ABSTRACT
Amyloidogenesis is significant in both protein function and pathology. Amyloid formation of folded, globular proteins is commonly initiated by partial unfolding. However, how this unfolding event is triggered for proteins that are otherwise stable in their native environments is not well understood. The accumulation of the immunoglobulin protein β2-microglobulin (β2m) into amyloid plaques in the joints of long-term hemodialysis patients is the hallmark of Dialysis Related Amyloidosis (DRA). While β2m does not form amyloid unassisted near neutral pH in vitro, the localization of β2m deposits to joint spaces suggests a role for the local extracellular matrix (ECM) proteins, specifically collagens, in promoting amyloid formation. Indeed, collagen and other ECM components have been observed to facilitate β2m amyloid formation, but the large size and anisotropy of the complex, combined with the low affinity of these interactions, has limited atomic-level elucidation of the amyloid-promoting mechanism by these molecules. Using solution NMR approaches that uniquely probe weak interactions and large complexes, we are able to derive binding interfaces for collagen I on β2m and detect collagen I-induced µs–ms timescale dynamics in the β2m backbone. By combining solution NMR relaxation methods and 15N-dark state exchange saturation transfer experiments, we propose a model in which weak, multimodal collagen I–β2m interactions promote exchange with a minor population of an amyloid-competent species to induce fibrillogenesis. The results portray the intimate role of the environment in switching an innocuous protein into an amyloid-competent state, rationalizing the localization of amyloid deposits in DRA.
INTRODUCTION
Several proteins self-associate into amyloid fibrils, which in some cases have functional roles1-3, but for others are associated with debilitating human diseases4-6 including Alzheimer’s Disease, Parkinson’s Disease, Huntington’s Disease, type II diabetes, cataracts, and Dialysis Related Amyloidosis (DRA). The protein precursors of amyloid diseases have unrelated primary sequences and structures7, spanning natively unfolded (intrinsically disordered) states, such as α-synuclein, amyloid-β peptide, and tau8-10, to stable, globular proteins, such as β2-microglobulin (β2m), transthyretin, and immunoglobulin light chains11-13. Initiation of amyloid formation of the latter class of proteins requires unfolding or partial unfolding of monomeric precursors, which can transiently assume amyloid-competent state(s). This kinetic barrier may be lower for intrinsically disordered proteins. However, what triggers the initial unfolding and subsequent amyloidogenesis of natively folded globular proteins remains poorly understood.
Accumulation of β2m amyloid plaques in the joints of long-term hemodialysis patients leads to DRA and arthritic symptoms14-17. In healthy individuals, β2m dissociates from the major histocompatibility complex-I (MHC-I), is released into the plasma, and is carried to the kidneys for degradation18-19. However, when hemodialysis or peritoneal dialysis are required due to kidney failure, β2m is not efficiently removed from the plasma, leading to increased concentrations by up to 60-fold16, 20-21. Remarkably, despite being transported throughout the body, β2m accumulates into amyloid plaques specifically in skeletal tissues of dialysis patients16, 21-24. The mechanism(s) by which β2m fibrillizes in vivo is not well understood, since in isolation the wild-type protein (the major culprit of DRA) resists amyloid formation in physiological conditions, even at high (100 µM) concentrations25-26. It has been proposed that β2m amyloid localized in the joints could result, at least in part, from interactions with the major components of the extracellular matrix (ECM) in bone and cartilage: collagens I and II20-23 and glycosaminoglycans (GAGs)27-29. The binding affinities of β2m to these collagens have been shown to be in the µM–mM range30 with preference for collagen I27. Although the interaction is weak, it is nonetheless pathologically significant, as images of ex vivo DRA plaques reveal β2m amyloid covering the surface of collagen I fibrils21. Indeed, recent kinetics studies have revealed that ECM components, such as collagens21, 28-29 and GAGs28-29, 31-32, as well as pre-formed fibril seeds and other co-factors25-26, 28, 31-49, induce and modulate β2m amyloid formation. However, atomic details of how these components interact with, and induce, the amyloid formation of β2m have remained an open question.
The weak nature of the interaction and large, anisotropic shape of the β2m–collagen I complex create a challenge for deriving atomic-level information on how collagen I–β2m interactions initiate β2m amyloidogenesis. The immunoglobulin fold of monomeric β2m has dimensions of ∼ 4 nm × 2 nm × 2 nm, whereas the simplest triple helical unit of collagen I has strikingly larger dimensions of 300 nm × 1.5 nm × 1.5 nm. Collagen I triple helices assemble into even larger, structured fibrils that have diameters ranging from 10–500 nm and lengths on the µm-scale. Collagen I therefore presents as a large surface with numerous reactive groups for β2m interactions. These challenges are not insurmountable, however, as powerful solution nuclear magnetic resonance (NMR) spectroscopy methods can indirectly probe large, lowly populated complexes in site-specific detail that are invisible by other biophysical techniques.
In this study, by utilizing NMR spectroscopy experiments designed to probe large complexes, we are able to pinpoint the binding interface of wild-type β2m for collagen I at physiological pH and have shown it to involve both β-sheets of the native protein, suggestive of different binding modes for the same molecular complex. Residues identified at the binding interface include both hydrophobic and hydrophilic sidechains. Through 15N relaxation experiments, we have also found that collagen I increases the number of residues in β2m involved in conformational exchange on the µs–ms timescale. These regions include residues 6–11 (β-strand A), 36–39 (β-strand C), 51 (β-strand D), and 91–94 (β-strand G) in the edge β-strands and loop residues 15–20 (loop AB), 35 (loop BC), 52-53 (loop DE), 63 (loop DE), and 78 (loop EF), the dynamics and conformations of which are known to be important for β2m amyloid formation31, 38, 50-51. We propose that the weak interactions of collagen I with the β2m β-sheets promote exchange of the native protein with a minor population of amyloid-competent species that induce fibrillogenesis. This study illuminates how a protein component, collagen I, local to the environment in which β2m plaques are found, can interact with a stable, globular protein to initiate debilitating amyloid formation.
RESULTS
Collagen I induces β2m amyloid formation in a concentration-dependent manner
Since the direct interaction of β2m with collagen in the joint space has been proposed to induce β2m amyloid formation21, 27, we probed the β2m–collagen I interaction under physiological pH conditions (pH 7.4) using a solid-phase enzyme-linked immunosorbent assays (ELISA) (Figure 1A). Importantly, the results suggest a dose-dependent interaction of the two proteins, consistent with previously published results27, under the conditions employed here. The adhesion of β2m to casein was monitored as a negative control, for which no significant binding was observed (Figure 1A).
Having verified the β2m–collagen I interaction under physiological pH conditions, we next monitored amyloid growth of β2m in the presence or absence of collagen I by thioflavin T (ThT) fluorescence (Figure 1B). In the presence of 3.4 mg/ml collagen I (1:0.1 molar ratio β2m:collagen I), β2m amyloid is formed within 12–21 days (Figure 1B, blue), as evident by enhanced ThT fluorescence. This is not observed in the absence of collagen I in the same conditions, and collagen I alone does not show ThT fluorescence enhancement (Figure 1B). Notably, at lower concentrations of collagen I, lower β2m concentrations, or shorter timescales fibrils are not observed29, 31. Atomic force microscopy (AFM) images also show that β2m interacts with collagen I fibrils, with β2m coating the collagen I fibril surface before detectable fibril formation by ThT fluorescence, obscuring the characteristic collagen I fibril D-banding that is clearly observed in the absence of β2m (Figure 1C,D), consistent with previous results21. β2m alone (Figure 1E) does not aggregate in the conditions employed, with no fibrils or high molecular weight assemblies observed by AFM. These data confirm that adhesion of collagen I to β2m induces β2m amyloid formation under physiological conditions in vitro, while the protein is not able to form amyloid in the absence of collagen I.
Weak, but specific β2m–collagen I interactions observed through 15N-R2 perturbations
In order to understand mechanistic details by which collagen I interacts with β2m to initiate amyloid formation, we used solution NMR methods, which provide an excellent toolbox of approaches able to characterize residue-specific features of weak protein–protein interactions on multiple timescales52-55. A titration of collagen I into a β2m monomer solution showed no significant chemical shift perturbations in 1H– 15N heteronuclear single quantum correlation (HSQC) spectra (Figure S1). However, a residue-specific attrition of the peak intensities observed with increasing collagen I concentrations (Figure 2A), suggests chemical exchange between the bound and free states of β2m consistent with the low affinity of the interaction in these conditions (Kd ≈ 410 µM30). To minimize collagen I aggregation during the NMR experiments and to capture the most specific interactions, we proceeded with low collagen I concentrations (0.6–1.2 mg/ml) that displayed consistent residue-specific perturbations and kept samples at 10°C, allowing NMR spectra to be acquired for over one week without visible alterations in spectral quality. Addition of 1.2 mg/ml collagen I to 300 µM β2m resulted in a reduction in resonance intensity of all peaks, consistent with transient formation of a high molecular weight complex (Figure 2A). However, the greatest reduction in peak intensities occurred for residues in the eight β-strands of the wild-type protein (Figure 2A). These peak intensity losses are in part due to increased 15N-transverse relaxation rates (R2), which are sensitive to changes in internal motions on the ps–ns timescale and conformational exchange on the µs–ms timescale. Indeed, at these concentrations, we observe an overall increase in 15N-R2, but importantly, the increase is not uniform across all residues, but is residue specific, involving predominantly residues 2–3 (N-terminus), 7–11 (β-strand A), 16–19 (loop AB), 23–26 (β-strand B), 35–39 (β-strand C), 50–52 (β-strand D), 64, 66–69 (β-strand E), 79–82 (β-strand F), 85, 87 (loop FG), and 91–94 (C-terminal β-strand G) (Figure 2B-C). The increased 15N-R2 at these specific sites could have multiple origins, arising due to reduced backbone mobility upon direct interaction with collagen I and/or to line broadening due to exchange between species with different chemical shifts, especially since the observed 15N-ΔR2 is dependent on magnetic field (700 MHz vs. 900 MHz, Fig. S2). In order to disentangle these contributions to the increase in 15N-R2, we proceeded with two sets of NMR experiments: 15N-dark state exchange saturation transfer (DEST) experiments, which can identify residues interacting with the large complex, and in-phase Hahn-echo experiments, which detect conformational exchange on the µs–ms timescale.
Pinpointing the collagen I interaction interface on β2m through 15N-DEST
In order to determine which residues of β2m interact most intimately with collagen I, we used 15N-DEST experiments56-57. This experiment is optimal when there is a measurable increase in R2 due to formation of a transient, large complex that is NMR-invisible because of its high R2 and detects the exchange between an observable ‘light’ state (free monomeric β2m) and the NMR-invisible ‘dark’ state (the high molecular weight collagen I–β2m complex). In the DEST experiment, high molecular weight species with high R2 values, such as the collagen I–β2m complex, can be partially saturated by weak radiofrequency (RF) fields at frequency offsets where monomeric β2m is not saturated. Saturation transfer to the observable monomeric species by chemical exchange is detected as a loss in monomeric β2m signal intensity. The broadening of these DEST saturation profiles (reduced signal intensities at further frequency offsets) in the presence of collagen I, relative to in its absence, is therefore indicative of residues at the interaction interface (Figure 3A–B). The ‘broadness’ of the profiles was measured by calculating the DEST difference (Θ) for each residue, which is a measure of the relative effects of on-resonance and off-resonance 15N saturation. Using a saturation frequency of 350 Hz, we measured Θ as:, where ±30 kHz were the most off-resonance 15N offsets, and 15N offsets of ±4 kHz provide enough saturation transfer from bound to unbound β2m to show significant intensity loss without eliminating the signal in most cases. A substantial change in Θ (ΔΘ) upon addition of collagen I is reflective of residues at the binding interface (Figure 3A). Notably, we observe that the broadening of the DEST saturation profiles is residue-specific and not uniform across all β2m residues, with some residues showing no change in the DEST difference in the presence of collagen I (Figure 3A–B). Examples of DEST profiles in the presence or absence of collagen I for a residue that shows DEST due to collagen I binding (V82 in β-strand F) and one that does not (K41 in the C–C’ loop) are given in Figure 3B. In Figure 3A, those residues with ΔΘ larger than the mean, and likely have the most direct contacts with the collagen in the β2m–collagen I complex (shaded red), include residues 6–11 (β-strand A), 15–20 (loop AB), 21–26 (β-strand B), 35 (loop BC), 36–39 (β-strand C), 51 (β-strand D), 52–53 (loop DE), 63 (loop DE), 64–69 (β-strand E), 78 (loop EF), 79–83 (β-strand F), and 91–94 (β-strand G).
In addition, the full DEST profiles can be used to quantify residue-specific transverse relaxation values of β2m in the collagen I-bound state (R2bound) and exchange kinetics between the bound and unbound β2m. Since the ΔR2 may be due to more complex processes than collagen I binding alone, such as an overall increased viscosity due to the presence of the large collagen I molecules, we fit only the 15N DEST profiles of each residue with 150 Hz and 350 Hz RF saturation to the McConnell equations56-57. Fitting to a simple two-state model, the population of the unbound, monomeric β2m was determined to be 94 +/- 2% with an apparent first-order rate constant for the conversion of β2m from unbound to collagen I-bound conformation (konapp) of 6.4 +/- 0.8 s-1. We interpret the direct binding interface to be the residues with the highest R2bound. The 15N-R2bound profile shows a similar trend to the ΔΘ profile (Figure 3A, C), and suggests that binding interfaces for collagen I on β2m occur on both β-sheets. Examples of fitting to the experimental values of residues V82 (in a binding region) and K41 (away from interface) are shown in Figure 3D.
Collagen I induced conformational exchange in β2m revealed by 15N relaxation
The enhanced 15N-R2 of β2m may not only be due to binding with a high molecular weight species (such as in a large complex), but also to an increase in conformational exchange dynamics of β2m on the µs–ms timescale, since the 15N-ΔR2 is dependent on the magnetic field (Figure S2). In order to determine which residues in β2m are in conformational exchange in the presence of collagen I, we use 15N in-phase Hahn echo experiments (R2HE) to estimate the relaxation exchange rates. At pH 7.4 and 10°C, few residues in β2m have Rex values greater than 10s-1 in the absence of collagen I as measured by the in-phase Hahn echo experiments (Figure 4A). The N-terminus and residues in the BC and DE loops (for which several signals are unobservable) are natively in conformational exchange (Figure 4A). Upon addition of 0.6 mg/ml collagen I, the regions with high Rex are expanded to include the full N-terminal β-strand A, part of β-strand B to part of β-strand C, including the connecting BC loop, β-strand D, the DE loop, the C-terminal end of β-strand F into the FG loop, and the C-terminal β-strand G (Figure 4B). Conformational dynamics in specific regions of β2m, including the N-terminal region and the BC loop that contains cis Pro32, have been shown to be crucial in controlling the amyloidogenicity of the protein49, 58. Thus, the enhanced conformational exchange induced by the presence of collagen I may facilitate minor populations of amyloid-component states of β2m.
DISCUSSION
A novel collagen I binding surface on β2m
Amyloid formation of β2m at physiological pH in vitro requires assistance by co-factors21, 25-26, 28-29, 31-49. In particular, ECM molecules, such as collagens and GAGs have been targeted as amyloid-inducing co-factors, since β2m amyloid formation has been localized to musculoskeletal tissues16, 22-24. While previous experiments have focused on the kinetics of amyloid formation in the presence of these molecules21, 28-29, 31, 59, a detailed atomistic description of the interactions involved and how these may enhance β2m conformational dynamics and amyloid formation had not been elucidated. Here, we have used complementary NMR relaxation-based experiments to pinpoint residues of β2m involved in the collagen I binding interface and collagen I-induced dynamics that lead to enhanced β2m amyloid formation at neutral pH in vitro. The 15N-DEST experiments indicate that residues in β-strands A, B, C, D, E, F, and G form interaction surfaces with collagen I. These provide two surfaces of mixed hydrophilic and hydrophobic composition (Fig. S3). Both contain hydrophobic patches with the ABED β-sheet displaying several aromatic residues on the interaction surface (Fig. S3). Since both β-sheets on opposite sides of the molecule were determined to interact with the collagen I surface, binding must be multimodal involving interaction surfaces formed by K6, Q8, Y10, F22, N24, Y26, S52, Y63, L65, Y67, and E69 on the ABED β-sheet and E36, D38, L40, A79, R81, N83, I92, and K94 on the GFC β-sheet (Figure 3, S3). Comparison of the molecular dimensions of the interacting molecules (4 x 2 x 2 nm for β2m, 300 nm x 1.5 nm for a collagen I triple helix, and microns in length x up to 500 nm in diameter for mature collagen I fibrils) highlights the potential for a myriad of binding modes, enabling independent binding of several β2m molecules to the same collagen molecule (Figure 5A–B). Importantly, the collagen I triple helix surface is interspersed with numerous hydrophilic and hydrophobic residues along its length (Figure 5A). The collagen I fibril surface maintains this repeating pattern of surface chemistries (Figure 5B), enhancing the potential for multiple binding modes to complementary surfaces in β2m (Figure 5C).
Collagen I is known to interact with multiple immunoglobulin-like protein folds through binding interfaces that include both hydrophobic and hydrophilic residues. Interactions of collagen I with osteoclast-associated receptor (OSCAR), leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), and glycoprotein VI (GPVI), play functional roles in immune system regulation61-64 and platelet activation65-67. Similar to the β-sheet binding interface on β2m for collagen I identified here, the collagen I binding sites on OSCAR and LAIR-1 are also found in β-sheet regions68-69. In the case of the OSCAR–collagen I interactions, Tyr and Arg residues that line the interacting β-sheet of OSCAR have been suggested to play a primary role68. LAIR-1 binds primarily to collagen fragments rich in Gly, Pro and hydroxyproline (GPO) content, but also has been shown to interact with multiple binding motifs in collagen II and III toolkit peptides, some of which are not GPO rich69. NMR and mutagenesis studies on LAIR-1 have shown that depletion of Arg or Glu at the putative β-sheet interface showed decreased collagen binding, suggesting a role for electrostatic interactions70. GPVI, also recognizes GPO rich collagen motifs, however through a unique hydrophobic groove formed by a β-strand connecting loop that is flanked by hydrophilic residues71-73. Thus, although these proteins all share a similar immunoglobulin fold, each shows a unique binding interface to collagen, interacting in grooves formed by β-sheets or loops and having both hydrophobic and hydrophilic residues that each play fundamental roles in binding.
Collagen-induced conformational dynamics in β2m reflect amyloid prone dynamics
Beyond the structured collagen I-binding interface of β2m, using 15N relaxation experiments, we observe enhanced dynamics in the N- and C-termini, BC and FG loops, and the β-strand D of β2m upon complex formation. Enhanced dynamics in each of these regions has been proposed to play key roles in the aggregation mechanism of wild-type β2m38, 49-51, 74-84. Amyloid formation of β2m is nucleation dependent and proceeds through a near native folding intermediate, IT, that is in part defined by a non-native trans-His31-Pro32 peptide bond in the BC loop38, 50-51, 85-86. The cis-trans isomerization of Pro32 is aided by displacement of the N-terminal six residues, which destabilizes the BC loop, allowing β2m to sample multiple amyloidogenic conformations that enhance the rate of aggregation38, 50-51, 74, 79-81, 85-86. Deletion of the first six N-terminal residues in the naturally occurring variant, ΔN6, enhances the propensity for amyloid formation, and aggregation occurs in the absence of additional cofactors at physiological pH in vitro49, 74, 87-88. In addition, NMR relaxation experiments show enhanced dynamics in β-strand D and the DE loop of amyloidogenic ΔN649, which have been proposed to contribute to its higher aggregation propensity. NMR studies of a P32G-β2m variant, which inherently has a trans-His31-Gly32 peptide bond, showed significant line broadening in β-strands A and D and the BC and FG loops relative to WT-β2m38. This was interpreted to result from conformational conversion between the native and IT conformations38. The observation of increased Rex of these same regions upon addition of collagen I to WT-β2m, in this study, is consistent with the same regions undergoing conformational exchange from the native state to an amyloidogenic precursor consistent with the IT state, to enhance amyloid formation. Such a model provides a mechanism to enhance cis-trans Pro isomerization to initiate assembly into amyloid without the involvement of a prolyl isomerase.
A proposed mechanism of collagen I-driven β2m amyloidogenesis
With the new insights into the binding interface of collagen I on β2m and its impact on β2m dynamics described here, we propose a mechanistic view of how collagen I drives amyloidogenesis of β2m. In the presence of collagen I, the β-sheets of β2m are available for binding to the collagen I surface, with both β-sheets providing potential binding interfaces, indicative of multiple binding modes, rather that a unique and specific binding interface. The interaction between the two molecules is mediated by hydrophobic and electrostatic interactions (Figures 3, S3). In its native state, high transverse relaxation rates are observed in the apical loops of β2m, including the BC loop that contains cis Pro32 and the adjacent DE loop (Figure 2B). Additional dynamics upon collagen I binding are imposed on the N-terminus, β-strands B and C, BC loop, β-strand D, FG loop, and the C-terminal β-strand G (Figure 4B). Through modification of the dynamics of β2m in these sites, the probability of cis-trans isomerization of Pro32, known to be a key step in β2m fibril formation85-86, will be increased, with concomitant sampling of amyloid-competent species, including the IT state, known to promote amyloid formation38, 50-51 (Figure 6). The results provide a molecular explanation for the mechanism of deposition of β2m in collagenous-rich joints in dialysis patients16, 21-24. More generally, they also serve as an exemplar of the key role of the physiological environment in amyloid formation, by rationalizing the often remarkably specific deposition of amyloid to different tissues1, and in some cases, of different variants of the same protein in different tissues89-90. The methods used here to interrogate the weak-transient interaction of the large, β2m–collagen I complex can be extended to future studies to gain atomic-level insight into how other physiologically relevant cofactors promote amyloid formation of globular proteins involved in other amyloid diseases.
MATERIALS AND METHODS
Expression and purification of β2m
Wild-type β2m was expressed recombinantly in Escherichia coli BL21(DE3) pLysS cells by induction with 1 mM IPTG overnight at 37°C, following methods described previously39. Cells were lysed in 25 mM Tris-HCl buffer, pH 8.0 and with an Avestin Emulsiflex-C5 homogenizer. β2m is accumulated in inclusion bodies. To extract the β2m from inclusion bodies, the cell pellet was washed five times with 25 mM Tris-HCl buffer, pH 8.0 and solubilized in 25 mM Tris-HCl, pH 8.0 buffer containing 8 M urea, rocking overnight at room temperature. The protein was verified to be in the soluble fraction by SDS-PAGE. β2m was refolded by dialyzing against 25 mM Tris-HCl buffer, pH 8.0 at 4°C and purifying by anion exchange (HiTrap Q HP, GE Healthcare). The protein was further purified by size exclusion chromatography with a Superdex 75 gel filtration column (GE Life Sciences). Protein purity was verified by SDS-PAGE, and concentrations for experiments were determined by measuring the absorbance at 280 nm using a molar extinction coefficient of 19,060 M-1cm-1. [U-15N]-enriched β2m was expressed recombinantly for NMR using the same protocol in HCDM1 minimal media supplemented with 15N-ammonium chloride.
ELISA
Relative adhesion of variable concentrations of β2m to collagen I was determined by ELISA experiments. Nunc Maxisorp 96-well plates (Thermo Scientific) were coated with 100 µl of collagen I from rat tail tendon (BD Biosciences; 10 µg/ml in 10 mM acetic acid) overnight at 4°C. Uncoated areas on the plates were blocked with 200 µl of 0.5% w/v casein in binding buffer at room temperature for 1 hr. The binding and washing buffer consisted of PBS at pH 7.4 with 0.05% v/v Tween 20 (PBS-T) and 0.05% w/v casein as a non-specific blocking agent. After washing the wells three times with 200 µl washing buffer, 100 µl β2m in PBS-T and 0.05% w/v casein (10 µg/ml, 40 µg/ml, or 80 µg/ml) was added to the wells and incubated for 1 hr at room temperature. After three washes with 200 µl washing buffer, 100 µl mouse anti-β2m monoclonal antibody (1:2000 v/v in PBS-T and 0.05% w/v casein, Millipore Sigma) was bound to β2m in each well by incubating at room temperature for 1 hr. Subsequently, following three washes with 200 µl washing buffer, 100 µl of goat HRP-conjugated anti-mouse secondary antibody (1:5000 v/v dilution in PBS-T and 0.05% w/v casein, Genscript) was incubated in the wells at room temperature for 30 min. After washing for a final four times with 200 µl washing buffer, the binding of β2m to collagen I was detected through a colorimetric assay using a 3,3’,5,5’-tetramethylbenzidine substrate kit (Pierce) according to the manufacturer’s protocol, and measuring the absorbance at 450 nm using a Tecan Infinite F50 plate reader with Magellan software.
ThT fluorescence
Amyloid fibril formation was monitored by ThT fluorescence assays of β2m in the presence or absence of collagen I fibrils. Purified recombinant β2m lyophilized powder was dissolved in 100 µl of 10 mM sodium phosphate buffer, pH 7.4 to 1 mg/ml (85 µM). Collagen I fibrils were prepared by incubating 3.4 mg/ml collagen I (BD Biosciences) in PBS, pH 7.4 at 37°C for 1 hr. The fibril suspension was sonicated in a bath sonicator for 10 min and centrifuged at 16,500 rpm for 10 min to isolate fibrils. Collagen fibril pellets were resuspended in 100 µl of 10 mM sodium phosphate buffer, pH 7.4 in the presence or absence of β2m. Three or four samples were prepared for each condition and were transferred to a 96-well plate. ThT was added to each sample to a final concentration of 10 µM. ThT fluorescence was monitored over 22 days at 37°C with shaking at 600 rpm in a POLARstar Omega fluorimeter (BMG Labtech).
NMR
For all NMR experiments, purified recombinant [U-15N]-labeled β2m was diluted to 300 µM in TBS, pH 7.4 with 0.5 mg/ml casein and 10% v/v D2O. Before mixing, collagen I from rat tail tendon was dialyzed against TBS, pH 7.4. The concentration of collagen I after dialysis was determined by bicinchoninic acid assay (Pierce). All experiments were performed at 10°C. All data were collected on a 700 MHz Bruker AVIII or 900 MHz AVI NMR spectrometers equipped with TCI-cryo-probes. Data were processed in NMRPipe91 and analyzed in Sparky92.
1H-15N HSQC spectra
1H-15N HSQC spectra93-94 of [U-15N]-labeled β2m were acquired with different concentrations of collagen I (0, 0.12 mg/ml, and 1.2 mg/ml) in TBS, pH 7.4 with 0.5 mg/ml casein and 10% D2O at 10°C. The intensity ratio is taken as the intensity of a given cross-peak in the 1H-15N HSQC spectrum of β2m in the presence of collagen I relative to the intensity of the same cross-peak in the absence of collagen I, determined in Sparky92. The errors were propagated from the signal to noise ratio in each spectra.
15N-R2 and 15N-R2HE
[U-15N]-labeled β2m 15N transverse relaxation rates (R2) were measured from a series of HSQC-based 2D 1H-15N spectra using the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence95 with varying relaxation delays: in the absence of collagen I at 700 MHz-0, 16, 16, 32, 48, 64, 64, 80, 96, and 112 ms and 900 MHz-0, 16, 32, 32, 32, 48, 64, 80, 96, 112, and 128 ms and in the presence of 1.2 mg/ml collagen I at 700 mHz-0, 16, 16, 32, 48, 48, 64, and 80 ms and at 900 MHz-0, 16, 32, 32, 32, 48, 64, 80, and 96 ms. Relaxation delays used to quantify 15N-R2 rates of β2m in the presence of 0.6 mg/ml collagen I at 700 MHz were: 0, 8, 8, 16, 24, 32, and 56 ms and in the absence of collagen I: 0, 8, 8, 16, 24, 40, and 56 ms. 15N-R HE informs on the chemical exchange contribution to R2 by using an in-phase Hahn echo experiment96. Relaxation delays used in the R2HE experiment both in the presence and absence of 0.6 mg/ml collagen I were: 0.768, 7.68, 7.68, 15.4, 23, 38.5, and 61.4 ms. In each case, the R2 rates were determined by fitting peak intensities to a single exponential decay function. The chemical exchange contribution (Rex) for each β2m residue in the absence and presence of 0.6 mg/ml collagen I was determined as: Rex = R2HE-R2.
DEST experiments
The 15N-DEST experiment56-57 was applied to [U-15N]-labeled β2m in the presence or absence of 0.6 mg/ml collagen I. In this experiment, an 15N saturation pulse of 150 or 350 Hz was applied for 0.9 ms at different 15N frequency offsets: 0, ±1, ±2, ±4, ±8, ±14, ±21, and ±30 kHz. An experiment in which the 15N saturation pulse was set to 0 Hz with an offset of 30 kHz was also included as a reference. The 15N-DEST profiles were extracted for each residue as the peak intensity at each 15N saturation offset and were fitted to a two-state model using the destfit program by Clore and co-workers to obtain R2bound, pbound, and konapp56-57. The ΔΘ profile was obtained by measuring Θ for each β2m residue in the presence and absence of 0.6 mg/ml collagen I as: , and taking ΔΘ = Θ+col − Θ−col
ASSOCIATED CONTENT
Supporting Information
The Supporting Information includes figures that show an overlay of 1H-15N HSQC spectra of [U-15N]-β2m in the presence of 0 mg/ml, 0.12 mg/ml, or 1.2 mg/ml collagen I (Figure S1); 15N-R2 measurements of [U-15N]-β2m in the absence or presence of collagen I at 700 MHz or 900 MHz (Figure S2); and a model showing the amino acid composition of the interacting β2m β-sheets (Figure S3). (PDF)
AUTHOR INFORMATION
Notes
The authors declare no competing financial interests.
Funding Sources
This work was supported by American Heart Association Postdoctoral Fellowship 17POST33410326 to CLH and NIH grant GM45302 to JB. Additional support was provided by the Wellcome Trust (204963 and 092896) and the European Research Council (ERC) under European Union’s Seventh Framework Programme (FP7/2007-2013) ERC grant agreement no. 322408 to SER. Some of the work presented here was conducted at the Center on Macromolecular Dynamics by NMR Spectroscopy located at the New York Structural Biology Center, supported by a grant from the NIH NIGMS (P41 GM118302) and ORIP/NIH facility improvement grant CO6RR015495. The 900 MHz NMR spectrometers were purchased with funds from NIH grant P41 GM066354, the Keck Foundation, New York State Assembly, and U.S. Dept. of Defense.
ACKNOWLEDGMENT
We acknowledge Arthur Palmer for helpful discussions. We also acknowledge with thanks the many discussions with our group members. We thank Ana Monica Nunes for contributions in the beginning of this project and Nuria Benseny-Cases, who provided critical insights in the early stages of the work.
ABBREVIATIONS
- β2m
- β2-microglobulin;
- DRA
- Dialysis Related Amyloidosis;
- ECM
- extracellular matrix;
- MHC-I
- major histocompatibility complex-I;
- GAG
- glycosaminoglycan;
- NMR
- nuclear magnetic resonance;
- ELISA
- enzyme-linked immunosorbent assay;
- ThT
- thioflavin T;
- AFM
- atomic force microscopy;
- HSQC
- heteronuclear single quantum correlation;
- DEST
- dark-state exchange saturation transfer;
- OSCAR
- osteoclast associated receptor;
- LAIR-1
- leukocyte associated immunoglobulin-like receptor-1;
- GPVI
- glycoprotein VI;
- CPMG
- Carr-Purcell-Meiboom-Gill.
REFERENCES
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- 3.↵
- 4.↵
- 5.
- 6.↵
- 7.↵
- 8.↵
- 9.
- 10.↵
- 11.↵
- 12.
- 13.↵
- 14.↵
- 15.↵
- 16.↵
- 17.↵
- 18.↵
- 19.↵
- 20.↵
- 21.↵
- 22.↵
- 23.↵
- 24.↵
- 25.↵
- 26.↵
- 27.↵
- 28.↵
- 29.↵
- 30.↵
- 31.↵
- 32.↵
- 33.
- 34.
- 35.
- 36.
- 37.
- 38.↵
- 39.↵
- 40.
- 41.
- 42.
- 43.
- 44.
- 45.
- 46.
- 47.
- 48.
- 49.↵
- 50.↵
- 51.↵
- 52.↵
- 53.
- 54.
- 55.↵
- 56.↵
- 57.↵
- 58.↵
- 59.↵
- 60.↵
- 61.↵
- 62.
- 63.
- 64.↵
- 65.↵
- 66.
- 67.↵
- 68.↵
- 69.↵
- 70.↵
- 71.↵
- 72.
- 73.↵
- 74.↵
- 75.
- 76.
- 77.
- 78.
- 79.↵
- 80.
- 81.↵
- 82.
- 83.
- 84.↵
- 85.↵
- 86.↵
- 87.↵
- 88.↵
- 89.↵
- 90.↵
- 91.↵
- 92.↵
- 93.↵
- 94.↵
- 95.↵
- 96.↵