cryo-electron-tomography-remote-data-collection-subtomogram

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved ...

Amyloid and the Cross-Beta Architecture

Amyloid fibrils are made up of a misfolded, self-assembled protein organised into a well-defined, repetitive structure. They are well known for their ...

Diamond Light Source Ltd, Harwell Science & Innovation Campus

University of Sussex

Clathrin-coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the β2 subunit of AP2, and several binding sites have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of β2 hinge-appendage (β2HA). We find that the β2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, β2-appendage cross-links two adjacent terminal domains from different triskelia. Functional analysis of β2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the "sandwich site" on the appendage. We propose that β2-appendage binding to more than one triskelion is a key feature of the system and likely explains why assembly is driven by AP2.

Multi-modal adaptor-clathrin contacts drive coated vesicle assembly.

Protein and peptide self-assembly is integral to a growing number of misfolding diseases that involve the ordered assembly and deposition of misfolded proteins as amyloid fibrils. However, it is becoming clear that these highly stable assemblies can be exploited as potential bionanomaterials. Recent work reveals that amyloid-like assembly is carefully controlled by a number of organisms to form functional amyloid. It is essential to gain an understanding of how sequence influences the assembly mechanism, as well as the final structure, to enable the control that is required to fully exploit these materials. In this tutorial review, we will discuss the different types of fibrous protein assembly from structural and cytoskeletal proteins through to misfolded proteins and finally designed self-assembling peptides. In the second part, we will discuss the advances in structure determination, with particular reference to the use of X-ray fibre diffraction.

From natural to designer self-assembling biopolymers, the structural characterisation of fibrous proteins & peptides using fibre diffraction.

Amyloid-like fibrous crystals formed by the peptide KFFEAAAKKFFE have been previously characterized and provide an ideal model system to examine the importance of specific interactions by introducing specific substitutions. We find that the removal of any phenylalanine residue completely abrogates assembly ability, while charged residues modulate interactions within the structure resulting in alternative fibrillar morphologies. X-ray fiber diffraction analysis reveals that the essential backbone packing of the peptide molecules is maintained, while small changes accommodate differences in side chain size in the variants. We conclude that even very short peptides are adaptable and add to the growing knowledge regarding amyloid polymorphisms. Additionally, this work impacts on our understanding of the importance of residue composition for amyloidogenic peptides, in particular the roles of electrostatic, aromatic, and hydrophobic interactions in amyloid assembly.

Hydrophobic, aromatic, and electrostatic interactions play a central role in amyloid fibril formation and stability.

Amyloid fibrils are polymeric assemblies of normally soluble proteins or peptides. To investigate their structure, it is generally not possible to use conventional methods of crystallography and solution nuclear magnetic resonance. To examine the repeating crystalline structure along the fibre axis, X-ray fibre diffraction has been a useful tool. Here we discuss the methods by which amyloid-like fibrils may be prepared to form a sample suitable for structural analysis and describe how data may be collected and then analysed to arrive at a potential model structure.

X-ray fibre diffraction studies of amyloid fibrils.

Amyloid fibril formation is associated with misfolding diseases, as well as fulfilling a functional role. The cross-β molecular architecture has been reported in increasing numbers of amyloid-like fibrillar systems. The Waltz algorithm is able to predict ordered self-assembly of amyloidogenic peptides by taking into account the residue type and position. This algorithm has expanded the amyloid sequence space, and in the present study we characterize the structures of amyloid-like fibrils formed by three peptides identified by Waltz that form fibrils but not crystals. The structural challenge is met by combining electron microscopy, linear dichroism, CD and X-ray fibre diffraction. We propose structures that reveal a cross-β conformation with 'steric-zipper' features, giving insights into the role for side chains in peptide packing and stability within fibrils. The amenity of these peptides to structural characterization makes them compelling model systems to use for understanding the relationship between sequence, self-assembly, stability and structure of amyloid fibrils.

Exploring the sequence-structure relationship for amyloid peptides.

The structure of cross-β tapes and tubes formed by an octapeptide, αSβ1.

Controlling the order and spatial distribution of self-assembly in multicomponent supramolecular systems could underpin exciting new functional materials, but it is extremely challenging. When a solution of different components self-assembles, the molecules can either coassemble, or self-sort, where a preference for like-like intermolecular interactions results in coexisting, homomolecular assemblies. A challenge is to produce generic and controlled 'one-pot' fabrication methods to form separate ordered assemblies from 'cocktails' of two or more self-assembling species, which might have relatively similar molecular structures and chemistry. Self-sorting in supramolecular gel phases is hence rare. Here we report the first example of the pH-controlled self-sorting of gelators to form self-assembled networks in water. Uniquely, the order of assembly can be predefined. The assembly of each component is preprogrammed by the pK(a) of the gelator. This pH-programming method will enable higher level, complex structures to be formed that cannot be accessed by simple thermal gelation.

Chemically programmed self-sorting of gelator networks.

Alzheimer's disease (AD) is characterized by the deposition of insoluble amyloid plaques in the neuropil composed of highly stable, self-assembled Amyloid-beta (Aβ) fibrils. Copper has been implicated to play a role in Alzheimer's disease. Dimers of Aβ have been isolated from AD brain and have been shown to be neurotoxic.

A central role for dityrosine crosslinking of Amyloid-β in Alzheimer's disease.

Low molecular weight hydrogels are formed by molecules that form a matrix that immobilises water to form a self-supporting gel. Such gels have uses as biomaterials such as molecular scaffolds and structures for tissue engineering. One class of low molecular weight gelator (LMWG), naphthalene-conjugated dipeptides, has been shown to form hydrogels via self-assembly following a controlled drop in pH. A library of naphthalene-dipeptides has been generated previously although the relationship between the precursor sequence and the resulting self-assembled structures remained unclear. Here, we have investigated the structural details of a set of dipeptide sequences containing alanine (A) and valine (V) conjugated to naphthalene groups substituted with a Br, CN or H at the 6-position. Electron microscopy, circular dichroism and X-ray fibre diffraction shows that these LMWG may be structurally classified by their composition: the molecular packing is determined by the class of conjugate, whilst the chirality of the self-assemblies can be attributed to the dipeptide sequence. This provides insights into the relationship between the precursor sequence and the macromolecular and molecular structures of the fibres that make up the resulting hydrogels.

Structural determinants in a library of low molecular weight gelators.

Structural analysis of protein fibrillation is inherently challenging. Given the crucial role of fibrils in amyloid diseases, method advancement is urgently needed. A hybrid modelling approach is presented enabling detailed analysis of a highly ordered and hierarchically organized fibril of the GNNQQNY peptide fragment of a yeast prion protein. Data from small-angle X-ray solution scattering, fibre diffraction and electron microscopy are combined with existing high-resolution X-ray crystallographic structures to investigate the fibrillation process and the hierarchical fibril structure of the peptide fragment. The elongation of these fibrils proceeds without the accumulation of any detectable amount of intermediate oligomeric species, as is otherwise reported for, for example, glucagon, insulin and α-synuclein. Ribbons constituted of linearly arranged protofilaments are formed. An additional hierarchical layer is generated via the pairing of ribbons during fibril maturation. Based on the complementary data, a quasi-atomic resolution model of the protofilament peptide arrangement is suggested. The peptide structure appears in a β-sheet arrangement reminiscent of the β-zipper structures evident from high-resolution crystal structures, with specific differences in the relative peptide orientation. The complexity of protein fibrillation and structure emphasizes the need to use multiple complementary methods.

The architecture of amyloid-like peptide fibrils revealed by X-ray scattering, diffraction and electron microscopy.

Amyloidogenic peptides are well known for their involvement in diseases such as type 2 diabetes and Alzheimer's disease. However, more recently, amyloid fibrils have been shown to provide scaffolding and protection as functional materials in a range of organisms from bacteria to humans. These roles highlight the incredible tensile strength of the cross-β amyloid architecture. Many amino acid sequences are able to self-assemble to form amyloid with a cross-β core. Here we describe our recent advances in understanding how sequence contributes to amyloidogenicity and structure. For example, we describe penta- and hexapeptides that assemble to form different morphologies; a 12mer peptide that forms fibrous crystals; and an eight-residue peptide originating from α-synuclein that has the ability to form nanotubes. This work provides a wide range of peptides that may be exploited as fibrous bionanomaterials. These fibrils provide a scaffold upon which functional groups may be added, or templated assembly may be performed.

ArticleTotal : 2	Year
Publication title Cited by 1	2022
Publication title	2026

Article	Year
Multi-modal adaptor-clathrin contacts drive coated vesicle assembly. The EMBO journal\| PubMed ID: 34487371	2021
From natural to designer self-assembling biopolymers, the structural characterisation of fibrous proteins & peptides using fibre diffraction. Chemical Society reviews\| PubMed ID: 20668734	2010
Hydrophobic, aromatic, and electrostatic interactions play a central role in amyloid fibril formation and stability. Biochemistry\| PubMed ID: 21288003	2011
X-ray fibre diffraction studies of amyloid fibrils. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 22528087	2012
Exploring the sequence-structure relationship for amyloid peptides. The Biochemical journal\| PubMed ID: 23252554	2013
The structure of cross-β tapes and tubes formed by an octapeptide, αSβ1. Angewandte Chemie (International ed. in English)\| PubMed ID: 23307646	2013
Chemically programmed self-sorting of gelator networks. Nature communications\| PubMed ID: 23403581	2013
A central role for dityrosine crosslinking of Amyloid-β in Alzheimer's disease. Acta neuropathologica communications\| PubMed ID: 24351276	2013
Structural determinants in a library of low molecular weight gelators. Soft matter\| PubMed ID: 25562785	2015
The architecture of amyloid-like peptide fibrils revealed by X-ray scattering, diffraction and electron microscopy. Acta crystallographica. Section D, Biological crystallography\| PubMed ID: 25849399	2015
The diversity and utility of amyloid fibrils formed by short amyloidogenic peptides. Interface focus\| PubMed ID: 29147557	2017

Kyle Morris

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