results from an impaired formation of lamellipodia, focal adhesions and stress fibres, which in part could be linked to an impaired mDia1‐mediated actin filament elongation. Neither an actin nor a poly‐proline binding‐deficient profilin 1 is able to rescue the defects. Taken together, our results demonstrate that profilin 1 is not required for actomyosin ring formation in dividing chondrocytes but necessary to generate sufficient force for abscission during late cytokinesis.

Signature Amyloid β Profiles Are Produced by Different γ-Secretase Complexes

Background: γ-Secretase complexes generate amyloid-β (Aβ) in Alzheimer disease. Results: Aβ profiles of the four γ-secretase complexes expressed in humans show that PSEN regulates total peptide levels and the Aβ38 pathway, whereas APH1 affects mainly the efficiency of the carboxypeptidase-like activity. Conclusion: γ-Secretase subunit composition regulates Aβ generation. Significance: These intrinsic differences could be used to advance AD therapeutic development. γ-Secretase complexes are involved in the generation of amyloid-β (Aβ) in the brain. Therefore, γ-secretase has been proposed as a potential therapeutic target in Alzheimer disease (AD). Targeting γ-secretase activity in AD requires the pharmacological dissociation of the processing of physiological relevant substrates and the generation of “toxic” Aβ. Previous reports suggest the differential targeting of γ-secretase complexes, based on their subunit composition, as a valid strategy. However, little is known about the biochemical properties of the different complexes, and key questions regarding their Aβ product profiles should be first addressed. Here, we expressed, purified, and analyzed, under the same conditions, the endopeptidase and carboxypeptidase-like activities of the four γ-secretase complexes present in humans. We find that the nature of the catalytic subunit in the complex affects both activities. Interestingly, PSEN2 complexes discriminate between the Aβ40 and Aβ38 production lines, indicating that Aβ generation in one or the other pathway can be dissociated. In contrast, the APH1 subunit mainly affects the carboxypeptidase-like activity, with APH1B complexes favoring the generation of longer Aβ peptides. In addition, we determined that expression of a single human γ-secretase complex in cell lines retains the intrinsic attributes of the protease while present in the membrane, providing validation for the in vitro studies. In conclusion, our data show that each γ-secretase complex produces a characteristic Aβ signature. The qualitative and quantitative differences between different γ-secretase complexes could be used to advance drug development in AD and other disorders.

Microtubule organization in the final stages of cytokinesis as revealed by cryo-electron tomography

The completion of cytokinesis is dominated by the midbody, a tightly-packed microtubule (MT)-based bridge that transiently connects the two daughter cells. Assembled from condensed, spindle-MTs and numerous associated proteins, the midbody gradually narrows down until daughter cell partitioning occurs at this site. Although described many years ago, detailed understanding of the abscission process remains lacking. Applying cryo-electron tomography to purified midbodies, in combination with fluorescence microscopy, we present here new insight into MT organization within the midbody. We find that the midbody is spatially divided into a core bundle of MTs that traverses the electron-dense overlap region (continuous MTs), surrounded by MTs that terminate within the overlap region (polar MTs). Residual continuous MTs remained intact up to the verge of abscission, whereas the residual polar MTs lost their organization and retreated from the overlap region at late cytokinesis stages. A detailed localization of the microtubule-bundling protein PRC1 supports the above notion. Our study thus provides a detailed account of the abscission process and suggests that the midbody, having acquired a distinct MT architecture as compared to the preceding central spindle, actively facilitates the final stage of cytokinesis.

The dynamic conformational landscape of γ-secretase

ABSTRACT The structure and function of the &ggr;-secretase proteases are of great interest because of their crucial roles in cellular and disease processes. We established a novel purification protocol for the &ggr;-secretase complex that involves a conformation- and complex-specific nanobody, yielding highly pure and active enzyme. Using single particle electron microscopy, we analyzed the &ggr;-secretase structure and its conformational variability. Under steady-state conditions, the complex adopts three major conformations, which differ in overall compactness and relative position of the nicastrin ectodomain. Occupancy of the active or substrate-binding sites by inhibitors differentially stabilizes subpopulations of particles with compact conformations, whereas a mutation linked to familial Alzheimer disease results in enrichment of extended-conformation complexes with increased flexibility. Our study presents the &ggr;-secretase complex as a dynamic population of interconverting conformations, involving rearrangements at the nanometer scale and a high level of structural interdependence between subunits. The fact that protease inhibition or clinical mutations, which affect amyloid &bgr; (A&bgr;) generation, enrich for particular subpopulations of conformers indicates the functional relevance of the observed dynamic changes, which are likely to be instrumental for highly allosteric behavior of the enzyme.

Improved Doxorubicin Encapsulation and Pharmacokinetics of Ferritin–Fusion Protein Nanocarriers Bearing Proline, Serine, and Alanine Elements

A novel human ferritin-based nanocarrier, composed of 24 modified monomers able to auto-assemble into a modified protein cage, was produced and used as selective carrier of anti-tumor payloads. Each modified monomer derives from the genetic fusion of two distinct modules, namely the heavy chain of human ferritin (HFt) and a stabilizing/protective PAS polypeptide sequence rich in proline (P), serine (S), and alanine (A) residues. Two genetically fused protein constructs containing PAS polymers with 40- and 75-residue lengths, respectively, were compared. They were produced and purified as recombinant proteins in Escherichia coli at high yields. Both preparations were highly soluble and stable in vitro as well as in mouse plasma. Size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy results indicated that PASylated ferritins are fully assembled and highly monodispersed. In addition, yields and stability of encapsulated doxorubicin were significantly better for both HFt-PAS proteins than for wild-type HFt. Importantly, PAS sequences considerably prolonged the half-life of HFt in the mouse bloodstream. Finally, our doxorubicin-loaded nanocages preserved the pharmacological activity of the drug. Taken together, these results indicate that both of the developed HFt-PAS fusion proteins are promising nanocarriers for future applications in cancer therapy.

The role of integrin-linked kinase in the molecular architecture of focal adhesions

Summary Integrin-mediated focal adhesions (FAs) are large, multi-protein complexes that link the actin cytoskeleton to the extracellular matrix and take part in adhesion-mediated signaling. These adhesions are highly complex and diverse at the molecular level; thus, assigning particular structural or signaling functions to specific components is highly challenging. Here, we combined functional, structural and biophysical approaches to assess the role of a major FA component, namely, integrin-linked kinase (ILK), in adhesion formation. We show here that ILK plays a key role in the formation of focal complexes, early forms of integrin adhesions, and confirm its involvement in the assembly of fibronectin-bound fibrillar adhesions. Examination of ILK-null fibroblasts by cryo-electron tomography pointed to major structural changes in their FAs, manifested as disarray of the associated actin filaments and an increase in the packing density of FA-related particles. Interestingly, adhesion of the mutant cells to the substrate required a higher ligand density than in control cells. These data indicate that ILK has a key role in integrin adhesion assembly and sub-structure, and in the regulation of the FA-associated cytoskeleton.

Proteins evolve on the edge of supramolecular self-assembly

The self-association of proteins into symmetric complexes is ubiquitous in all kingdoms of life. Symmetric complexes possess unique geometric and functional properties, but their internal symmetry can pose a risk. In sickle-cell disease, the symmetry of haemoglobin exacerbates the effect of a mutation, triggering assembly into harmful fibrils. Here we examine the universality of this mechanism and its relation to protein structure geometry. We introduced point mutations solely designed to increase surface hydrophobicity among 12 distinct symmetric complexes from Escherichia coli. Notably, all responded by forming supramolecular assemblies in vitro, as well as in vivo upon heterologous expression in Saccharomyces cerevisiae. Remarkably, in four cases, micrometre-long fibrils formed in vivo in response to a single point mutation. Biophysical measurements and electron microscopy revealed that mutants self-assembled in their folded states and so were not amyloid-like. Structural examination of 73 mutants identified supramolecular assembly hot spots predictable by geometry. A subsequent structural analysis of 7,471 symmetric complexes showed that geometric hot spots were buffered chemically by hydrophilic residues, suggesting a mechanism preventing mis-assembly of these regions. Thus, point mutations can frequently trigger folded proteins to self-assemble into higher-order structures. This potential is counterbalanced by negative selection and can be exploited to design nanomaterials in living cells.