Integrative determination of the atomic structure of mutant huntingtin exon 1 fibrils from Huntington's disease.

Neurodegeneration in Huntington's disease (HD) is accompanied by the aggregation of fragments of the mutant huntingtin protein, a biomarker of disease progression. A particular pathogenic role has been attributed to the aggregation-prone huntingtin exon 1 (HttEx1) fragment, whose polyglutamine (polyQ) segment is expanded. Unlike amyloid fibrils from Parkinson's and Alzheimer's diseases, the atomic-level structure of HttEx1 fibrils has remained unknown, limiting diagnostic and treatment efforts. We present and analyze the structure of fibrils formed by polyQ peptides and polyQ-expanded HttEx1. Atomic-resolution perspectives are enabled by an integrative analysis and unrestrained all-atom molecular dynamics (MD) simulations incorporating experimental data from electron microscopy (EM), solid-state NMR, and other techniques. Visualizing the HttEx1 subdomains in atomic detail helps explaining the biological properties of these protein aggregates, as well as paves the way for targeting them for detection and degradation.

Selective excitation with recoupling pulse schemes uncover properties of disordered mineral phases in bone-like apatite grown with bone proteins.

Bone construction has been under intensive scrutiny for many years using numerous techniques. Solid-state NMR spectroscopy helped unravel key characteristics of the mineral structure in bone owing to its capability of analyzing crystalline and disordered phases at high-resolution. This has invoked new questions regarding the roles of persistent disordered phases in structural integrity and mechanical function of mature bone as well as regarding regulation of early events in formation of apatite by bone proteins which interact intimately with the different mineral phases to exert biological control. Here, spectral editing tethered to standard NMR techniques is employed to analyze bone-like apatite minerals prepared synthetically in the presence and absence of two non-collagenous bone proteins, osteocalcin and osteonectin. A 1H spectral editing block allows excitation of species from the crystalline and disordered phases selectively, facilitating analysis of phosphate or carbon species in each phase by magnetization transfer via cross polarization. Further characterization of phosphate proximities using SEDRA dipolar recoupling, cross-phase magnetization transfer using DARR and T1/T2 relaxation times demonstrate that the mineral phases formed in the presence of bone proteins are more complex than bimodal. They reveal disparities in the physical properties of the mineral layers, indicate the layers in which the proteins reside and highlight the effect that each protein imparts across the mineral layers.

Selective observation of semi-rigid non-core residues in dynamically complex mutant huntingtin protein fibrils.

Many amyloid-forming proteins, which are normally intrinsically disordered, undergo a disorder-to-order transition to form fibrils with a rigid ß-sheet core flanked by disordered domains. Solid-state NMR (ssNMR) and cryogenic electron microscopy (cryoEM) excel at resolving the rigid structures within amyloid cores but studying the dynamically disordered domains remains challenging. This challenge is exemplified by mutant huntingtin exon 1 (HttEx1), which self-assembles into pathogenic neuronal inclusions in Huntington disease (HD). The mutant protein's expanded polyglutamine (polyQ) segment forms a fibril core that is rigid and sequestered from the solvent. Beyond the core, solvent-exposed surface residues mediate biological interactions and other properties of fibril polymorphs. Here we deploy magic angle spinning ssNMR experiments to probe for semi-rigid residues proximal to the fibril core and examine how solvent dynamics impact the fibrils' segmental dynamics. Dynamic spectral editing (DYSE) 2D ssNMR based on a combination of cross-polarization (CP) ssNMR with selective dipolar dephasing reveals the weak signals of solvent-mobilized glutamine residues, while suppressing the normally strong background of rigid core signals. This type of 'intermediate motion selection' (IMS) experiment based on cross-polarization (CP) ssNMR, is complementary to INEPT- and CP-based measurements that highlight highly flexible or highly rigid protein segments, respectively. Integration of the IMS-DYSE element in standard CP-based ssNMR experiments permits the observation of semi-rigid residues in a variety of contexts, including in membrane proteins and protein complexes. We discuss the relevance of semi-rigid solvent-facing residues outside the fibril core to the latter's detection with specific dyes and positron emission tomography tracers.

Regulatory inter-domain interactions influence Hsp70 recruitment to the DnaJB8 chaperone.

The Hsp40/Hsp70 chaperone families combine versatile folding capacity with high substrate specificity, which is mainly facilitated by Hsp40s. The structure and function of many Hsp40s remain poorly understood, particularly oligomeric Hsp40s that suppress protein aggregation. Here, we used a combination of biochemical and structural approaches to shed light on the domain interactions of the Hsp40 DnaJB8, and how they may influence recruitment of partner Hsp70s. We identify an interaction between the J-Domain (JD) and C-terminal domain (CTD) of DnaJB8 that sequesters the JD surface, preventing Hsp70 interaction. We propose a model for DnaJB8-Hsp70 recruitment, whereby the JD-CTD interaction of DnaJB8 acts as a reversible switch that can control the binding of Hsp70. These findings suggest that the evolutionarily conserved CTD of DnaJB8 is a regulatory element of chaperone activity in the proteostasis network.

Osteopontin regulates biomimetic calcium phosphate crystallization from disordered mineral layers covering apatite crystallites.

Details of apatite formation and development in bone below the nanometer scale remain enigmatic. Regulation of mineralization was shown to be governed by the activity of non-collagenous proteins with many bone diseases stemming from improper activity of these proteins. Apatite crystal growth inhibition or enhancement is thought to involve direct interaction of these proteins with exposed faces of apatite crystals. However, experimental evidence of the molecular binding events that occur and that allow these proteins to exert their functions are lacking. Moreover, recent high-resolution measurements of apatite crystallites in bone have shown that individual crystallites are covered by a persistent layer of amorphous calcium phosphate. It is therefore unclear whether non-collagenous proteins can interact with the faces of the mineral crystallites directly and what are the consequences of the presence of a disordered mineral layer to their functionality. In this work, the regulatory effect of recombinant osteopontin on biomimetic apatite is shown to produce platelet-shaped apatite crystallites with disordered layers coating them. The protein is also shown to regulate the content and properties of the disordered mineral phase (and sublayers within it). Through solid-state NMR atomic carbon-phosphorous distance measurements, the protein is shown to be located in the disordered phases, reaching out to interact with the surfaces of the crystals only through very few sidechains. These observations suggest that non-phosphorylated osteopontin acts as regulator of the coating mineral layers and exerts its effect on apatite crystal growth processes mostly from afar with a limited number of contact points with the crystal.

Protofilament Structure and Supramolecular Polymorphism of Aggregated Mutant Huntingtin Exon 1.

Huntington's disease is a progressive neurodegenerative disease caused by expansion of the polyglutamine domain in the first exon of huntingtin (HttEx1). The extent of expansion correlates with disease progression and formation of amyloid-like protein deposits within the brain. The latter display polymorphism at the microscopic level, both in cerebral tissue and in vitro. Such polymorphism can dramatically influence cytotoxicity, leading to much interest in the conditions and mechanisms that dictate the formation of polymorphs. We examine conditions that govern HttEx1 polymorphism in vitro, including concentration and the role of the non-polyglutamine flanking domains. Using electron microscopy, we observe polymorphs that differ in width and tendency for higher-order bundling. Strikingly, aggregation yields different polymorphs at low and high concentrations. Narrow filaments dominate at low concentrations that may be more relevant in vivo. We dissect the role of N- and C-terminal flanking domains using protein with the former (httNT or N17) largely removed. The truncated protein is generated by trypsin cleavage of soluble HttEx1 fusion protein, which we analyze in some detail. Dye binding and solid-state NMR studies reveal changes in fibril surface characteristics and flanking domain mobility. Higher-order interactions appear facilitated by the C-terminal tail, while the polyglutamine forms an amyloid core resembling those of other polyglutamine deposits. Fibril-surface-mediated branching, previously attributed to secondary nucleation, is reduced in absence of httNT. A new model for the architecture of the HttEx1 filaments is presented and discussed in context of the assembly mechanism and biological activity.

Structural Fingerprinting of Protein Aggregates by Dynamic Nuclear Polarization-Enhanced Solid-State NMR at Natural Isotopic Abundance.

A pathological hallmark of Huntington's disease (HD) is the formation of neuronal protein deposits containing mutant huntingtin fragments with expanded polyglutamine (polyQ) domains. Prior studies have shown the strengths of solid-state NMR (ssNMR) to probe the atomic structure of such aggregates, but have required in vitro isotopic labeling. Herein, we present an approach for the structural fingerprinting of fibrils through ssNMR at natural isotopic abundance (NA). These methods will enable the spectroscopic fingerprinting of unlabeled (e.g., ex vivo) protein aggregates and the extraction of valuable new long-range 13C-13C distance constraints.

Engineering l-asparaginase for spontaneous formation of calcium phosphate bioinspired microreactors.

Active bioinspired materials are appealing biotechnological targets, and their study is gaining momentum. These materials, which comprise of an inorganic matrix and one or more biomolecules, are extremely variable and therefore may result difficult to characterize in their intimate structure. In this work we have prepared a hydroxyapatite-l-asparaginase composite, with the perspective of using it in acute leukemia treatment. We demonstrate that the use of electron microscopy and powder X-ray diffraction, combined with the atomic-resolution information coming from solid-state NMR, allows us to understand the topology of the material and how the different components interplay to obtain an active composite.

Hidden motions and motion-induced invisibility: Dynamics-based spectral editing in solid-state NMR.

Solid-state nuclear magnetic resonance (ssNMR) spectroscopy enables the structural characterization of a diverse array of biological assemblies that include amyloid fibrils, non-amyloid aggregates, membrane-associated proteins and viral capsids. Such biological samples feature functionally relevant molecular dynamics, which often affect different parts of the sample in different ways. Solid-state NMR experiments' sensitivity to dynamics represents a double-edged sword. On the one hand, it offers a chance to measure dynamics in great detail. On the other hand, certain types of motion lead to signal loss and experimental inefficiencies that at first glance interfere with the application of ssNMR to overly dynamic proteins. Dynamics-based spectral editing (DYSE) ssNMR methods leverage motion-dependent signal losses to simplify spectra and enable the study of sub-structures with particular motional properties.

Changes to the Disordered Phase and Apatite Crystallite Morphology during Mineralization by an Acidic Mineral Binding Peptide from Osteonectin.

Noncollagenous proteins regulate the formation of the mineral constituent in hard tissue. The mineral formed contains apatite crystals coated by a functional disordered calcium phosphate phase. Although the crystalline phase of bone mineral was extensively investigated, little is known about the disordered layer's composition and structure, and less is known regarding the function of noncollagenous proteins in the context of this layer. In the current study, apatite was prepared with an acidic peptide (ON29) derived from the bone/dentin protein osteonectin. The mineral formed comprises needle-shaped hydroxyapatite crystals like in dentin and a stable disordered phase coating the apatitic crystals as shown using X-ray diffraction, transmission electron microscopy, and solid-state NMR techniques. The peptide, embedded between the mineral particles, reduces the overall phosphate content in the mineral formed as inferred from inductively coupled plasma and elemental analysis results. Magnetization transfers between disordered phase species and apatitic phase species are observed for the first time using 2D (1)H-(31)P heteronuclear correlation NMR measurements. The dynamics of phosphate magnetization transfers reveal that ON29 decreases significantly the amount of water molecules in the disordered phase and increases slightly their content at the ordered-disordered interface. The peptide decreases hydroxyl to disordered phosphate transfers within the surface layer but does not influence transfer within the bulk crystalline mineral. Overall, these results indicate that control of crystallite morphology and properties of the inorganic component in hard tissue by biomolecules is more involved than just direct interaction between protein functional groups and mineral crystal faces. Subtler mechanisms such as modulation of the disordered phase composition and structural changes at the ordered-disordered interface may be involved.

Trapping RNase A on MCM41 pores: effects on structure stability, product inhibition and overall enzymatic activity.

Catalytic activity of enzymes can be drastically modified by immobilization on surfaces of different materials. It is particularly effective when the dimensions of the biomolecules and adsorption sites on the material surfaces are commensurate. This can be utilized to hinder the biological activity of degradation enzymes and switch off undesired biological processes. Ribonucleases are particularly attractive targets for complete sequestration being efficient at disintegrating viable RNA molecules. Here we show that efficient quenching of ribonuclease A activity can be achieved by immobilization on the surface of MCM41 porous silica. Electron microscopy, isothermal titration calorimetry, differential scanning calorimetry and adsorption isotherm measurements of ribonuclease A on the MCM41 surface are used to demonstrate that the enzyme adsorbs on the external surface of the porous silica through electrostatic interactions that overcome the unfavorable entropy change as the protein gets trapped on the surface, and that immobilization shifts up its denaturation temperature by 20-25 °C. Real-time kinetic measurements, using single injection titration calorimetry, demonstrate that enzymatic activity towards hydrolysis of cyclic nucleotides is lowered by nearly two orders of magnitude on MCM41 and that active inhibition by the formed product is much less effective on the surface than in solution.