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Cryo-EM structure of a light chain-derived amyloid fibril from a patient with systemic AL amyloidosis

Cryo-EM structure of a light chain-derived amyloid fibril from a patient with systemic AL... ARTICLE https://doi.org/10.1038/s41467-019-09032-0 OPEN Cryo-EM structure of a light chain-derived amyloid fibril from a patient with systemic AL amyloidosis 1 1 1 2 3 Lynn Radamaker , Yin-Hsi Lin , Karthikeyan Annamalai , Stefanie Huhn , Ute Hegenbart , 3 4,5 1 1 Stefan O. Schönland , Günter Fritz , Matthias Schmidt & Marcus Fändrich Amyloid fibrils derived from antibody light chains are key pathogenic agents in systemic AL amyloidosis. They can be deposited in multiple organs but cardiac amyloid is the major risk factor of mortality. Here we report the structure of a λ1 AL amyloid fibril from an explanted human heart at a resolution of 3.3 Å which we determined using cryo-electron microscopy. The fibril core consists of a 91-residue segment presenting an all-beta fold with ten mutagenic changes compared to the germ line. The conformation differs substantially from natively folded light chains: a rotational switch around the intramolecular disulphide bond being the crucial structural rearrangement underlying fibril formation. Our structure provides insight into the mechanism of protein misfolding and the role of patient-specific mutations in pathogenicity. 1 2 Institute of Protein Biochemistry, Ulm University, 89081 Ulm, Germany. Medical Department V, Section of Multiple Myeloma, Heidelberg University 3 4 Hospital, 69120 Heidelberg, Germany. Medical Department V, Amyloidosis Center, Heidelberg University Hospital, 69120 Heidelberg, Germany. Institute of Microbiology, University of Hohenheim, 70599 Stuttgart, Germany. Institute for Neuropathology, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany. Correspondence and requests for materials should be addressed to M.F. (email: marcus.faendrich@uni-ulm.de) NATURE COMMUNICATIONS | (2019) 10:1103 | https://doi.org/10.1038/s41467-019-09032-0 | www.nature.com/naturecommunications 1 1234567890():,; ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09032-0 ntibodies are protein structures of utmost importance to fibril morphology, which we present here, is informative about human health. They underlie the humoral immune sys- the mechanism of LC misfolding and illuminates the role of Atem and many top-selling biopharmaceutical agents; yet, patient-specific mutations for the development of amyloidosis. they can be the basis of devastating human diseases with systemic AL amyloidosis (i.e. the amyloidosis caused by immunoglobulin Results light chains) being a particularly important one . Moreover, Structural rigidity of the extracted fibrils. Using a previously antibodies or antibody fragments can misfold during bio- established protocol to extract amyloid fibrils from diseased tis- pharmaceutical production, leading to a great need to improve 2 sue , we obtained AL amyloid fibrils from heart muscle tissue of our understanding of the misfolding of these proteins . Systemic a patient who underwent a heart transplantation as a con- AL amyloidosis belongs to the most common forms of systemic 3 sequence of severe cardiac AL amyloidosis (Supplementary amyloidosis in industrialized countries . In the USA it occurs Table 1). The fibrils are derived from the germ line segments with an incidence of ~9–14 patients per 1 million inhabitants . IGLV1-44, IGLJ3, and IGLC2, demonstrating that the fibrils are The misfolding of immunoglobulin light chains (LCs), which are representative for a λ-subtype causing cardiac involvement. The constituents of natural antibodies , gives rise to the disease. dominant fibril morphology in the extract is relatively straight, Precondition is a clonal B cell disorder, such as a multiple mye- indicating its resistance to bending deformations. Quantitatively, loma, which elevates the concentration of one monoclonal LC in measurement of the fibril contour length and its end to end the serum. distance yielded values of 6.7 ± 0.5 μm for the persistence length The clinical and pathological disease manifestations are diverse −26 2 and 2.78 ± 0.21 × 10 Nm for the bending rigidity (Supple- and AL amyloid deposits can be found in different tissues and 6 mentary Figure 1). organs . Especially important are those variants of the disease that are associated with cardiac amyloidosis. Cardiac involvement is a major cause of mortality . Untreated patients show a median Fibril topology obtained by cryo-EM. Cryo-EM imaging of the survival of 7 months after initial diagnosis . The current treat- extracted fibrils at 300 kV (Fig. 1a) allowed us to reconstruct the ment standard is to stop the production of LCs with che- dominant fibril morphology at 3.3 Å resolution (Fig. 1b, Table 1) motherapy directed against the underlying B cell clone. In case of based on the 0.143 Fourier-shell correlation (FSC) criterion advanced heart involvement, patients may additionally have to (Supplementary Figure 2). The two-dimensional (2D) class 9,10 undergo a heart transplantation , which provides access to averages cover the entire fibril (Supplementary Figure 3). The large quantities of amyloid fibrils for research purposes. reconstructed density shows a width of ~12 nm (Fig. 1b–d), in Several studies demonstrate that the properties of the precursor agreement with measurements from negatively stained samples . LCs predispose patients to develop the disease or a specific disease The fibril consists of a single protein stack, termed here proto- variant. There is a preponderance of λ-LCs versus κ-LCs (λ:κ = filament. It contains parallel cross-β sheets with intramolecular 3:1) in patients with AL amyloidosis, while κ-LCs are more backbone hydrogen bonds (Fig. 2a, b). The fibril cross-section is abundant (λ:κ = 1:2) in healthy individuals and in patients with asymmetrical (Fig. 1b), therefore a C1 symmetry was assumed multiple myeloma . Mutations in LC domains can destabilize the during reconstruction. The fibril helix is left-hand twisted as protein and/or accelerate the fibrillation of model proteins confirmed by map-inversion and shows a pitch of ~300 nm as 12–15 in vitro . The presence of the IGLV1-44 germ line segment in well as a polar topology (Fig. 1d). the LCs correlates positively with cardiomyopathy, while the IGLV6-57 germ line segment correlates positively with kidney 7,16–18 involvement . Fold of the fibril protein. The three-dimensional (3D) cryo-EM Amyloid fibrils are much better established as pathogenic map was fitted with a continuous polypeptide segment (Supple- agents in systemic amyloidosis than in many neurodegenerative mentary Table 2), corresponding to residues Gly15–Thr105 of the amyloid diseases that rather depend on toxic amyloid oligo- AL fibril protein (Fig. 2c, d). The protein N-terminal and C- mers . Although free LCs or LC oligomers can make patholo- terminal ends are juxtaposed in the structure and form a pro- gical contributions to systemic AL amyloidosis , cardiac truding stalk. The remaining part of the protein roughly outlines pathology arises largely from massive amyloid fibril deposits that the shape of a ram head (Supplementary Figure 4). Head region impair the natural ability of the heart to pump and to contract. So and stalk lie on either side of an intramolecular disulfide bond far, little is known about the structure of pathogenic LC aggre- that is formed between residues Cys22 and Cys89 (Fig. 2c, see gates. AL fibrils have generic structural characteristics of amyloid below). The N-terminal and C-terminal ends of the stalk are fibrils, such as a cross-β structure, a width of ~15 nm and a surrounded by diffuse density (Supplementary Figure 4), indi- twisted fibril architecture leading to regularly spaced cross- cating structural disorder of the first and last 12 residues (Fig. 2c). 21–23 overs . A deeper understanding of the mechanism of LC The fibril protein belongs to the all-beta class of protein folds, misfolding and consequent disease pathology is hampered by a consisting of 12 β-strands (β1–β12). The strands vary in length lack of detailed structural information. from two to eight residues. The folded structure shows several In this study, we used electron cryo microscopy (cryo-EM) to non-local contacts, such as between segments Gln16–Val18 and determine the molecular structure of an amyloid fibril underlying Asn97–Trp99, Cys22–Arg25 and Glu84–Cys89, Trp36–Gly58 the pathology in an AL patient with severe cardiac amyloidosis. and Thr70–Gln80, as well as Arg62–Lys67 and Asp83–Trp92. The fibrils were previously shown to consist of a LC fragment that The polypeptide chain changes height by 8.2 Å along the fibril corresponds to residues Val3–Ser118 of a λ-LC and they match main axis (Supplementary Figure 5a), interdigitating the fibril 22,23 the size of other AL fibril proteins in cardiac amyloidosis . proteins in the direction of the main axis. The intermolecular The fibril protein is not glycosylated and encompasses mainly the interactions in the fibril rarely extend beyond the next molecular variable light (V ) domain, which is typical for λ-AL layer. For example, strand β4 from layer i is in contact with strand 13,22,23 amyloidosis . Using negatively stained transmission elec- β8 in layers i and i + 1 (Supplementary Figure 5b). Residue Arg25 tron microscopy (TEM), we recently demonstrated that the fibrils interacts with residues Glu84 and Asp86 from layer i + 1 from this patient contain a dominant fibril morphology, exhi- (Supplementary Figure 5c). The height change produces different biting a width of 13.6 ± 0.9 nm, and a minor morphology with a tip structures at the two fibril ends (Supplementary Figure 5d), 23 24–28 width of 20.4 ± 0.4 nm . The cryo-EM structure of this dominant resembling other cross-β fibrils . It was suggested that this 2 NATURE COMMUNICATIONS | (2019) 10:1103 | https://doi.org/10.1038/s41467-019-09032-0 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09032-0 ARTICLE ad b Fig. 1 Cryo-EM structure of an amyloid fibril from systemic AL amyloidosis. a Raw cryo-EM image. Scale bar: 100 nm. b Cross-section of the reconstruction superimposed with a molecular model. Three internal cavities are labeled A–C. Scale bar: 1 nm. c Side view of the reconstructed density. Scale bar: 1 nm. d Side views of the molecular model. A segment corresponding to the reconstruction (c) is boxed. Scale bar: 50 nm fact may give rise to different mechanisms/kinetics of fibril Table 1 Cryo-EM data collection and image processing 26,29 outgrowth . The protein fold encloses three major cavities, labeled A–C Microscope Titan Krios (Thermo Fisher (Fig. 1b). Cavities A and B are hydrophilic and occur within the Scientific) head region. They are lined with many polar and ionic amino acid Camera K2 Summit (Gatan) side chains, suggesting the presence of water. Cavity C is Acceleration voltage (kV) 300 hydrophobic and located within the stalk region. This cavity is Magnification 130,000 lined with hydrophobic side chains and the intramolecular Defocus range (μm) 0.4–4.6 − −1 −1 disulfide bond. It also contains a small density that cannot be Dose rate (e pixel s ) 5.78 Number of movie frames 30 assigned to the polypeptide chain (Fig. 1b), indicating the Exposure time (s) 6 presence of a molecular inclusion of low polarity. − −2 Total electron dose (e Å)32 Pixel size (Å) 1.041 Gatan imaging filter 20 eV Location of sequence elements in the fibril protein. The most Mode Counting mode aggregation-prone segments of the protein exist at residues Box size (pixel) 320 Asn97–Phe101, Lys46–Tyr50, Tyr87–Ala91, and Val34–Gln39 Inter box distance (Å) 28.8 (Fig. 3a, Supplementary Table 3), similar to other λ1-LCs . These Number of extracted segments 119,395 Number of segments after 2D 62,250 segments do not correspond well with the β-strands in the fibril classification (Fig. 2a, c). Three segments are solvent-exposed, while residues Number of segments after 3D 32,677 Tyr87–Ala91 are buried (Fig. 3b). The fibril protein lacks a typical classification hydrophobic core (Fig. 2d). One of its most central structural Resolution, 0.143 FSC criterion (Å) 3.3 elements is stand β9, which contains a highly acidic motif Map sharpening B-Factor (Å ) 119.6 (Fig. 2d, Fig. 3c). The charges of this segment are only partially Helical rise (Å) 4.8 compensated by buried residues of the opposite charge (Arg25 Helical twist (°) 0.58 and Lys73) (Fig. 2d). The complementarity determining regions Symmetry imposed C1 (CDRs) are located on the fibril surface (Fig. 3d) and there are 10 mutations compared with the amino acid sequence encoded by the IGLV1-44 germ line segment (Fig. 3d). NATURE COMMUNICATIONS | (2019) 10:1103 | https://doi.org/10.1038/s41467-019-09032-0 | www.nature.com/naturecommunications 3 Aggregation score ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09032-0 a b d β5 β4 β3 45 43 41 β6 N C 49 β8 47 β7 β9 77 37 N C β10 β11 β12 E 79 83 69 67 N C 85 E β2 59 65 63 29 N C β1 87 Q16 I20 Native LC β1 β2 β3 β4 β5 β6 Fibril protein 3 10 20 30 40 50 60 17 G VLTQPPSASGTPGQRVTISCSGRSSNIGRNLVKWYQQFPGTAPKLLIYSNDQRPSGVP Acidic (D, E) β5 Basic (K, R) Native LC β7 β8 β9 β10 β11 β12 Polar (C, N, Q, S, T, Y) Fibril protein 70 80 90 100 110 118 Hydrophobic (A, F, I, L, V, W) C 105 DRFSGSKSGTSASLAVSGLQSEDEADYYCAAWDATLNAWVFGGGTKLTVLSQPKAAPS Fig. 2 Fibril protein β-sheet structure. a Ribbon representation of a stack of five fibril proteins rainbow colored from N-terminus to C-terminus. b Close-up of the parallel cross-β sheet β1. c Position of the β-strands (arrows) in the fibril protein and in the natively folded LC as defined in PDB entry 1BJM . Residue numbers refer to the native LC without signal sequence. The color of the β-strands in the fibril corresponds to panel a. Dotted gray line refers to the part of the protein that is disordered in the fibril. d Schematic representation of the fibril protein packing 5.0 2.5 0.0 –2.5 –5.0 0 GQRVTISCSGRSSNIGRNLVKWYQQFPGTAPKLLIYSNDQRPSGVPDRFSGSKSGTSASLAVSGLQSEDEADYYCAAWDATLNAWVFGGGT 15 25 35 45 55 65 75 85 95 105 b c d e L40F CDR2 I76V N53D N35K T33L S25R S31R CDR1 25 D94A 94 31 CDR3 95 S95T G98A Fig. 3 Location of specific sequence elements in the structure. a Hydrophobicity (gray) and aggregation score (brown) of the ordered part of the fibril protein. Magenta letters: mutations compared to the IGLV1-44 germ line segment. Boxes: CDRs. Residue numbers refer to the native LC without signal sequence. b Fibril protein showing the residue-specific aggregation score (0–5). c Electrostatic surface representation of the fibril protein. d Fibril protein with CDRs (black) and mutations (magenta) highlighted. e Ribbon diagram of a native V domain (PDB entry 1BJM) showing residues 3–113. CDRs are colored black; mutations are colored in red if they affect the core (residue 76), purple (surface residues with potential relevance for domain–domain interactions), or magenta (other surface residues) 4 NATURE COMMUNICATIONS | (2019) 10:1103 | https://doi.org/10.1038/s41467-019-09032-0 | www.nature.com/naturecommunications Hydrophobicity score NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09032-0 ARTICLE a b Native Fibril Native Fibril Native Fibril 90° N C 180° 12 3 Fig. 4 Comparison of the fibril structure and the native V domain fold. a Comparison of the native V domain fold (PDB entry 1BJM) with the fibril state. L L Residues 15–105 are shown in rainbow color. The native conformation is truncated at residue 118, corresponding to our fibril protein. The diffuse N-terminal and C-terminal tails of the fibril structure are schematically added with a gray line. b Part of the native structure and of the fibril state showing the conformational switch of segments 86–93 and 16–23 relative to one another around the protein disulfide bond. c Schematic representation of the hypothetical misfolding reaction consisting of an unfolding reaction (1), the rotational switch (2) and the assembly into the fibril structure (3) Three of these mutations add a surface charge to the fibril content, but there are differences in the number and position of (Ser31Arg, Asn35Lys, Asn53Asp) (Fig. 2d). One mutation the β-strands within the sequence (Fig. 2c). The fibril conforma- removes a charge from the non-polar cavity C (Asp94Ala). tion is more extended and flattened compared to the native state Ser25Arg inserts a basic residue into the polar cavity B, where it (Fig. 4a), enabling the polypeptide chain to form one molecular helps to compensate the charges of Glu84 and Asp86 and to fibril layer. A particularly substantial structural rearrangement interdigitate two molecular layers of the fibril Supplementary happens in the region around the disulfide bond which cross- Figure 5c). Gly98Ala occurs in the highly aggregation-prone links the N-terminal and C-terminal segments of the protein. segment at residues Asn97–Phe101 (Fig. 3a). Thr33Leu, These segments show a parallel N to C orientation in the native Leu40Phe, Ile76Val, and Ser95Thr have no obvious structural state and an antiparallel orientation in the fibril (Fig. 4b). effect on the fibril. None of the replacements is clearly Therefore, misfolding induces a 180° rotational switch of one unfavorable to the fibril structure. No mutational change occurs segment relative to the other around the disulfide bond, placing within the IGLJ3 and IGLC2 germ line segments, nor within the stalk on one side of the disulfide bond and the head region on residues Gln1–Thr13, a previously described mutational hot spot the other (Fig. 4c). These stark structural differences between the of some amyloidogenic λ-LCs . This segment is conformation- native and misfolded state are consistent with our previous ally disordered or missing in the fibril (Supplementary Figure 4), observation that AL amyloid fibrils and refolded fibril proteins which implies that at least for some patients this segment is not differ in several structural features, such as their infrared spectral relevant to fibril formation. Indeed, mutational changes to the N- characteristics and their affinity for the amyloid-binding dyes terminus were found to be more relevant to a λ6-LC-derived V Thioflavin T and Congo red . domain, which is destabilized upon mutation , and λ6-LCs may 29,33 form fibrils with an ordered N-terminus . Discussion In this study, we have analyzed the molecular structure of Comparison with native LC conformations. The majority of the an amyloid fibril that was purified from patient tissue and is mutations occur at surface positions in the globular V domain therefore directly relevant to disease. Interestingly, we previously (Fig. 3e). Four of these changes (Thr33Leu, Asn35Lys, Ser95Thr, found that the fibril protein constituting this fibril is able to form Gly98Ala) may potentially impact the interactions with other amyloid-like fibrils in vitro that possess a different morphology, immunoglobulin domains. Only Ile76Val affects a buried residue. and possibly also a different protofilament substructure, than the This mutation removes a methylene group from the protein core, bona fide pathogenic aggregates studied here . These observa- which typically destabilizes a protein by 6 kJ/mol . Taken toge- tions indicate that it is essential to investigate patient-derived ther, we find one out of 10 mutations in this patient to be rather than in vitro formed fibrils when scrutinizing the mole- unfavorable to the native state, while two to six changes make the cular basis of a protein misfolding disease. LC more compatible with the fibril structure than the germ line The reconstructed fibril has an elongated and rigid structure, segment. consisting of a single protofilament. Values measured for the −26 2 The fibril structure is profoundly different from a natively bending rigidity of the AL fibrils (2.78 ± 0.21 × 10 Nm ) folded V domain. Both protein states contain a high β-sheet correspond to previously reported values, which were on the NATURE COMMUNICATIONS | (2019) 10:1103 | https://doi.org/10.1038/s41467-019-09032-0 | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09032-0 −28 −25 2 35 order of 10 –10 Nm for different amyloid-like fibrils . unfavorable to the native protein conformation (Fig. 3e). The The stiffness of the fibrils could explain why amyloid fibril analyzed fibril is representative for amyloid fibrils from cardiac deposits impair the natural function of the heart. Cryo-EM and AL amyloidosis as it is derived from an IGLV1-44 germ line reconstruction of the 3D map enabled us to reveal the molecular segment, the major germ line segment leading to heart 7,16–18 structure underlying these effects. We could show that residues involvement . The mass of the fibril protein corresponds to Gly15–Thr105 adopt a stable conformation in the fibril, while the that of other AL fibril proteins and consists mainly of a V 22,23 first and last 12 amino acid residues are not resolved in our domain . The observed fibril morphology resembles fibril 22,23 structure and are conformationally disordered (Supplementary morphologies from other AL patients with cardiomyopathy , Figure 4). The fibril proteins are interdigitated along the main despite clear patient-specific differences. However, systemic AL axis of the fibril, providing resilience to mechanical stress (Sup- amyloidosis is an extraordinarily variable disease and other LC plementary Figure 1). The fold of the AL fibril protein is novel subtypes may be associated with different structural properties, and differs from previously published fibril protein structures. It particularly at the protein N-terminus, as was indicated recently 29,33 consists of a head and stalk region and encompasses one non- for fibrils derived from λ6-LCs . polar and two polar cavities (Figs. 1b, 2d). The current data from cryo-EM sheds light onto the misfolding The single-protofilament architecture of this AL amyloid fibril of proteins and their pathogenicity, representing a solid basis for contrasts with the majority of previously described cross-β fibrils, further investigation of molecular mechanisms underlying human 24,26–28,36 which consist of multiple protofilaments . However, we pathology, for example by in vitro aggregation studies. Detailed cannot exclude the formation of multi-protofilament AL amyloid knowledge of the molecular structure of pathogenic protein states fibrils, as our sample contains a second fibril morphology that is may lead to the development of novel ligands which recognize thicker than the presently studied one . Furthermore, the pre- these structures and form the basis of new detection methods or sently investigated fibril contains surface-exposed charge pairs, therapeutic strategies. However, due to the heterogeneity of sys- for example at residues Asp53 and Arg55, which were previously temic AL amyloidosis further work will be necessary to dissect the identified as protofilament–protofilament interaction sites in structural characteristics of fibrils from different groups of murine AA amyloid fibrils . However, the majority of the fibrils patients and to identify common structural themes between dif- in the AL fibril sample does not contain multiple protofilaments, ferent cohorts of patients as well as systematic variations. indicating that the assembly into higher-order structures is unfavorable for this fibril protein. Methods The protein fold provides insight into the mechanism of LC Source of AL fibrils. AL amyloid fibrils were extracted from the heart of a woman (Supplementary Table 1), suffering from advanced heart failure due to AL misfolding. It does not support mechanisms that assume of the 22,23 amyloidosis . First symptoms (dyspnea, fatigue) started 1 year before diagnosis initial formation of fibril segments consisting of dimeric proteins of AL amyloidosis. A monoclonal plasma cell disorder (smoldering myeloma) was 13,37,38 or peptides . Nor is there evidence for an assembly of diagnosed at the same time as AL amyloidosis. Bone marrow cytology showed 19% 37,39 domain-swapped molecules, consistent with other studies . plasma cells (<5% λ-positive in bone marrow histology) and interphase fluores- cence in situ-hybridization analysis of CD138+ enriched plasma cells showed the t The substantial conformational differences between the fibril (11;14) translocation and the 13q14 deletion. The patient received 5 months of structure and the native state imply instead that the native con- treatment with bortezomib and dexamethasone, and achieved a serological com- formation must be largely, if not entirely, unfolded to allow fibril plete remission of the smoldering myeloma. Ten months later, free λ-LCs increased formation to occur (Fig. 4c). In particular, we identified a rota- and treatment with lenalidomide and dexamethasone was started but stopped after 2 months due to cardiac decompensation. 1 month later high urgency listing was tional switch of the polypeptide chain around the disulfide bond done and the transplantation was performed 2 months later. Informed consent was that is only possible if most of the native strand–strand interac- obtained from the patient for the analysis of the amyloid deposits. tions are lost. Unfolding and the rotational switch are crucial for The fibril extraction was performed from heart muscle tissue as described fibril formation as unfolding is the prerequisite for the rotational previously . In brief, 250 mg of tissue were diced and washed five times with 0.5 mL Tris calcium buffer (20 mM Tris, 138 mM NaCl, 2 mM CaCl , 0.1 % NaN , switch, which in turn represents the basis for the formation of a 2 3 pH 8.0). Each washing step consisted of gentle vortexing and centrifugation at flat protein structure that lacks chain crossings (Fig. 4c). This 3100 × g for 1 min at 4 °C. The supernatant was discarded and the pellet was conformation is then able to associate into the intermolecular −1 resuspended in 1 mL of freshly prepared 5 mg mL Clostridium histolyticum hydrogen bond network of a cross-β sheet. collagenase (Sigma) in Tris calcium buffer. After incubation overnight at 37 °C the tissue material was centrifuged at 3100 × g for 30 min at 4 °C. The retained pellet As the disulfide bond occurs at the same position in the fibril as was resuspended in 0.5 mL buffer containing 20 mM Tris, 140 mM NaCl, 10 mM in the native V domain, previous research suggested that the ethylenediaminetetraacetic acid, 0.1 % NaN , pH 8.0, and subjected to 10 cycles of misfolding of the LC occurs under oxidizing conditions and homogenization in fresh buffer and centrifugation for 5 min at 3100 × g at 4 °C. maintains the cysteine disulfide bond . Our structure lends The remaining pellet was homogenized in 0.5 mL ice cold water, centrifuged for further support to this view, as we reveal a number of notable 5 min at 3100 × g at 4 °C and the fibril-containing supernatant was analyzed. The study was approved by the ethical committees of the University of Heidelberg (123/ imperfections in the fibril packing. There are three major internal 2006) and of Ulm University (210/13) cavities (Fig. 1b) and the protein is packed inside-out or outside- in. A highly acidic segment of low aggregation propensity is Measurement of the persistence length. Patient-derived fibrils were dried onto a buried in the core and only partially compensated by basic carbon-coated grid and negatively stained with uranyl acetate as described pre- charges, whereas segments that are much more hydrophobic and 22 viously . Images were taken at 120 kV using a JEM-1400Plus microscopy (Jeol) aggregation-prone are exposed to the solvent (Fig. 3b, c). The equipped with a TemCam-F216 camera (TVIPS). The contour length L and end- to-end distance D of 124 well-resolved fibrils was determined using the program situation looks drastically different in systemic AA amyloidosis, Fiji (http://fiji.sc). The plot of the squared end-to-end distance was fit with Eq. (1) where the most aggregation-prone and hydrophobic segments are to obtain the persistence length P: buried in the fibril core and where buried acidic residues are 2P compensated by an equal number of buried basic residues .AA 2 ðÞ L=2P D ¼ 4PL  1  1  e ð1Þ amyloid fibrils, which contain no disulfide bond, accomplish a tighter packing than AL amyloid fibrils and form much smaller The persistence length can be converted into the bending rigidity B as described by: internal cavities. The detailed information provided by our structure helps to B ¼ P  k  T ð2Þ explain the effects of mutational variants in systemic AL amy- loidosis. Several mutational changes have a beneficial effect on the In this equation, k refers to the Boltzmann constant and T to the temperature fibril structure (Fig. 3d), while only one mutation is clearly (300 K). 6 NATURE COMMUNICATIONS | (2019) 10:1103 | https://doi.org/10.1038/s41467-019-09032-0 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09032-0 ARTICLE Cryo-EM. A 3.5 μL aliquot was applied to a glow-discharged holey carbon-coated Reporting summary. Further information on experimental design is available in grid (C-flat 1.2/1.3 400 mesh) blotted from the back side after an incubation time of the Nature Research Reporting Summary linked to this article. 4 s at a humidity of >80% and plunge-frozen in liquid ethane using a Cryoplunge 3 System (Gatan). For image acquisition a K2-Summit detector (Gatan) in counting Data availability mode on a Titan Krios transmission electron microscope (Thermo Fisher Scien- The reconstructed cryo-EM map was deposited in the Electron Microscopy Data Bank tific) at 300 kV was used, using a Gatan imaging filter with a 20 eV slit. Table 1 lists with the accession code EMD-4452. The coordinates of the fitted atomic model were the data acquisition parameters. The raw data have been deposited at https://www. deposited in the PDB under the accession code 6IC3. The Cryo-EM data were deposited ebi.ac.uk/pdbe/emdb/ with the accession code EMPIAR-10245. on EMPIAR with the accession code EMPIAR-10245. The datasets and materials used during the current study are available from the corresponding author on reasonable Helical reconstruction. The raw data movie frames were gain-corrected with request. The data underlying the Supplementary Figures 1 and 2 are provided as a Source IMOD and aligned, motion-corrected and dose-weighted using MOTION- Data File. 41 42 COR2 . Gctf was used to estimate the contrast transfer function from the aligned and motion-corrected images. RELION 2.1 was used for the helical Received: 6 December 2018 Accepted: 15 February 2019 reconstruction of the fibril density. After manual selection of the fibrils from the aligned, motion-corrected micrographs, segments were extracted with a box size of ~333 Å and an inter-box distance of ~9% of the box length. Reference-free 2D classification with a regularization value of T = 3 produced class averages showing the helical repeat along the fibril axis. Class averages were selected based on a manual arrangement into a fibril structure. From the selected class averages, 200 randomly picked particles per class were used to generate an initial 3D model using References the Stochastic Gradient Descent algorithm implemented in RELION. These initial 1. Ramirez-Alvarado, M. et al. Systemic misfolding of immunoglobulins in the models were low-pass filtered to 60 Å and used to generate a single-fibril model of test tube and in the cell. FASEB J. 32, abstr. 247.3 (2018). two selected fibrils with clearly visible cross-overs. The single-fibril model showed 2. van der Kant, R. et al. Prediction and reduction of the aggregation of the general shape of the fibril, and was then used as an initial model for 3D monoclonal antibodies. J. Mol. Biol. 429, 1244–1261 (2017). classification with K = 4. Two out of four classes were selected. The ~30k particles 3. Gertz, M. A. et al. 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Methods 14, 331–332 reprintsandpermissions/ (2017). 42. Zhang, K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. Journal peer review information: Nature Communications thanks D. Alejandro 193,1–12 (2016). Fernández-Velasco, Henning Stahlberg and the other anonymous reviewer(s) for their contribution to the peer review of this work. 43. He, S. & Scheres, S. H. W. Helical reconstruction in RELION. J. Struct. Biol. 198, 163–176 (2017). 44. Afonine, P. V. et al. New tools for the analysis and validation of cryo-EM Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in maps and atomic models. Acta Crystallogr. D 74, 814–840 (2018). published maps and institutional affiliations. 45. Langer, G., Cohen, S. X., Lamzin, V. S. & Perrakis, A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nat. Protoc. 3, 1171–1179 (2008). Open Access This article is licensed under a Creative Commons 46. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development Attribution 4.0 International License, which permits use, sharing, of coot. Acta Crystallogr. D 66, 486–501 (2010). adaptation, distribution and reproduction in any medium or format, as long as you give 47. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and appropriate credit to the original author(s) and the source, provide a link to the Creative crystallography. Acta Crystallogr. D 74, 531–544 (2018). Commons license, and indicate if changes were made. The images or other third party 48. Retter, I., Althaus, H. H., Munch, R. & Muller, W. VBASE2, an integrative V material in this article are included in the article’s Creative Commons license, unless gene database. Nucleic Acids Res. 33, D671–D674 (2005). indicated otherwise in a credit line to the material. If material is not included in the 49. Kent, W. J. BLAT–the BLAST-like alignment tool. Genome Res. 12, 656–664 article’s Creative Commons license and your intended use is not permitted by statutory (2002). regulation or exceeds the permitted use, you will need to obtain permission directly from 50. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local the copyright holder. To view a copy of this license, visit http://creativecommons.org/ alignment search tool. J. Mol. Biol. 215, 403–410 (1990). licenses/by/4.0/. 51. Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729 (2013). © The Author(s) 2019 8 NATURE COMMUNICATIONS | (2019) 10:1103 | https://doi.org/10.1038/s41467-019-09032-0 | www.nature.com/naturecommunications http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Communications Springer Journals

Cryo-EM structure of a light chain-derived amyloid fibril from a patient with systemic AL amyloidosis

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Abstract

ARTICLE https://doi.org/10.1038/s41467-019-09032-0 OPEN Cryo-EM structure of a light chain-derived amyloid fibril from a patient with systemic AL amyloidosis 1 1 1 2 3 Lynn Radamaker , Yin-Hsi Lin , Karthikeyan Annamalai , Stefanie Huhn , Ute Hegenbart , 3 4,5 1 1 Stefan O. Schönland , Günter Fritz , Matthias Schmidt & Marcus Fändrich Amyloid fibrils derived from antibody light chains are key pathogenic agents in systemic AL amyloidosis. They can be deposited in multiple organs but cardiac amyloid is the major risk factor of mortality. Here we report the structure of a λ1 AL amyloid fibril from an explanted human heart at a resolution of 3.3 Å which we determined using cryo-electron microscopy. The fibril core consists of a 91-residue segment presenting an all-beta fold with ten mutagenic changes compared to the germ line. The conformation differs substantially from natively folded light chains: a rotational switch around the intramolecular disulphide bond being the crucial structural rearrangement underlying fibril formation. Our structure provides insight into the mechanism of protein misfolding and the role of patient-specific mutations in pathogenicity. 1 2 Institute of Protein Biochemistry, Ulm University, 89081 Ulm, Germany. Medical Department V, Section of Multiple Myeloma, Heidelberg University 3 4 Hospital, 69120 Heidelberg, Germany. Medical Department V, Amyloidosis Center, Heidelberg University Hospital, 69120 Heidelberg, Germany. Institute of Microbiology, University of Hohenheim, 70599 Stuttgart, Germany. Institute for Neuropathology, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany. Correspondence and requests for materials should be addressed to M.F. (email: marcus.faendrich@uni-ulm.de) NATURE COMMUNICATIONS | (2019) 10:1103 | https://doi.org/10.1038/s41467-019-09032-0 | www.nature.com/naturecommunications 1 1234567890():,; ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09032-0 ntibodies are protein structures of utmost importance to fibril morphology, which we present here, is informative about human health. They underlie the humoral immune sys- the mechanism of LC misfolding and illuminates the role of Atem and many top-selling biopharmaceutical agents; yet, patient-specific mutations for the development of amyloidosis. they can be the basis of devastating human diseases with systemic AL amyloidosis (i.e. the amyloidosis caused by immunoglobulin Results light chains) being a particularly important one . Moreover, Structural rigidity of the extracted fibrils. Using a previously antibodies or antibody fragments can misfold during bio- established protocol to extract amyloid fibrils from diseased tis- pharmaceutical production, leading to a great need to improve 2 sue , we obtained AL amyloid fibrils from heart muscle tissue of our understanding of the misfolding of these proteins . Systemic a patient who underwent a heart transplantation as a con- AL amyloidosis belongs to the most common forms of systemic 3 sequence of severe cardiac AL amyloidosis (Supplementary amyloidosis in industrialized countries . In the USA it occurs Table 1). The fibrils are derived from the germ line segments with an incidence of ~9–14 patients per 1 million inhabitants . IGLV1-44, IGLJ3, and IGLC2, demonstrating that the fibrils are The misfolding of immunoglobulin light chains (LCs), which are representative for a λ-subtype causing cardiac involvement. The constituents of natural antibodies , gives rise to the disease. dominant fibril morphology in the extract is relatively straight, Precondition is a clonal B cell disorder, such as a multiple mye- indicating its resistance to bending deformations. Quantitatively, loma, which elevates the concentration of one monoclonal LC in measurement of the fibril contour length and its end to end the serum. distance yielded values of 6.7 ± 0.5 μm for the persistence length The clinical and pathological disease manifestations are diverse −26 2 and 2.78 ± 0.21 × 10 Nm for the bending rigidity (Supple- and AL amyloid deposits can be found in different tissues and 6 mentary Figure 1). organs . Especially important are those variants of the disease that are associated with cardiac amyloidosis. Cardiac involvement is a major cause of mortality . Untreated patients show a median Fibril topology obtained by cryo-EM. Cryo-EM imaging of the survival of 7 months after initial diagnosis . The current treat- extracted fibrils at 300 kV (Fig. 1a) allowed us to reconstruct the ment standard is to stop the production of LCs with che- dominant fibril morphology at 3.3 Å resolution (Fig. 1b, Table 1) motherapy directed against the underlying B cell clone. In case of based on the 0.143 Fourier-shell correlation (FSC) criterion advanced heart involvement, patients may additionally have to (Supplementary Figure 2). The two-dimensional (2D) class 9,10 undergo a heart transplantation , which provides access to averages cover the entire fibril (Supplementary Figure 3). The large quantities of amyloid fibrils for research purposes. reconstructed density shows a width of ~12 nm (Fig. 1b–d), in Several studies demonstrate that the properties of the precursor agreement with measurements from negatively stained samples . LCs predispose patients to develop the disease or a specific disease The fibril consists of a single protein stack, termed here proto- variant. There is a preponderance of λ-LCs versus κ-LCs (λ:κ = filament. It contains parallel cross-β sheets with intramolecular 3:1) in patients with AL amyloidosis, while κ-LCs are more backbone hydrogen bonds (Fig. 2a, b). The fibril cross-section is abundant (λ:κ = 1:2) in healthy individuals and in patients with asymmetrical (Fig. 1b), therefore a C1 symmetry was assumed multiple myeloma . Mutations in LC domains can destabilize the during reconstruction. The fibril helix is left-hand twisted as protein and/or accelerate the fibrillation of model proteins confirmed by map-inversion and shows a pitch of ~300 nm as 12–15 in vitro . The presence of the IGLV1-44 germ line segment in well as a polar topology (Fig. 1d). the LCs correlates positively with cardiomyopathy, while the IGLV6-57 germ line segment correlates positively with kidney 7,16–18 involvement . Fold of the fibril protein. The three-dimensional (3D) cryo-EM Amyloid fibrils are much better established as pathogenic map was fitted with a continuous polypeptide segment (Supple- agents in systemic amyloidosis than in many neurodegenerative mentary Table 2), corresponding to residues Gly15–Thr105 of the amyloid diseases that rather depend on toxic amyloid oligo- AL fibril protein (Fig. 2c, d). The protein N-terminal and C- mers . Although free LCs or LC oligomers can make patholo- terminal ends are juxtaposed in the structure and form a pro- gical contributions to systemic AL amyloidosis , cardiac truding stalk. The remaining part of the protein roughly outlines pathology arises largely from massive amyloid fibril deposits that the shape of a ram head (Supplementary Figure 4). Head region impair the natural ability of the heart to pump and to contract. So and stalk lie on either side of an intramolecular disulfide bond far, little is known about the structure of pathogenic LC aggre- that is formed between residues Cys22 and Cys89 (Fig. 2c, see gates. AL fibrils have generic structural characteristics of amyloid below). The N-terminal and C-terminal ends of the stalk are fibrils, such as a cross-β structure, a width of ~15 nm and a surrounded by diffuse density (Supplementary Figure 4), indi- twisted fibril architecture leading to regularly spaced cross- cating structural disorder of the first and last 12 residues (Fig. 2c). 21–23 overs . A deeper understanding of the mechanism of LC The fibril protein belongs to the all-beta class of protein folds, misfolding and consequent disease pathology is hampered by a consisting of 12 β-strands (β1–β12). The strands vary in length lack of detailed structural information. from two to eight residues. The folded structure shows several In this study, we used electron cryo microscopy (cryo-EM) to non-local contacts, such as between segments Gln16–Val18 and determine the molecular structure of an amyloid fibril underlying Asn97–Trp99, Cys22–Arg25 and Glu84–Cys89, Trp36–Gly58 the pathology in an AL patient with severe cardiac amyloidosis. and Thr70–Gln80, as well as Arg62–Lys67 and Asp83–Trp92. The fibrils were previously shown to consist of a LC fragment that The polypeptide chain changes height by 8.2 Å along the fibril corresponds to residues Val3–Ser118 of a λ-LC and they match main axis (Supplementary Figure 5a), interdigitating the fibril 22,23 the size of other AL fibril proteins in cardiac amyloidosis . proteins in the direction of the main axis. The intermolecular The fibril protein is not glycosylated and encompasses mainly the interactions in the fibril rarely extend beyond the next molecular variable light (V ) domain, which is typical for λ-AL layer. For example, strand β4 from layer i is in contact with strand 13,22,23 amyloidosis . Using negatively stained transmission elec- β8 in layers i and i + 1 (Supplementary Figure 5b). Residue Arg25 tron microscopy (TEM), we recently demonstrated that the fibrils interacts with residues Glu84 and Asp86 from layer i + 1 from this patient contain a dominant fibril morphology, exhi- (Supplementary Figure 5c). The height change produces different biting a width of 13.6 ± 0.9 nm, and a minor morphology with a tip structures at the two fibril ends (Supplementary Figure 5d), 23 24–28 width of 20.4 ± 0.4 nm . The cryo-EM structure of this dominant resembling other cross-β fibrils . It was suggested that this 2 NATURE COMMUNICATIONS | (2019) 10:1103 | https://doi.org/10.1038/s41467-019-09032-0 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09032-0 ARTICLE ad b Fig. 1 Cryo-EM structure of an amyloid fibril from systemic AL amyloidosis. a Raw cryo-EM image. Scale bar: 100 nm. b Cross-section of the reconstruction superimposed with a molecular model. Three internal cavities are labeled A–C. Scale bar: 1 nm. c Side view of the reconstructed density. Scale bar: 1 nm. d Side views of the molecular model. A segment corresponding to the reconstruction (c) is boxed. Scale bar: 50 nm fact may give rise to different mechanisms/kinetics of fibril Table 1 Cryo-EM data collection and image processing 26,29 outgrowth . The protein fold encloses three major cavities, labeled A–C Microscope Titan Krios (Thermo Fisher (Fig. 1b). Cavities A and B are hydrophilic and occur within the Scientific) head region. They are lined with many polar and ionic amino acid Camera K2 Summit (Gatan) side chains, suggesting the presence of water. Cavity C is Acceleration voltage (kV) 300 hydrophobic and located within the stalk region. This cavity is Magnification 130,000 lined with hydrophobic side chains and the intramolecular Defocus range (μm) 0.4–4.6 − −1 −1 disulfide bond. It also contains a small density that cannot be Dose rate (e pixel s ) 5.78 Number of movie frames 30 assigned to the polypeptide chain (Fig. 1b), indicating the Exposure time (s) 6 presence of a molecular inclusion of low polarity. − −2 Total electron dose (e Å)32 Pixel size (Å) 1.041 Gatan imaging filter 20 eV Location of sequence elements in the fibril protein. The most Mode Counting mode aggregation-prone segments of the protein exist at residues Box size (pixel) 320 Asn97–Phe101, Lys46–Tyr50, Tyr87–Ala91, and Val34–Gln39 Inter box distance (Å) 28.8 (Fig. 3a, Supplementary Table 3), similar to other λ1-LCs . These Number of extracted segments 119,395 Number of segments after 2D 62,250 segments do not correspond well with the β-strands in the fibril classification (Fig. 2a, c). Three segments are solvent-exposed, while residues Number of segments after 3D 32,677 Tyr87–Ala91 are buried (Fig. 3b). The fibril protein lacks a typical classification hydrophobic core (Fig. 2d). One of its most central structural Resolution, 0.143 FSC criterion (Å) 3.3 elements is stand β9, which contains a highly acidic motif Map sharpening B-Factor (Å ) 119.6 (Fig. 2d, Fig. 3c). The charges of this segment are only partially Helical rise (Å) 4.8 compensated by buried residues of the opposite charge (Arg25 Helical twist (°) 0.58 and Lys73) (Fig. 2d). The complementarity determining regions Symmetry imposed C1 (CDRs) are located on the fibril surface (Fig. 3d) and there are 10 mutations compared with the amino acid sequence encoded by the IGLV1-44 germ line segment (Fig. 3d). NATURE COMMUNICATIONS | (2019) 10:1103 | https://doi.org/10.1038/s41467-019-09032-0 | www.nature.com/naturecommunications 3 Aggregation score ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09032-0 a b d β5 β4 β3 45 43 41 β6 N C 49 β8 47 β7 β9 77 37 N C β10 β11 β12 E 79 83 69 67 N C 85 E β2 59 65 63 29 N C β1 87 Q16 I20 Native LC β1 β2 β3 β4 β5 β6 Fibril protein 3 10 20 30 40 50 60 17 G VLTQPPSASGTPGQRVTISCSGRSSNIGRNLVKWYQQFPGTAPKLLIYSNDQRPSGVP Acidic (D, E) β5 Basic (K, R) Native LC β7 β8 β9 β10 β11 β12 Polar (C, N, Q, S, T, Y) Fibril protein 70 80 90 100 110 118 Hydrophobic (A, F, I, L, V, W) C 105 DRFSGSKSGTSASLAVSGLQSEDEADYYCAAWDATLNAWVFGGGTKLTVLSQPKAAPS Fig. 2 Fibril protein β-sheet structure. a Ribbon representation of a stack of five fibril proteins rainbow colored from N-terminus to C-terminus. b Close-up of the parallel cross-β sheet β1. c Position of the β-strands (arrows) in the fibril protein and in the natively folded LC as defined in PDB entry 1BJM . Residue numbers refer to the native LC without signal sequence. The color of the β-strands in the fibril corresponds to panel a. Dotted gray line refers to the part of the protein that is disordered in the fibril. d Schematic representation of the fibril protein packing 5.0 2.5 0.0 –2.5 –5.0 0 GQRVTISCSGRSSNIGRNLVKWYQQFPGTAPKLLIYSNDQRPSGVPDRFSGSKSGTSASLAVSGLQSEDEADYYCAAWDATLNAWVFGGGT 15 25 35 45 55 65 75 85 95 105 b c d e L40F CDR2 I76V N53D N35K T33L S25R S31R CDR1 25 D94A 94 31 CDR3 95 S95T G98A Fig. 3 Location of specific sequence elements in the structure. a Hydrophobicity (gray) and aggregation score (brown) of the ordered part of the fibril protein. Magenta letters: mutations compared to the IGLV1-44 germ line segment. Boxes: CDRs. Residue numbers refer to the native LC without signal sequence. b Fibril protein showing the residue-specific aggregation score (0–5). c Electrostatic surface representation of the fibril protein. d Fibril protein with CDRs (black) and mutations (magenta) highlighted. e Ribbon diagram of a native V domain (PDB entry 1BJM) showing residues 3–113. CDRs are colored black; mutations are colored in red if they affect the core (residue 76), purple (surface residues with potential relevance for domain–domain interactions), or magenta (other surface residues) 4 NATURE COMMUNICATIONS | (2019) 10:1103 | https://doi.org/10.1038/s41467-019-09032-0 | www.nature.com/naturecommunications Hydrophobicity score NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09032-0 ARTICLE a b Native Fibril Native Fibril Native Fibril 90° N C 180° 12 3 Fig. 4 Comparison of the fibril structure and the native V domain fold. a Comparison of the native V domain fold (PDB entry 1BJM) with the fibril state. L L Residues 15–105 are shown in rainbow color. The native conformation is truncated at residue 118, corresponding to our fibril protein. The diffuse N-terminal and C-terminal tails of the fibril structure are schematically added with a gray line. b Part of the native structure and of the fibril state showing the conformational switch of segments 86–93 and 16–23 relative to one another around the protein disulfide bond. c Schematic representation of the hypothetical misfolding reaction consisting of an unfolding reaction (1), the rotational switch (2) and the assembly into the fibril structure (3) Three of these mutations add a surface charge to the fibril content, but there are differences in the number and position of (Ser31Arg, Asn35Lys, Asn53Asp) (Fig. 2d). One mutation the β-strands within the sequence (Fig. 2c). The fibril conforma- removes a charge from the non-polar cavity C (Asp94Ala). tion is more extended and flattened compared to the native state Ser25Arg inserts a basic residue into the polar cavity B, where it (Fig. 4a), enabling the polypeptide chain to form one molecular helps to compensate the charges of Glu84 and Asp86 and to fibril layer. A particularly substantial structural rearrangement interdigitate two molecular layers of the fibril Supplementary happens in the region around the disulfide bond which cross- Figure 5c). Gly98Ala occurs in the highly aggregation-prone links the N-terminal and C-terminal segments of the protein. segment at residues Asn97–Phe101 (Fig. 3a). Thr33Leu, These segments show a parallel N to C orientation in the native Leu40Phe, Ile76Val, and Ser95Thr have no obvious structural state and an antiparallel orientation in the fibril (Fig. 4b). effect on the fibril. None of the replacements is clearly Therefore, misfolding induces a 180° rotational switch of one unfavorable to the fibril structure. No mutational change occurs segment relative to the other around the disulfide bond, placing within the IGLJ3 and IGLC2 germ line segments, nor within the stalk on one side of the disulfide bond and the head region on residues Gln1–Thr13, a previously described mutational hot spot the other (Fig. 4c). These stark structural differences between the of some amyloidogenic λ-LCs . This segment is conformation- native and misfolded state are consistent with our previous ally disordered or missing in the fibril (Supplementary Figure 4), observation that AL amyloid fibrils and refolded fibril proteins which implies that at least for some patients this segment is not differ in several structural features, such as their infrared spectral relevant to fibril formation. Indeed, mutational changes to the N- characteristics and their affinity for the amyloid-binding dyes terminus were found to be more relevant to a λ6-LC-derived V Thioflavin T and Congo red . domain, which is destabilized upon mutation , and λ6-LCs may 29,33 form fibrils with an ordered N-terminus . Discussion In this study, we have analyzed the molecular structure of Comparison with native LC conformations. The majority of the an amyloid fibril that was purified from patient tissue and is mutations occur at surface positions in the globular V domain therefore directly relevant to disease. Interestingly, we previously (Fig. 3e). Four of these changes (Thr33Leu, Asn35Lys, Ser95Thr, found that the fibril protein constituting this fibril is able to form Gly98Ala) may potentially impact the interactions with other amyloid-like fibrils in vitro that possess a different morphology, immunoglobulin domains. Only Ile76Val affects a buried residue. and possibly also a different protofilament substructure, than the This mutation removes a methylene group from the protein core, bona fide pathogenic aggregates studied here . These observa- which typically destabilizes a protein by 6 kJ/mol . Taken toge- tions indicate that it is essential to investigate patient-derived ther, we find one out of 10 mutations in this patient to be rather than in vitro formed fibrils when scrutinizing the mole- unfavorable to the native state, while two to six changes make the cular basis of a protein misfolding disease. LC more compatible with the fibril structure than the germ line The reconstructed fibril has an elongated and rigid structure, segment. consisting of a single protofilament. Values measured for the −26 2 The fibril structure is profoundly different from a natively bending rigidity of the AL fibrils (2.78 ± 0.21 × 10 Nm ) folded V domain. Both protein states contain a high β-sheet correspond to previously reported values, which were on the NATURE COMMUNICATIONS | (2019) 10:1103 | https://doi.org/10.1038/s41467-019-09032-0 | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09032-0 −28 −25 2 35 order of 10 –10 Nm for different amyloid-like fibrils . unfavorable to the native protein conformation (Fig. 3e). The The stiffness of the fibrils could explain why amyloid fibril analyzed fibril is representative for amyloid fibrils from cardiac deposits impair the natural function of the heart. Cryo-EM and AL amyloidosis as it is derived from an IGLV1-44 germ line reconstruction of the 3D map enabled us to reveal the molecular segment, the major germ line segment leading to heart 7,16–18 structure underlying these effects. We could show that residues involvement . The mass of the fibril protein corresponds to Gly15–Thr105 adopt a stable conformation in the fibril, while the that of other AL fibril proteins and consists mainly of a V 22,23 first and last 12 amino acid residues are not resolved in our domain . The observed fibril morphology resembles fibril 22,23 structure and are conformationally disordered (Supplementary morphologies from other AL patients with cardiomyopathy , Figure 4). The fibril proteins are interdigitated along the main despite clear patient-specific differences. However, systemic AL axis of the fibril, providing resilience to mechanical stress (Sup- amyloidosis is an extraordinarily variable disease and other LC plementary Figure 1). The fold of the AL fibril protein is novel subtypes may be associated with different structural properties, and differs from previously published fibril protein structures. It particularly at the protein N-terminus, as was indicated recently 29,33 consists of a head and stalk region and encompasses one non- for fibrils derived from λ6-LCs . polar and two polar cavities (Figs. 1b, 2d). The current data from cryo-EM sheds light onto the misfolding The single-protofilament architecture of this AL amyloid fibril of proteins and their pathogenicity, representing a solid basis for contrasts with the majority of previously described cross-β fibrils, further investigation of molecular mechanisms underlying human 24,26–28,36 which consist of multiple protofilaments . However, we pathology, for example by in vitro aggregation studies. Detailed cannot exclude the formation of multi-protofilament AL amyloid knowledge of the molecular structure of pathogenic protein states fibrils, as our sample contains a second fibril morphology that is may lead to the development of novel ligands which recognize thicker than the presently studied one . Furthermore, the pre- these structures and form the basis of new detection methods or sently investigated fibril contains surface-exposed charge pairs, therapeutic strategies. However, due to the heterogeneity of sys- for example at residues Asp53 and Arg55, which were previously temic AL amyloidosis further work will be necessary to dissect the identified as protofilament–protofilament interaction sites in structural characteristics of fibrils from different groups of murine AA amyloid fibrils . However, the majority of the fibrils patients and to identify common structural themes between dif- in the AL fibril sample does not contain multiple protofilaments, ferent cohorts of patients as well as systematic variations. indicating that the assembly into higher-order structures is unfavorable for this fibril protein. Methods The protein fold provides insight into the mechanism of LC Source of AL fibrils. AL amyloid fibrils were extracted from the heart of a woman (Supplementary Table 1), suffering from advanced heart failure due to AL misfolding. It does not support mechanisms that assume of the 22,23 amyloidosis . First symptoms (dyspnea, fatigue) started 1 year before diagnosis initial formation of fibril segments consisting of dimeric proteins of AL amyloidosis. A monoclonal plasma cell disorder (smoldering myeloma) was 13,37,38 or peptides . Nor is there evidence for an assembly of diagnosed at the same time as AL amyloidosis. Bone marrow cytology showed 19% 37,39 domain-swapped molecules, consistent with other studies . plasma cells (<5% λ-positive in bone marrow histology) and interphase fluores- cence in situ-hybridization analysis of CD138+ enriched plasma cells showed the t The substantial conformational differences between the fibril (11;14) translocation and the 13q14 deletion. The patient received 5 months of structure and the native state imply instead that the native con- treatment with bortezomib and dexamethasone, and achieved a serological com- formation must be largely, if not entirely, unfolded to allow fibril plete remission of the smoldering myeloma. Ten months later, free λ-LCs increased formation to occur (Fig. 4c). In particular, we identified a rota- and treatment with lenalidomide and dexamethasone was started but stopped after 2 months due to cardiac decompensation. 1 month later high urgency listing was tional switch of the polypeptide chain around the disulfide bond done and the transplantation was performed 2 months later. Informed consent was that is only possible if most of the native strand–strand interac- obtained from the patient for the analysis of the amyloid deposits. tions are lost. Unfolding and the rotational switch are crucial for The fibril extraction was performed from heart muscle tissue as described fibril formation as unfolding is the prerequisite for the rotational previously . In brief, 250 mg of tissue were diced and washed five times with 0.5 mL Tris calcium buffer (20 mM Tris, 138 mM NaCl, 2 mM CaCl , 0.1 % NaN , switch, which in turn represents the basis for the formation of a 2 3 pH 8.0). Each washing step consisted of gentle vortexing and centrifugation at flat protein structure that lacks chain crossings (Fig. 4c). This 3100 × g for 1 min at 4 °C. The supernatant was discarded and the pellet was conformation is then able to associate into the intermolecular −1 resuspended in 1 mL of freshly prepared 5 mg mL Clostridium histolyticum hydrogen bond network of a cross-β sheet. collagenase (Sigma) in Tris calcium buffer. After incubation overnight at 37 °C the tissue material was centrifuged at 3100 × g for 30 min at 4 °C. The retained pellet As the disulfide bond occurs at the same position in the fibril as was resuspended in 0.5 mL buffer containing 20 mM Tris, 140 mM NaCl, 10 mM in the native V domain, previous research suggested that the ethylenediaminetetraacetic acid, 0.1 % NaN , pH 8.0, and subjected to 10 cycles of misfolding of the LC occurs under oxidizing conditions and homogenization in fresh buffer and centrifugation for 5 min at 3100 × g at 4 °C. maintains the cysteine disulfide bond . Our structure lends The remaining pellet was homogenized in 0.5 mL ice cold water, centrifuged for further support to this view, as we reveal a number of notable 5 min at 3100 × g at 4 °C and the fibril-containing supernatant was analyzed. The study was approved by the ethical committees of the University of Heidelberg (123/ imperfections in the fibril packing. There are three major internal 2006) and of Ulm University (210/13) cavities (Fig. 1b) and the protein is packed inside-out or outside- in. A highly acidic segment of low aggregation propensity is Measurement of the persistence length. Patient-derived fibrils were dried onto a buried in the core and only partially compensated by basic carbon-coated grid and negatively stained with uranyl acetate as described pre- charges, whereas segments that are much more hydrophobic and 22 viously . Images were taken at 120 kV using a JEM-1400Plus microscopy (Jeol) aggregation-prone are exposed to the solvent (Fig. 3b, c). The equipped with a TemCam-F216 camera (TVIPS). The contour length L and end- to-end distance D of 124 well-resolved fibrils was determined using the program situation looks drastically different in systemic AA amyloidosis, Fiji (http://fiji.sc). The plot of the squared end-to-end distance was fit with Eq. (1) where the most aggregation-prone and hydrophobic segments are to obtain the persistence length P: buried in the fibril core and where buried acidic residues are 2P compensated by an equal number of buried basic residues .AA 2 ðÞ L=2P D ¼ 4PL  1  1  e ð1Þ amyloid fibrils, which contain no disulfide bond, accomplish a tighter packing than AL amyloid fibrils and form much smaller The persistence length can be converted into the bending rigidity B as described by: internal cavities. The detailed information provided by our structure helps to B ¼ P  k  T ð2Þ explain the effects of mutational variants in systemic AL amy- loidosis. Several mutational changes have a beneficial effect on the In this equation, k refers to the Boltzmann constant and T to the temperature fibril structure (Fig. 3d), while only one mutation is clearly (300 K). 6 NATURE COMMUNICATIONS | (2019) 10:1103 | https://doi.org/10.1038/s41467-019-09032-0 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09032-0 ARTICLE Cryo-EM. A 3.5 μL aliquot was applied to a glow-discharged holey carbon-coated Reporting summary. Further information on experimental design is available in grid (C-flat 1.2/1.3 400 mesh) blotted from the back side after an incubation time of the Nature Research Reporting Summary linked to this article. 4 s at a humidity of >80% and plunge-frozen in liquid ethane using a Cryoplunge 3 System (Gatan). For image acquisition a K2-Summit detector (Gatan) in counting Data availability mode on a Titan Krios transmission electron microscope (Thermo Fisher Scien- The reconstructed cryo-EM map was deposited in the Electron Microscopy Data Bank tific) at 300 kV was used, using a Gatan imaging filter with a 20 eV slit. Table 1 lists with the accession code EMD-4452. The coordinates of the fitted atomic model were the data acquisition parameters. The raw data have been deposited at https://www. deposited in the PDB under the accession code 6IC3. The Cryo-EM data were deposited ebi.ac.uk/pdbe/emdb/ with the accession code EMPIAR-10245. on EMPIAR with the accession code EMPIAR-10245. The datasets and materials used during the current study are available from the corresponding author on reasonable Helical reconstruction. The raw data movie frames were gain-corrected with request. The data underlying the Supplementary Figures 1 and 2 are provided as a Source IMOD and aligned, motion-corrected and dose-weighted using MOTION- Data File. 41 42 COR2 . Gctf was used to estimate the contrast transfer function from the aligned and motion-corrected images. RELION 2.1 was used for the helical Received: 6 December 2018 Accepted: 15 February 2019 reconstruction of the fibril density. After manual selection of the fibrils from the aligned, motion-corrected micrographs, segments were extracted with a box size of ~333 Å and an inter-box distance of ~9% of the box length. Reference-free 2D classification with a regularization value of T = 3 produced class averages showing the helical repeat along the fibril axis. Class averages were selected based on a manual arrangement into a fibril structure. 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Genome Res. 12, 656–664 article’s Creative Commons license and your intended use is not permitted by statutory (2002). regulation or exceeds the permitted use, you will need to obtain permission directly from 50. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local the copyright holder. To view a copy of this license, visit http://creativecommons.org/ alignment search tool. J. Mol. Biol. 215, 403–410 (1990). licenses/by/4.0/. 51. Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729 (2013). © The Author(s) 2019 8 NATURE COMMUNICATIONS | (2019) 10:1103 | https://doi.org/10.1038/s41467-019-09032-0 | www.nature.com/naturecommunications

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