Dimers of D76N-ß2-microglobulin display potent antiamyloid aggregation activity.

Self-association of WT ß2-microglobulin (WT-ß2m) into amyloid fibrils is associated with the disorder dialysis related amyloidosis. In the familial variant D76N-ß2m, the single amino acid substitution enhances the aggregation propensity of the protein dramatically and gives rise to a disorder that is independent of renal dysfunction. Numerous biophysical and structural studies on WT- and D76N-ß2m have been performed in order to better understand the structure and dynamics of the native proteins and their different potentials to aggregate into amyloid. However, the structural properties of transient D76N-ß2m oligomers and their role(s) in assembly remained uncharted. Here, we have utilized NMR methods, combined with photo-induced crosslinking, to detect, trap, and structurally characterize transient dimers of D76N-ß2m. We show that the crosslinked D76N-ß2m dimers have different structures from those previously characterized for the on-pathway dimers of ΔN6-ß2m and are unable to assemble into amyloid. Instead, the crosslinked D76N-ß2m dimers are potent inhibitors of amyloid formation, preventing primary nucleation and elongation/secondary nucleation when added in substoichiometric amounts with D76N-ß2m monomers. The results highlight the specificity of early protein-protein interactions in amyloid formation and show how mapping these interfaces can inform new strategies to inhibit amyloid assembly.

Chasing long-range evolutionary couplings in the AlphaFold era.

Coevolution between protein residues is normally interpreted as direct contact. However, the evolutionary record of a protein sequence contains rich information that may include long-range functional couplings, couplings that report on homo-oligomeric states or even conformational changes. Due to the complexity of the sequence space and the lack of structural information on various members of a protein family, it has been difficult to effectively mine the additional information encoded in a multiple sequence alignment (MSA). Here, taking advantage of the recent release of the AlphaFold (AF) database we attempt to identify coevolutionary couplings that cannot be explained simply by spatial proximity. We propose a simple computational method that performs direct coupling analysis on a MSA and searches for couplings that are not satisfied in any of the AF models of members of the identified protein family. Application of this method on 2012 protein families suggests that ~12% of the total identified coevolving residue pairs are spatially distant and more likely to be disordered than their contacting counterparts. We expect that this analysis will help improve the quality of coevolutionary distance restraints used for structure determination and will be useful in identifying potentially functional/allosteric cross-talk between distant residues.

An S/T motif controls reversible oligomerization of the Hsp40 chaperone DNAJB6b through subtle reorganization of a ß sheet backbone.

Chaperone oligomerization is often a key aspect of their function. Irrespective of whether chaperone oligomers act as reservoirs for active monomers or exhibit a chaperoning function themselves, understanding the mechanism of oligomerization will further our understanding of how chaperones maintain the proteome. Here, we focus on the class-II Hsp40, human DNAJB6b, a highly efficient inhibitor of protein self-assembly in vivo and in vitro that forms functional oligomers. Using single-quantum methyl-based relaxation dispersion NMR methods we identify critical residues for DNAJB6b oligomerization in its C-terminal domain (CTD). Detailed solution NMR studies on the structure of the CTD showed that a serine/threonine-rich stretch causes a backbone twist in the N-terminal ß strand, stabilizing the monomeric form. Quantitative analysis of an array of NMR relaxation-based experiments (including Carr-Purcell-Meiboom-Gill relaxation dispersion, off-resonance R1ρ profiles, lifetime line broadening, and exchange-induced shifts) on the CTD of both wild type and a point mutant (T142A) within the S/T region of the first ß strand delineates the kinetics of the interconversion between the major twisted-monomeric conformation and a more regular ß strand configuration in an excited-state dimer, as well as exchange of both monomer and dimer species with high-molecular-weight oligomers. These data provide insights into the molecular origins of DNAJB6b oligomerization. Further, the results reported here have implications for the design of ß sheet proteins with tunable self-assembling properties and pave the way to an atomic-level understanding of amyloid inhibition.

Visualization of transient protein-protein interactions that promote or inhibit amyloid assembly.

In the early stages of amyloid formation, heterogeneous populations of oligomeric species are generated, the affinity, specificity, and nature of which may promote, inhibit, or define the course of assembly. Despite the importance of the intermolecular interactions that initiate amyloid assembly, our understanding of these events remains poor. Here, using amyloidogenic and nonamyloidogenic variants of ß2-microglobulin, we identify the interactions that inhibit or promote fibril formation in atomic detail. The results reveal that different outcomes of assembly result from biomolecular interactions involving similar surfaces. Specifically, inhibition occurs via rigid body docking of monomers in a head-to-head orientation to form kinetically trapped dimers. By contrast, the promotion of fibrillation involves relatively weak protein association in a similar orientation, which results in conformational changes in the initially nonfibrillogenic partner. The results highlight the complexity of interactions early in amyloid assembly and reveal atomic-level information about species barriers in amyloid formation.

Unraveling the structure and dynamics of the human DNAJB6b chaperone by NMR reveals insights into Hsp40-mediated proteostasis.

J-domain chaperones are involved in the efficient handover of misfolded/partially folded proteins to Hsp70 but also function independently to protect against cell death. Due to their high flexibility, the mechanism by which they regulate the Hsp70 cycle and how specific substrate recognition is performed remains unknown. Here we focus on DNAJB6b, which has been implicated in various human diseases and represents a key player in protection against neurodegeneration and protein aggregation. Using a variant that exists mainly in a monomeric form, we report the solution structure of an Hsp40 containing not only the J and C-terminal substrate binding (CTD) domains but also the functionally important linkers. The structure reveals a highly dynamic protein in which part of the linker region masks the Hsp70 binding site. Transient interdomain interactions via regions crucial for Hsp70 binding create a closed, autoinhibited state and help retain the monomeric form of the protein. Detailed NMR analysis shows that the CTD (but not the J domain) self-associates to form an oligomer comprising ∼35 monomeric units, revealing an intricate balance between intramolecular and intermolecular interactions. The results shed light on the mechanism of autoregulation of the Hsp70 cycle via conserved parts of the linker region and reveal the mechanism of DNAJB6b oligomerization and potentially antiaggregation.

Microsecond Backbone Motions Modulate the Oligomerization of the DNAJB6 Chaperone.

DNAJB6 is a prime example of an anti-aggregation chaperone that functions as an oligomer. DNAJB6 oligomers are dynamic and subunit exchange is critical for inhibiting client protein aggregation. The T193A mutation in the C-terminal domain (CTD) of DNAJB6 reduces both chaperone self-oligomerization and anti-aggregation of client proteins, and has recently been linked to Parkinson's disease. Here, we show by NMR, including relaxation-based methods, that the T193A mutation has minimal effects on the structure of the ß-stranded CTD but increases the population and rate of formation of a partially folded state. The results can be rationalized in terms of ß-strand peptide plane flips that occur on a timescale of ≈100 µs and lead to global changes in the overall pleat/flatness of the CTD, thereby altering its ability to oligomerize. These findings help forge a link between chaperone dynamics, oligomerization and anti-aggregation activity which may possibly lead to new therapeutic avenues tuned to target specific substrates.

Extracellular matrix components modulate different stages in ß2-microglobulin amyloid formation.

Amyloid deposition of WT human ß2-microglobulin (WT-hß2m) in the joints of long-term hemodialysis patients is the hallmark of dialysis-related amyloidosis. In vitro, WT-hß2m does not form amyloid fibrils at physiological pH and temperature unless co-solvents or other reagents are added. Therefore, understanding how fibril formation is initiated and maintained in the joint space is important for elucidating WT-hß2m aggregation and dialysis-related amyloidosis onset. Here, we investigated the roles of collagen I and the commonly administered anticoagulant, low-molecular-weight (LMW) heparin, in the initiation and subsequent aggregation phases of WT-hß2m in physiologically relevant conditions. Using thioflavin T fluorescence to study the kinetics of amyloid formation, we analyzed how these two agents affect specific stages of WT-hß2m assembly. Our results revealed that LMW-heparin strongly promotes WT-hß2m fibrillogenesis during all stages of aggregation. However, collagen I affected WT-hß2m amyloid formation in contrasting ways: decreasing the lag time of fibril formation in the presence of LMW-heparin and slowing the rate at higher concentrations. We found that in self-seeded reactions, interaction of collagen I with WT-hß2m amyloid fibrils attenuates surface-mediated growth of WT-hß2m fibrils, demonstrating a key role of secondary nucleation in WT-hß2m amyloid formation. Interestingly, collagen I fibrils did not suppress surface-mediated assembly of WT-hß2m monomers when cross-seeded with fibrils formed from the N-terminally truncated variant ΔN6-hß2m. Together, these results provide detailed insights into how collagen I and LMW-heparin impact different stages in the aggregation of WT-hß2m into amyloid, which lead to dramatic effects on the time course of assembly.

Modulation of Amyloidogenic Protein Self-Assembly Using Tethered Small Molecules.

Protein-protein interactions (PPIs) are involved in many of life's essential biological functions yet are also an underlying cause of several human diseases, including amyloidosis. The modulation of PPIs presents opportunities to gain mechanistic insights into amyloid assembly, particularly through the use of methods which can trap specific intermediates for detailed study. Such information can also provide a starting point for drug discovery. Here, we demonstrate that covalently tethered small molecule fragments can be used to stabilize specific oligomers during amyloid fibril formation, facilitating the structural characterization of these assembly intermediates. We exemplify the power of covalent tethering using the naturally occurring truncated variant (ΔN6) of the human protein ß2-microglobulin (ß2m), which assembles into amyloid fibrils associated with dialysis-related amyloidosis. Using this approach, we have trapped tetramers formed by ΔN6 under conditions which would normally lead to fibril formation and found that the degree of tetramer stabilization depends on the site of the covalent tether and the nature of the protein-fragment interaction. The covalent protein-ligand linkage enabled structural characterization of these trapped, off-pathway oligomers using X-ray crystallography and NMR, providing insight into why tetramer stabilization inhibits amyloid assembly. Our findings highlight the power of "post-translational chemical modification" as a tool to study biological molecular mechanisms.

Optimized selection of slow-relaxing 13C transitions in methyl groups of proteins: application to relaxation dispersion.

Optimized selection of the slow-relaxing components of single-quantum 13C magnetization in 13CH3 methyl groups of proteins using acute (< 90°) angle 1H radio-frequency pulses, is described. The optimal selection scheme is more relaxation-tolerant and provides sensitivity gains in comparison to the experiment where the undesired (fast-relaxing) components of 13C magnetization are simply 'filtered-out' and only 90° 1H pulses are employed for magnetization transfer to and from 13C nuclei. When applied to methyl 13C single-quantum Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments for studies of chemical exchange, the selection of the slow-relaxing 13C transitions results in a significant decrease in intrinsic (exchange-free) transverse spin relaxation rates of all exchanging species. For exchanging systems involving high-molecular-weight species, the lower transverse relaxation rates translate into an increase in the information content of the resulting relaxation dispersion profiles.

Determining methyl sidechain conformations in a CS-ROSETTA model using methyl 1H-13C residual dipolar couplings.

Modelling of protein structures based on backbone chemical shifts, using programs such as CS-ROSETTA, is becoming increasingly popular, especially for systems where few restraints are available or where homologous structures are already known. While the reliability of CS-ROSETTA calculations can be improved by incorporation of some additional backbone NMR data such as those afforded by residual dipolar couplings or minimal NOE data sets involving backbone amide protons, the sidechain conformations are largely modelled by statistical energy terms. Here, we present a simple method based on methyl residual dipolar couplings that can be used to determine the rotameric state of the threefold symmetry axis of methyl groups that occupy a single rotamer, determine rotameric distributions, and identify regions of high flexibility. The method is demonstrated for methyl side chains of a deletion variant of the human chaperone DNAJB6b.

Optimized NMR Experiments for the Isolation of I=1/2 Manifold Transitions in Methyl Groups of Proteins.

Optimized NMR experiments are developed for isolating magnetization belonging to the I=1/2 manifolds of 13 CH3 methyl groups in proteins, enabling the manipulation of the magnetization of a 13 CH3 moiety as if it were an AX (1 H-13 C) spin-system. These experiments result in the same 'simplification' of a 13 CH3 spin-system that would be obtained from the production of {13 CHD2 }-methyl-labeled protein samples. The sensitivity of I=1/2 manifold-selection experiments is a factor of approximately 2 less than that of the corresponding experiments acquired on {13 CHD2 }-labeled methyl groups. The methodology described here is primarily intended for small-to-medium sized proteins, where the losses in sensitivity associated with the isolation of I=1/2 manifold transitions can be tolerated. Several NMR applications that benefit from simplification of the 13 CH3 (AX3 ) spin-systems are described, with an emphasis on the measurements of methyl 1 H-13 C residual dipolar couplings in a {13 CH3 }-methyl-labeled deletion mutant of the human chaperone DNAJB6b, where modulation of NMR signal intensities due to evolution of methyl 1 H-13 C scalar and dipolar couplings follows a simple cosine function characteristic of an AX (1 H-13 C) spin-system, significantly simplifying data analysis.

Magic-Angle-Pulse Driven Separation of Degenerate 1 H Transitions in Methyl Groups of Proteins: Application to Studies of Methyl Axis Dynamics.

Dynamics of protein side chains is one of the principal determinants of conformational entropy in protein structures and molecular recognition events. We describe NMR experiments that rely on the use of magic-angle pulses for efficient isolation of degenerate 1 H transitions of the I=3/2 manifold of 13 CH3 methyl groups, and serve as 'building blocks' for the measurement of transverse spin relaxation rates of the fast- and slow-relaxing 1 H transitions - the primary quantitative reporters of methyl axis dynamics in selectively {13 CH3 }-methyl-labelled, highly deuterated proteins. The magic-angle-pulse driven experiments are technically simpler and, in the absence of relaxation, predicted to be 2.3-fold more sensitive than previously developed analogous schemes. Validation of the methodology on a sample of {13 CH3 }-labeled ubiquitin demonstrates quantitative agreement between order parameters of methyl three-fold symmetry axis obtained with magic-angle-pulse driven experiments and other established NMR techniques, paving the way for studies of methyl axis dynamics in human DNAJB6b chaperone, a protein that undergoes exchange with high-molecular-weight oligomeric species.

Identification of a novel site of interaction between ataxin-3 and the amyloid aggregation inhibitor polyglutamine binding peptide 1.

Amyloid diseases represent a growing social and economic burden in the developed world. Understanding the assembly pathway and the inhibition of amyloid formation is key to developing therapies to treat these diseases. The neurodegenerative condition Machado-Joseph disease is characterised by the self-aggregation of the protein ataxin-3. Ataxin-3 consists of a globular N-terminal Josephin domain, which can aggregate into curvilinear protofibrils, and an unstructured, dynamically disordered C-terminal domain containing three ubiquitin interacting motifs separated by a polyglutamine stretch. Upon expansion of the polyglutamine region above 50 residues, ataxin-3 undergoes a second stage of aggregation in which long, straight amyloid fibrils form. A peptide inhibitor of polyglutamine aggregation, known as polyQ binding peptide 1, has been shown previously to prevent the maturation of ataxin-3 fibrils. However, the mechanism of this inhibition remains unclear. Using nanoelectrospray ionisation-mass spectrometry, we demonstrate that polyQ binding peptide 1 binds to monomeric ataxin-3. By investigating the ability of polyQ binding peptide 1 to bind to truncated ataxin-3 constructs lacking one or more domains, we localise the site of this interaction to a 39-residue sequence immediately C-terminal to the Josephin domain. The results suggest a new mechanism for the inhibition of polyglutamine aggregation by polyQ binding peptide 1 in which binding to a region outside of the polyglutamine tract can prevent fibril formation, highlighting the importance of polyglutamine flanking regions in controlling aggregation and disease.

pH-induced molecular shedding drives the formation of amyloid fibril-derived oligomers.

Amyloid disorders cause debilitating illnesses through the formation of toxic protein aggregates. The mechanisms of amyloid toxicity and the nature of species responsible for mediating cellular dysfunction remain unclear. Here, using ß2-microglobulin (ß2m) as a model system, we show that the disruption of membranes by amyloid fibrils is caused by the molecular shedding of membrane-active oligomers in a process that is dependent on pH. Using thioflavin T (ThT) fluorescence, NMR, EM and fluorescence correlation spectroscopy (FCS), we show that fibril disassembly at pH 6.4 results in the formation of nonnative spherical oligomers that disrupt synthetic membranes. By contrast, fibril dissociation at pH 7.4 results in the formation of nontoxic, native monomers. Chemical cross-linking or interaction with hsp70 increases the kinetic stability of fibrils and decreases their capacity to cause membrane disruption and cellular dysfunction. The results demonstrate how pH can modulate the deleterious effects of preformed amyloid aggregates and suggest why endocytic trafficking through acidic compartments may be a key factor in amyloid disease.

A Population Shift between Sparsely Populated Folding Intermediates Determines Amyloidogenicity.

The balance between protein folding and misfolding is a crucial determinant of amyloid assembly. Transient intermediates that are sparsely populated during protein folding have been identified as key players in amyloid aggregation. However, due to their ephemeral nature, structural characterization of these species remains challenging. Here, using the power of nonuniformly sampled NMR methods we investigate the folding pathway of amyloidogenic and nonamyloidogenic variants of ß2-microglobulin (ß2m) in atomic detail. Despite folding via common intermediate states, we show that the decreased population of the aggregation-prone ITrans state and population of a less stable, more dynamic species ablate amyloid formation by increasing the energy barrier for amyloid assembly. The results show that subtle changes in conformational dynamics can have a dramatic effect in determining whether a protein is amyloidogenic, without perturbation of the mechanism of protein folding.

Distinguishing closely related amyloid precursors using an RNA aptamer.

Although amyloid fibrils assembled in vitro commonly involve a single protein, fibrils formed in vivo can contain multiple protein sequences. The amyloidogenic protein human ß2-microglobulin (hß2m) can co-polymerize with its N-terminally truncated variant (ΔN6) in vitro to form hetero-polymeric fibrils that differ from their homo-polymeric counterparts. Discrimination between the different assembly precursors, for example by binding of a biomolecule to one species in a mixture of conformers, offers an opportunity to alter the course of co-assembly and the properties of the fibrils formed. Here, using hß2m and its amyloidogenic counterpart, ΔΝ6, we describe selection of a 2'F-modified RNA aptamer able to distinguish between these very similar proteins. SELEX with a N30 RNA pool yielded an aptamer (B6) that binds hß2m with an EC50 of ∼200 nM. NMR spectroscopy was used to assign the (1)H-(15)N HSQC spectrum of the B6-hß2m complex, revealing that the aptamer binds to the face of hß2m containing the A, B, E, and D ß-strands. In contrast, binding of B6 to ΔN6 is weak and less specific. Kinetic analysis of the effect of B6 on co-polymerization of hß2m and ΔN6 revealed that the aptamer alters the kinetics of co-polymerization of the two proteins. The results reveal the potential of RNA aptamers as tools for elucidating the mechanisms of co-assembly in amyloid formation and as reagents able to discriminate between very similar protein conformers with different amyloid propensity.

Biomolecular NMR in the AI-assisted structural biology era: Old tricks and new opportunities.

Over the last 40 years nuclear magnetic resonance (NMR) spectroscopy has established itself as one of the most versatile techniques for the characterization of biomolecules, especially proteins. Given the molecular size limitations of NMR together with recent advances in cryo-electron microscopy and artificial intelligence-assisted protein structure prediction, the bright future of NMR in structural biology has been put into question. In this mini review we argue the contrary. We discuss the unique opportunities solution NMR offers to the protein chemist that distinguish it from all other experimental or computational methods, and how it can benefit from machine learning.

Understanding ß-strand mediated protein-protein interactions: tuning binding behaviour of intrinsically disordered sequences by backbone modification.

A significant challenge in chemical biology is to understand and modulate protein-protein interactions (PPIs). Given that many PPIs involve a folded protein domain and a peptide sequence that is intrinsically disordered in isolation, peptides represent powerful tools to understand PPIs. Using the interaction between small ubiquitin-like modifier (SUMO) and SUMO-interacting motifs (SIMs), here we show that N-methylation of the peptide backbone can effectively restrict accessible peptide conformations, predisposing them for protein recognition. Backbone N-methylation in appropriate locations results in faster target binding, and thus higher affinity, as shown by relaxation-based NMR experiments and computational analysis. We show that such higher affinities occur as a consequence of an increase in the energy of the unbound state, and a reduction in the entropic contribution to the binding and activation energies. Thus, backbone N-methylation may represent a useful modification within the peptidomimetic toolbox to probe ß-strand mediated interactions.

Monitoring the conformational ensemble and lipid environment of a mechanosensitive channel under cyclodextrin-induced membrane tension.

Membrane forces shift the equilibria of mechanosensitive channels enabling them to convert mechanical cues into electrical signals. Molecular tools to stabilize and methods to capture their highly dynamic states are lacking. Cyclodextrins can mimic tension through the sequestering of lipids from membranes. Here we probe the conformational ensemble of MscS by EPR spectroscopy, the lipid environment with NMR, and function with electrophysiology under cyclodextrin-induced tension. We show the extent of MscS activation depends on the cyclodextrin-to-lipid ratio, and that lipids are depleted slower when MscS is present. This has implications in MscS' activation kinetics when distinct membrane scaffolds such as nanodiscs or liposomes are used. We find MscS transits from closed to sub-conducting state(s) before it desensitizes, due to the lack of lipid availability in its vicinity required for closure. Our approach allows for monitoring tension-sensitive states in membrane proteins and screening molecules capable of inducing molecular tension in bilayers.

Large Chaperone Complexes Through the Lens of Nuclear Magnetic Resonance Spectroscopy.

Molecular chaperones are the guardians of the proteome inside the cell. Chaperones recognize and bind unfolded or misfolded substrates, thereby preventing further aggregation; promoting correct protein folding; and, in some instances, even disaggregating already formed aggregates. Chaperones perform their function by means of an array of weak protein-protein interactions that take place over a wide range of timescales and are therefore invisible to structural techniques dependent upon the availability of highly homogeneous samples. Nuclear magnetic resonance (NMR) spectroscopy, however, is ideally suited to study dynamic, rapidly interconverting conformational states and protein-protein interactions in solution, even if these involve a high-molecular-weight component. In this review, we give a brief overview of the principles used by chaperones to bind their client proteins and describe NMR methods that have emerged as valuable tools to probe chaperone-substrate and chaperone-chaperone interactions. We then focus on a few systems for which the application of these methods has greatly increased our understanding of the mechanisms underlying chaperone functions.