Bacterial Inclusion Bodies Contain Amyloid-Like Structure

Protein aggregation is a process in which identical proteins self-associate into imperfectly ordered macroscopic entities. Such aggregates are generally classified as amorphous, lacking any long-range order, or highly ordered fibrils. Protein fibrils can be composed of native globular molecules, such as the hemoglobin molecules in sickle-cell fibrils, or can be reorganized β-sheet–rich aggregates, termed amyloid-like fibrils. Amyloid fibrils are associated with several pathological conditions in humans, including Alzheimer disease and diabetes type II. We studied the structure of bacterial inclusion bodies, which have been believed to belong to the amorphous class of aggregates. We demonstrate that all three in vivo-derived inclusion bodies studied are amyloid-like and comprised of amino-acid sequence-specific cross-β structure. These findings suggest that inclusion bodies are structured, that amyloid formation is an omnipresent process both in eukaryotes and prokaryotes, and that amino acid sequences evolve to avoid the amyloid conformation

Cooperativity among Short Amyloid Stretches in Long Amyloidogenic Sequences

Amyloid fibrillar aggregates of polypeptides are associated with many neurodegenerative diseases. Short peptide segments in protein sequences may trigger aggregation. Identifying these stretches and examining their behavior in longer protein segments is critical for understanding these diseases and obtaining potential therapies. In this study, we combined machine learning and structure-based energy evaluation to examine and predict amyloidogenic segments. Our feature selection method discovered that windows consisting of long amino acid segments of ∼30 residues, instead of the commonly used short hexapeptides, provided the highest accuracy. Weighted contributions of an amino acid at each position in a 27 residue window revealed three cooperative regions of short stretch, resemble the β-strand-turn-β-strand motif in A-βpeptide amyloid and β-solenoid structure of HET-s(218–289) prion (C). Using an in-house energy evaluation algorithm, the interaction energy between two short stretches in long segment is computed and incorporated as an additional feature. The algorithm successfully predicted and classified amyloid segments with an overall accuracy of 75%. Our study revealed that genome-wide amyloid segments are not only dependent on short high propensity stretches, but also on nearby residues

Towards a Pharmacophore for Amyloid

Diagnosing and treating Alzheimer's and other diseases associated with amyloid fibers remains a great challenge despite intensive research. To aid in this effort, we present atomic structures of fiber-forming segments of proteins involved in Alzheimer's disease in complex with small molecule binders, determined by X-ray microcrystallography. The fiber-like complexes consist of pairs of β-sheets, with small molecules binding between the sheets, roughly parallel to the fiber axis. The structures suggest that apolar molecules drift along the fiber, consistent with the observation of nonspecific binding to a variety of amyloid proteins. In contrast, negatively charged orange-G binds specifically to lysine side chains of adjacent sheets. These structures provide molecular frameworks for the design of diagnostics and drugs for protein aggregation diseases

Diffusion, Crowding & Protein Stability in a Dynamic Molecular Model of the Bacterial Cytoplasm

A longstanding question in molecular biology is the extent to which the behavior of macromolecules observed in vitro accurately reflects their behavior in vivo. A number of sophisticated experimental techniques now allow the behavior of individual types of macromolecule to be studied directly in vivo; none, however, allow a wide range of molecule types to be observed simultaneously. In order to tackle this issue we have adopted a computational perspective, and, having selected the model prokaryote Escherichia coli as a test system, have assembled an atomically detailed model of its cytoplasmic environment that includes 50 of the most abundant types of macromolecules at experimentally measured concentrations. Brownian dynamics (BD) simulations of the cytoplasm model have been calibrated to reproduce the translational diffusion coefficients of Green Fluorescent Protein (GFP) observed in vivo, and “snapshots” of the simulation trajectories have been used to compute the cytoplasm's effects on the thermodynamics of protein folding, association and aggregation events. The simulation model successfully describes the relative thermodynamic stabilities of proteins measured in E. coli, and shows that effects additional to the commonly cited “crowding” effect must be included in attempts to understand macromolecular behavior in vivo

Structure-Based Design of Functional Amyloid Materials

Amyloid fibers, once exclusively associated with disease, are acquiring utility as a class of biological nanomaterials. Here we introduce a method that utilizes the atomic structures of amyloid peptides, to design materials with versatile applications. As a model application, we designed amyloid fibers capable of capturing carbon dioxide from flue gas, to address the global problem of excess anthropogenic carbon dioxide. By measuring dynamic separation of carbon dioxide from nitrogen, we show that fibers with designed amino acid sequences double the carbon dioxide binding capacity of the previously reported fiber formed by VQIVYK from Tau protein. In a second application, we designed fibers that facilitate retroviral gene transfer. By measuring lentiviral transduction, we show that designed fibers exceed the efficiency of polybrene, a commonly used enhancer of transduction. The same procedures can be adapted to the design of countless other amyloid materials with a variety of properties and uses

Common dynamical signatures of familial amyotrophic lateral sclerosis-associated structurally diverse Cu, Zn superoxide dismutase mutants

More than 100 structurally diverse point mutations leading to aggregation in the dimeric enzyme Cu, Zn superoxide dismutase (SOD1) are implicated in familial amyotrophic lateral sclerosis (FALS). Although SOD1 dimer dissociation is a known requirement for its aggregation, the common structural basis for diverse FALS mutations resulting in aggregation is not fully understood. In molecular dynamics simulations of wild-type SOD1 and three structurally diverse FALS mutants (A4V, G37R, and H46R), we find that a common effect of mutations on SOD1 dimer is the mutation-induced disruption of dynamic coupling between monomers. In the wild-type dimer, the principal coupled motion corresponds to a “breathing motion” of the monomers around an axis parallel to the dimer interface, and an opening–closing motion of the distal metal-binding loops. These coupled motions are disrupted in all three mutants independent of the mutation location. Loss of coupled motions in mutant dimers occurs with increased disruption of a key stabilizing structural element (the β-plug) leading to the de-protection of edge strands. To rationalize disruption of coupling, which is independent of the effect of the mutation on global SOD1 stability, we analyze the residue–residue interaction network formed in SOD1. We find that the dimer interface and metal-binding loops, both involved in coupled motions, are regions of high connectivity in the network. Our results suggest that independent of the effect on protein stability, altered protein dynamics, due to long-range communication within its structure, may underlie the aggregation of mutant SOD1 in FALS

Atomic structures of peptide self-assembly mimics

Although the β-rich self-assemblies are a major structural class for polypeptides and the focus of intense research, little is known about their atomic structures and dynamics due to their insoluble and noncrystalline nature. We developed a protein engineering strategy that captures a self-assembly segment in a water-soluble molecule. A predefined number of self-assembling peptide units are linked, and the β-sheet ends are capped to prevent aggregation, which yields a mono-dispersed soluble protein. We tested this strategy by using Borrelia outer surface protein (OspA) whose single-layer β-sheet located between two globular domains consists of two β-hairpin units and thus can be considered as a prototype of self-assembly. We constructed self-assembly mimics of different sizes and determined their atomic structures using x-ray crystallography and NMR spectroscopy. Highly regular β-sheet geometries were maintained in these structures, and peptide units had a nearly identical conformation, supporting the concept that a peptide in the regular β-geometry is primed for self-assembly. However, we found small but significant differences in the relative orientation between adjacent peptide units in terms of β-sheet twist and bend, suggesting their inherent flexibility. Modeling shows how this conformational diversity, when propagated over a large number of peptide units, can lead to a substantial degree of nanoscale polymorphism of self-assemblies

Cytochrome c polymerization by successive domain swapping at the C-terminal helix

Cytochrome c (cyt c) is a stable protein that functions in a monomeric state as an electron donor for cytochrome c oxidase. It is also released to the cytosol when permeabilization of the mitochondrial outer membrane occurs at the early stage of apoptosis. For nearly half a century, it has been known that cyt c forms polymers, but the polymerization mechanism remains unknown. We found that cyt c forms polymers by successive domain swapping, where the C-terminal helix is displaced from its original position in the monomer and Met-heme coordination is perturbed significantly. In the crystal structures of dimeric and trimeric cyt c, the C-terminal helices are replaced by the corresponding domain of other cyt c molecules and Met80 is dissociated from the heme. The solution structures of dimeric, trimeric, and tetrameric cyt c were linear based on small-angle X-ray scattering measurements, where the trimeric linear structure shifted toward the cyclic structure by addition of PEG and (NH4)2HPO4. The absorption and CD spectra of high-order oligomers (∼40 mer) were similar to those of dimeric and trimeric cyt c but different from those of monomeric cyt c. For dimeric, trimeric, and tetrameric cyt c, the ΔH of the oligomer dissociation to monomers was estimated to be about -20 kcal/mol per protomer unit, where Met-heme coordination appears to contribute largely to ΔH. The present results suggest that cyt c polymerization occurs by successive domain swapping, which may be a common mechanism of protein polymerization