Carsten Sachse

Aβ(1-40) Fibril Polymorphism Implies Diverse Interaction Patterns in Amyloid Fibrils

Amyloid fibrils characterize a diverse group of human diseases that includes Alzheimers disease, Creutzfeldt-Jakob and type II diabetes. Alzheimers amyloid fibrils consist of amyloid-beta (Abeta) peptide and occur in a range of structurally different fibril morphologies. The structural characteristics of 12 single Abeta(1-40) amyloid fibrils, all formed under the same solution conditions, were determined by electron cryo-microscopy and three-dimensional reconstruction. The majority of analyzed fibrils form a range of morphologies that show almost continuously altering structural properties. The observed fibril polymorphism implies that amyloid formation can lead, for the same polypeptide sequence, to many different patterns of inter- or intra-residue interactions. This property differs significantly from native, monomeric protein folding reactions that produce, for one protein sequence, only one ordered conformation and only one set of inter-residue interactions.

Directed selection of a conformational antibody domain that prevents mature amyloid fibril formation by stabilizing Aβ protofibrils

The formation of amyloid fibrils is a common biochemical characteristic that occurs in Alzheimers disease and several other amyloidoses. The unifying structural feature of amyloid fibrils is their specific type of β-sheet conformation that differentiates these fibrils from the products of normal protein folding reactions. Here we describe the generation of an antibody domain, termed B10, that recognizes an amyloid-specific and conformationally defined epitope. This antibody domain was selected by phage-display from a recombinant library of camelid antibody domains. Surface plasmon resonance, immunoblots, and immunohistochemistry show that this antibody domain distinguishes Aβ amyloid fibrils from disaggregated Aβ peptide as well as from specific Aβ oligomers. The antibody domain possesses functional activity in preventing the formation of mature amyloid fibrils by stabilizing Aβ protofibrils. These data suggest possible applications of B10 in the detection of amyloid fibrils or in the modulation of their formation.

Paired β-sheet structure of an Aβ(1-40) amyloid fibril revealed by electron microscopy

Alzheimers disease is a neurodegenerative disorder that is characterized by the cerebral deposition of amyloid fibrils formed by Aβ peptide. Despite their prevalence in Alzheimers and other neurodegenerative diseases, important details of the structure of amyloid fibrils remain unknown. Here, we present a three-dimensional structure of a mature amyloid fibril formed by Aβ(1-40) peptide, determined by electron cryomicroscopy at ≈8-Å resolution. The fibril consists of two protofilaments, each containing ≈5-nm-long regions of β-sheet structure. A local twofold symmetry within each region suggests that pairs of β-sheets are formed from equivalent parts of two Aβ(1-40) peptides contained in each protofilament. The pairing occurs via tightly packed interfaces, reminiscent of recently reported steric zipper structures. However, unlike these previous structures, the β-sheet pairing is observed within an amyloid fibril and includes significantly longer amino acid sequences.

Comparison of Alzheimer Aβ(1–40) and Aβ(1–42) amyloid fibrils reveals similar protofilament structures

We performed mass-per-length (MPL) measurements and electron cryomicroscopy (cryo-EM) with 3D reconstruction on an Aβ(1–42) amyloid fibril morphology formed under physiological pH conditions. The data show that the examined Aβ(1–42) fibril morphology has only one protofilament, although two protofilaments were observed with a previously studied Aβ(1–40) fibril. The latter fibril was resolved at 8 Å resolution showing pairs of β-sheets at the cores of the two protofilaments making up a fibril. Detailed comparison of the Aβ(1–42) and Aβ(1–40) fibril structures reveals that they share an axial twofold symmetry and a similar protofilament structure. Furthermore, the MPL data indicate that the protofilaments of the examined Aβ(1–40) and Aβ(1–42) fibrils have the same number of Aβ molecules per cross-β repeat. Based on this data and the previously studied Aβ(1–40) fibril structure, we describe a model for the arrangement of peptides within the Aβ(1–42) fibril.

Structure of a Bacterial Dynamin-like Protein Lipid Tube Provides a Mechanism For Assembly and Membrane Curving

Summary Proteins of the dynamin superfamily mediate membrane fission, fusion, and restructuring events by polymerizing upon lipid bilayers and forcing regions of high curvature. In this work, we show the electron cryomicroscopy reconstruction of a bacterial dynamin-like protein (BDLP) helical filament decorating a lipid tube at ∼11 Å resolution. We fitted the BDLP crystal structure and produced a molecular model for the entire filament. The BDLP GTPase domain dimerizes and forms the tube surface, the GTPase effector domain (GED) mediates self-assembly, and the paddle region contacts the lipids and promotes curvature. Association of BDLP with GMPPNP and lipid induces radical, large-scale conformational changes affecting polymerization. Nucleotide hydrolysis seems therefore to be coupled to polymer disassembly and dissociation from lipid, rather than membrane restructuring. Observed structural similarities with rat dynamin 1 suggest that our results have broad implication for other dynamin family members.

An atomic model of HIV-1 capsid-SP1 reveals structures regulating assembly and maturation

Maturation and inhibition of HIV-1 HIV-1 undergoes a two-step assembly process controlled largely by a single region of its Gag protein. Schur et al. determined a complete atomic model for this region within an assembled Gag protein lattice using cryo-electron tomography together with subtomogram averaging. Amino acids from different parts of multiple Gag molecules come together to form an intricate network of interactions that drive HIV-1 assembly. The final step of maturation into the infectious HIV-1 virus is controlled by structural changes in Gag that alter the accessibility of the final cleavage site to the viral protease. Science, this issue p. 506 Improved cryo-electron tomography and subtomogram averaging show how HIV maturation inhibitor drugs may work. Immature HIV-1 assembles at and buds from the plasma membrane before proteolytic cleavage of the viral Gag polyprotein induces structural maturation. Maturation can be blocked by maturation inhibitors (MIs), thereby abolishing infectivity. The CA (capsid) and SP1 (spacer peptide 1) region of Gag is the key regulator of assembly and maturation and is the target of MIs. We applied optimized cryo-electron tomography and subtomogram averaging to resolve this region within assembled immature HIV-1 particles at 3.9 angstrom resolution and built an atomic model. The structure reveals a network of intra- and intermolecular interactions mediating immature HIV-1 assembly. The proteolytic cleavage site between CA and SP1 is inaccessible to protease. We suggest that MIs prevent CA-SP1 cleavage by stabilizing the structure, and MI resistance develops by destabilizing CA-SP1.

Three-dimensional structure of TspO by electron cryomicroscopy of helical crystals.

Summary The 18 kDa TSPO protein is a polytopic mitochondrial outer membrane protein involved in a wide range of physiological functions and pathologies, including neurodegeneration and cancer. The pharmacology of TSPO has been extensively studied, but little is known about its biochemistry, oligomeric state, and structure. We have expressed, purified, and characterized a homologous protein, TspO from Rhodobacter sphaeroides, and reconstituted it as helical crystals. Using electron cryomicroscopy and single-particle helical reconstruction, we have determined a three-dimensional structure of TspO at 10 Å resolution. The structure suggests that monomeric TspO comprises five transmembrane α helices that form a homodimer, which is consistent with the dimeric state observed in detergent solution. Furthermore, the arrangement of transmembrane domains of individual TspO subunits indicates a possibility of two substrate translocation pathways per dimer. The structure provides the first insight into the molecular architecture of TSPO/PBR protein family that will serve as a framework for future studies.

Molecular structures of unbound and transcribing RNA polymerase III

Transcription of genes encoding small structured RNAs such as transfer RNAs, spliceosomal U6 small nuclear RNA and ribosomal 5S RNA is carried out by RNA polymerase III (Pol III), the largest yet structurally least characterized eukaryotic RNA polymerase. Here we present the cryo-electron microscopy structures of the Saccharomyces cerevisiae Pol III elongating complex at 3.9 Å resolution and the apo Pol III enzyme in two different conformations at 4.6 and 4.7 Å resolution, respectively, which allow the building of a 17-subunit atomic model of Pol III. The reconstructions reveal the precise orientation of the C82–C34–C31 heterotrimer in close proximity to the stalk. The C53–C37 heterodimer positions residues involved in transcription termination close to the non-template DNA strand. In the apo Pol III structures, the stalk adopts different orientations coupled with closed and open conformations of the clamp. Our results provide novel insights into Pol III-specific transcription and the adaptation of Pol III towards its small transcriptional targets.

The Selective Autophagy Receptor p62 Forms a Flexible Filamentous Helical Scaffold

The scaffold protein p62/SQSTM1 is involved in protein turnover and signaling and is commonly found in dense protein bodies in eukaryotic cells. In autophagy, p62 acts as a selective autophagy receptor that recognizes and shuttles ubiquitinated proteins to the autophagosome for degradation. The structural organization of p62 in cellular bodies and the interplay of these assemblies with ubiquitin and the autophagic marker LC3 remain to be elucidated. Here, we present a cryo-EM structural analysis of p62. Together with structures of assemblies from the PB1 domain, we show that p62 is organized in flexible polymers with the PB1 domain constituting a helical scaffold. Filamentous p62 is capable of binding LC3 and addition of long ubiquitin chains induces disassembly and shortening of filaments. These studies explain how p62 assemblies provide a large molecular scaffold for the nascent autophagosome and reveal how they can bind ubiquitinated cargo.

Nanoscale Flexibility Parameters of Alzheimer Amyloid Fibrils Determined by Electron Cryo-Microscopy

Amyloid fibrils are fibrillar polypeptide aggregates consisting of a cross-β structure.1, 2 The rigidity and stability of these fibrils contributes to their natural pathogenicity or functionality and has suggested potential applications in bionanotechnology.3–6 Yet, amyloid fibrils can occur in different morphologies with unique mechanical and flexible characteristics.7–9 Herein, we use electron cryo-microscopy (cryo-EM) to characterize these nanoscale structural properties. Cryo-EM images effectively represent snapshots of thermally fluctuating fibrils in solution; it is not necessary to micromanipulate or immobilize the fibrils on a solid surface. The amyloid fibrils analyzed here consist of Alzheimer’s Aβ(1–40) peptide. They are homogenous in width (w≍20 nm), although different fibrils can vary significantly in their crossover distances d (Figure 1). Figure 1 Global structural characteristics. Negatively stained micrographs (A) and cryo-micrographs (B, C) illustrate definitions of fibril length L, width w, crossover distance d, and normal distance δu. In addition to these interfibrillar differences, there are variations of d within each single fibril. However, the intrafibrillar standard deviations of d range mostly from 5 to 7 nm, while average d values of different fibrils vary from 100 to 160 nm (Figure 2 A). Hence, the encountered variations cannot be explained by purely thermally determined and stochastic fluctuations. Instead, they represent subtle, yet systematic, structural differences between the fibrils in the sample. Figure 2 A) Mean crossover distances of representative fibrils. B) Distribution of mean crossover distances of the entire fibril population. F120: light gray; F140: dark gray. To further analyze these structural differences, two subpopulations were defined, termed here F120 and F140 fibrils. F140 fibrils show mean d values of (140±10) nm (Figure 2 B), and their 3D structure was reconstructed previously at approximately 8 A resolution.10, 11 F120 fibrils possess an average d value of (120±10) nm (Figure 2 B). The structure of F120 fibrils is determined here at approximately 10 A resolution (Figure 3 A, B, Figure 2 in the Supporting Information). Whereas the distinction between F120 and F140 fibrils remains arbitrary, the two subpopulations consist of a sufficiently large data set for a medium-resolution 3D reconstruction and for measurement of the nanoscale elastic properties. Reconstructed F120 and F140 fibrils present effectively the same cross-section (Figure 3). Hence, the conformational differences of the peptides forming F120 and F140 fibrils are too small to be revealed at the current levels of structural resolution. These data imply that the systematic variations in the crossover distances of different fibrils (Figure 2 A) occur within fibrils that all belong to the same basic morphology. In other words, different fibrils of the same morphology can occur with different torsional properties. Figure 3 Cross-section of F120 (A) and F140 fibrils (B). C) Difference map F140−F120. Negative peaks: orange=2σ, red=3σ. Positive peaks: light blue=2σ, blue=3σ. Calculation of the nanoscale elastic properties is based on the measurement of variations of fibril twisting and bending. Assuming that the fibrils are made up of an isotropic homogeneous medium, variations of the fibril twist d enable computation of torsional persistence length lc and torsional rigidity c. Bending variations yield persistence length lp and bending rigidity κ (see the Supporting Information for details). Our measurements imply that F120 and F140 fibrils possess very similar, if not identical, torsional properties (torsional rigidity c and torsional persistence length lc; Table 2 in the Supporting Information). By contrast, the two fibril populations differ significantly in their bending properties (Table 3 in the Supporting Information). F120 fibrils possess a smaller bending rigidity κ (Table 3 in the Supporting Information) and a larger normalized bending fluctuation Δu than F140 fibrils (Figure 3 B in the Supporting Information). However, part of this difference may result from the different spacing of crossovers in these two populations (Figure 3 in the Supporting Information). The measured lp and κ values are within the reported range for other amyloid fibrils.12–14 They also comply with a fundamental relationship between lp and the molecular density (mass per length; Figure 4 A). Figure 4 Flexibility parameters of F120 and F140 fibrils in comparison with those of other filamentous protein assemblies. A) Persistence length increases with the increase in mass per unit of length.19 B) Comparison of moments of inertia and polar ... For several protein fibrils, the dependence of c and κ on shape- and material-specific factors has been analyzed.15–17 The physical formalism used in these analyses was developed for macroscopic objects. Thus, its general applicability to nanoscale protein fibrils remains to be established. According to this formalism, the torsional rigidity c depends on the shape-dependent polar moment of inertia Iz and the material-specific shear modulus G [Eq. (1)]. The bending rigidity κ depends on the material-specific Young’s modulus Y and the shape-dependent moment of inertia [Eq. (2)]. (1) (2) In contrast to previous approaches that had to use model estimates for the fibril cross-section, cryo-EM enables calculation of the two shape-dependent factors Iz and directly from the cross-section of the 3D fibril reconstructions. F120 and F140 fibrils effectively possess the same cross-sectional architecture (Figure 3) and therefore similar shape-specific factors Iz and (Tables 2 and 3 in the Supporting Information). The torsional rigidities of F120 and F140 fibrils are very similar and produce the same shear modulus G within error margins [Eq. (1), Table 2 in the Supporting Information). We have compared the calculated material moduli with literature data. Exact numeric values should be considered carefully, however, owing to possible effects of the method of analysis.14 The shear moduli G of F120 and F140 fibrils (12.7 MPa) are in close proximity to those of other protein assemblies, such as F-actin (9 MPa)16 and sickle-cell fibrils (SCF, 1 MPa).18 In comparison to macroscopic materials, these values fall in the range between plastics (ca. 100 MPa) and rubber (ca. 0.6 MPa).19 The Young’s moduli Y of F120 and F140 fibrils (90 and 320 MPa, respectively) are close to the observed values for filamentous proteins, such as SCF (50 MPa),18 but are somewhat lower than figures of microtubuli and actin (1 and 3 GPa, respectively).15 The material constants of F120 and F140 fibrils differ more profoundly from those reported recently for insulin amyloid fibrils (shear modulus G=130 MPa, Young’s modulus Y=6 GPa14). By contrast, the persistence length (42 μm) and bending rigidity (1.7×10−25 N m2) of insulin fibrils are remarkably similar to those of Aβ(1–40) fibrils. Since no 3D reconstruction of the analyzed insulin fibrils was reported, their cross-sectional structure cannot be compared easily with the structure of the Aβ(1–40) fibrils used here. While our data cannot confirm the existence of unusually high nanoscale material constants for the analyzed Aβ(1–40) fibrils, we find that the shape-dependent properties polar moment of inertia Iz and moment of inertia are significantly greater for the analyzed Aβ(1–40) fibrils than for area-normalized cross-sections of other protein filaments (Figure 4 B). Hence, the analyzed Aβ fibrils represent a very material-efficient way to construct proteinaceous filaments of high stability and structural flexibility. These observations are relevant for better estimating the potential applications of amyloid fibrils in the material sciences. In addition, our data contribute to understanding amyloid pathogenicity in vivo. The stability and flexibility of amyloid fibrils are similar to those of native protein filaments, such as F-actin or microtubules. However, growth and disassembly of the latter represent highly dynamic and regulated processes, and as such they are tightly controlled by specific sets of proteins. Therefore, an unregulated outgrowth of similarly stable amyloid fibrils will be much more difficult to tolerate within a biological environment. This conclusion is consistent with the fact that amyloid pathogenicity arises, at least partially, from the distortion or disruption of naturally elastic and flexible tissues, such as cardiac ventricles or blood vessel walls.20 Further work will be required, however, to delineate the cellular pathways by which these reactions result in the death of affected cells.