Geoffrey W. Platt

Fibril Growth Kinetics Reveal a Region of β2-microglobulin Important for Nucleation and Elongation of Aggregation

Amyloid is a highly ordered form of aggregate comprising long, straight and unbranched proteinaceous fibrils that are formed with characteristic nucleation-dependent kinetics in vitro. Currently, the structural molecular mechanism of fibril nucleation and elongation is poorly understood. Here, we investigate the role of the sequence and structure of the initial monomeric precursor in determining the rates of nucleation and elongation of human β2-microglobulin (β2m). We describe the kinetics of seeded and spontaneous (unseeded) fibril growth of wild-type β2m and 12 variants at pH 2.5, targeting specifically an aromatic-rich region of the polypeptide chain (residues 62–70) that has been predicted to be highly amyloidogenic. The results reveal the importance of aromatic residues in this part of the β2m sequence in fibril formation under the conditions explored and show that this region of the polypeptide chain is involved in both the nucleation and the elongation phases of fibril formation. Structural analysis of the conformational properties of the unfolded monomer for each variant using NMR relaxation methods revealed that all variants contain significant non-random structure involving two hydrophobic clusters comprising regions 29–51 and 58–79, the extent of which is critically dependent on the sequence. No direct correlation was observed, however, between the extent of non-random structure in the unfolded state and the rates of fibril nucleation and elongation, suggesting that the early stages of aggregation involve significant conformational changes from the initial unfolded state. Together, the data suggest a model for β2m amyloid formation in which structurally specific interactions involving the highly hydrophobic and aromatic-rich region comprising residues 62–70 provide a complementary interface that is key to the generation of amyloid fibrils for this protein at acidic pH.

Ligand binding to distinct states diverts aggregation of an amyloid-forming protein.

Although small molecules that modulate amyloid formation in vitro have been identified, significant challenges remain in determining precisely how these species act. Here we describe the identification of rifamycin SV as a potent inhibitor of β2m fibrillogenesis when added during the lag time of assembly or early during fibril elongation. Biochemical experiments demonstrate that the small molecule does not act by a colloidal mechanism. Exploiting the ability of electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) to resolve intermediates of amyloid assembly, we show instead that rifamycin SV inhibits β2m fibrillation by binding distinct monomeric conformers, disfavoring oligomer formation, and diverting the course of assembly to the formation of spherical aggregates. The results reveal the power of ESI-IMS-MS to identify specific protein conformers as targets for intervention in fibrillogenesis using small molecules and reveal a mechanism of action in which ligand binding diverts unfolded protein monomers towards alternative assembly pathways.

Magic Angle Spinning NMR Analysis of β2-Microglobulin Amyloid Fibrils in Two Distinct Morphologies

Beta(2)-microglobulin (beta(2)m) is the major structural component of amyloid fibrils deposited in a condition known as dialysis-related amyloidosis. Despite numerous studies that have elucidated important aspects of the fibril formation process in vitro, and a magic angle spinning (MAS) NMR study of the fibrils formed by a small peptide fragment, structural details of beta(2)m fibrils formed by the full-length 99-residue protein are largely unknown. Here, we present a site-specific MAS NMR analysis of fibrils formed by the full-length beta(2)m protein and compare spectra of fibrils prepared under two different conditions. Specifically, long straight (LS) fibrils are formed at pH 2.5, while a very different morphology denoted as worm-like (WL) fibrils is observed in preparations at pH 3.6. High-resolution MAS NMR spectra have allowed us to obtain (13)C and (15)N resonance assignments for 64 residues of beta(2)m in LS fibrils, including part of the highly mobile N-terminus. Approximately 25 residues did not yield observable signals. Chemical shift analysis of the sequentially assigned residues indicates that these fibrils contain an extensive beta-sheet core organized in a non-native manner, with a trans-P32 conformation. In contrast, WL fibrils exhibit more extensive dynamics and appear to have a smaller beta-sheet core than LS fibrils, although both cores seem to share some common elements. Our results suggest that the distinct macroscopic morphological features observed for the two types of fibrils result from variations in structure and dynamics at the molecular level.

Intermolecular Alignment in β2-Microglobulin Amyloid Fibrils

The deposition of amyloid-like fibrils, composed primarily of the 99-residue protein β2-microglobulin (β2m), is one of the characteristic symptoms of dialysis-related amyloidosis. Fibrils formed in vitro at low pH and low salt concentration share many properties with the disease related fibrils and have been extensively studied by a number of biochemical and biophysical methods. These fibrils contain a significant β-sheet core and have a complex cryoEM electron density profile. Here, we investigate the intrasheet arrangement of the fibrils by means of 15N−13C MAS NMR correlation spectroscopy. We utilize a fibril sample grown from a 50:50 mixture of 15N,12C- and 14N,13C-labeled β2m monomers, the latter prepared using 2-13C glycerol as the carbon source. Together with the use of ZF-TEDOR mixing, this sample allowed us to observe intermolecular 15N−13C backbone-to-backbone contacts with excellent resolution and good sensitivity. The results are consistent with a parallel, in-register arrangement of the protein subunits in the fibrils and suggest that a significant structural reorganization occurs from the native to the fibril state.

Competition between Intramolecular and Intermolecular Interactions in an Amyloid-Forming Protein

Despite much progress in understanding the folding and the aggregation processes of proteins, the rules defining their interplay have yet to be fully defined. This problem is of particular importance since many diseases are initiated by protein unfolding and hence the propensity to aggregate competes with intramolecular collapse and other folding events. Here, we describe the roles of intramolecular and intermolecular interactions in defining the length of the lag time and the apparent rate of elongation of the 100-residue protein human β2-microglobulin at pH 2.5, commencing from an acid-denatured state that lacks persistent structure but contains significant non-random hydrophobic interactions. Using a combination of site-directed mutagenesis, quantitative kinetic analysis and computational methods, we show that only a single region of about 10 residues in length, determines the rate of fibril formation, despite the fact that other regions exhibit a significant intrinsic propensity for aggregation. We rationalise these results by analysing the effect of incorporating the conformational properties of acid-unfolded β2-microglobulin and its variants at pH 2.5 as measured by NMR spectroscopy into the Zyggregator aggregation prediction algorithm. These results demonstrate that residual structure in the precursor state modulates the intrinsic propensity of the polypeptide chain to aggregate and that the algorithm developed here allows the key regions for aggregation to be more clearly identified and the rates of their self-association to be predicted. Given the common propensity of unfolded chains to form non-random intramolecular interactions as monomers and to self-assemble subsequently into amyloid fibrils, the approach developed should find widespread utility for the prediction of regions important in amyloid formation and their rates of self-assembly.

Stacked Sets of Parallel, In-register β-Strands of β2-Microglobulin in Amyloid Fibrils Revealed by Site-directed Spin Labeling and Chemical Labeling

β2-microglobulin (β2m) is a 99-residue protein with an immunoglobulin fold that forms β-sheet-rich amyloid fibrils in dialysis-related amyloidosis. Here the environment and accessibility of side chains within amyloid fibrils formed in vitro from β2m with a long straight morphology are probed by site-directed spin labeling and accessibility to modification with N-ethyl maleimide using 19 site-specific cysteine variants. Continuous wave electron paramagnetic resonance spectroscopy of these fibrils reveals a core predominantly organized in a parallel, in-register arrangement, by contrast with other β2m aggregates. A continuous array of parallel, in-register β-strands involving most of the polypeptide sequence is inconsistent with the cryoelectron microscopy structure, which reveals an architecture based on subunit repeats. To reconcile these data, the number of spins in close proximity required to give rise to spin exchange was determined. Systematic studies of a model protein system indicated that juxtaposition of four spin labels is sufficient to generate exchange narrowing. Combined with information about side-chain mobility and accessibility, we propose that the amyloid fibrils of β2m consist of about six β2m monomers organized in stacks with a parallel, in-register array. The results suggest an organization more complex than the accordion-like β-sandwich structure commonly proposed for amyloid fibrils.

Glimpses of the molecular mechanisms of β2-microglobulin fibril formation in vitro: Aggregation on a complex energy landscape

β2‐microglobulin (β2m) is a 99‐residue protein that aggregates to form amyloid fibrils in dialysis‐related amyloidosis. The protein provides a powerful model for exploration of the structural molecular mechanisms of fibril formation from a full‐length protein in vitro. Fibrils have been assembled from β2m under both low pH conditions, where the precursor is disordered, and at neutral pH where the protein is initially natively folded. Here we discuss the roles of sequence and structure in amyloid formation, the current understanding of the structural mechanisms of the early stages of aggregation of β2m at both low and neutral pH, and the common and distinct features of these assembly pathways.

Probing Dynamics within Amyloid Fibrils Using a Novel Capping Method

A host of diseases involve deposition of proteinaceous amyloid fibrils, which are highly ordered, noncovalent polymers that contain a cross-b architecture. Despite great interest in these fibers, knowledge of the atomic structure of amyloid is limited owing to the difficulty of studying these large heterogeneous biomolecules, especially those formed from long polypeptide chains, with any single biophysical method. Solid-state NMR spectroscopic methods have provided information on the arrangement of the polypeptide chain within amyloid-like structures, affording constraints for secondary, tertiary, and quaternary structure. Herein we study the manner in which the polypeptide chain of b2microglobulin (b2m), a 99-residue protein that forms amyloidlike fibrils in vitro and in vivo, is accommodated within its fibril architecture. By employing a novel method that decouples the interfering contributions of dynamic exchange between fibrillar and soluble material in structural analyses by solution NMR spectroscopy, we discern which regions of b2m are structured in the core of the fibrils, which are exposed, and which are dynamic. Limited proteolysis of b2m fibrils with pepsin has shown that the N-terminal nine residues are exposed to solvent and that digestion of this sample results in a homogeneous product in which 100% of the fibrils are cleaved at a single site (Val9) (Figure 1a and Supporting Information, Figure S1). These data are consistent with NMR spectroscopy hydrogen exchange experiments that reveal limited protection in the 20 N-terminal residues of these fibrils. However, little is known about the dynamics of the polypeptide chain when it is organized into the fibril structure. Recently solidstate NMR spectroscopy methods have identified flexible regions in amyloid fibrils, 7] and previous studies have indicated that mobile regions within large macromolecules can be observed by solution NMR spectroscopy, even though the size of the systems examined would usually prohibit the use of this technique. To better understand the structural organization of the polypeptide chain in b2m amyloid-like fibrils (Figure 1 b) and to identify possible mobile regions within this system, fibril formation of b2m was monitored in real time by H–N HSQC NMR spectroscopy (Figure 2a,b). In parallel, the progression of the fibrillation reaction was monitored by fluorescence of the amyloid-specific dye thioflavin-T as well as by imaging with TEM. Typical thioflavin-T-positive longstraight and twisted amyloid-like fibrils were observed at the conclusion of the reaction (Figure 1b). The initial NMR spectrum (Figure 2a), which was acquired as soon as the protein was placed under low-pH-value conditions, is typical of acid-unfolded b2m, in which a number of intense resonances are observed with limited chemical shift dispersion, indicative of a highly unfolded polypeptide chain. As the reaction proceeds, peak intensities throughout the protein sequence are decreased as monomeric protein is recruited to the fibrillar form, leading to broadened contributions to their linewidths. At the endpoint of the reaction (after 250 h), a surprising number of peaks remains visible in the spectrum (Figure 2b). Interestingly, no chemical-shift changes are Figure 1. a) Sequence of wild-type (WT) b2m and the variant with an extended N-terminal sequence. The fragments prone to pepsinolysis are highlighted within the dashed box and positions of secondary structure and the disulfide bond in the native state are indicated. b, c) Negative-stain TEM images of fibrils formed at pH 2.5 from WT b2m (b) and N-terminally extended b2m (c).