Structure of the preamyloid dimer of beta-2-microglobulin from covalent labeling and mass spectrometry.
ABSTRACT: Beta-2-microglobulin (beta2m) self-associates into fibrillar amyloid deposits in the musculoskeletal system of patients undergoing hemodialysis treatment. Previous studies have shown that stoichiometric amounts of Cu(II) at near physiological conditions can cause beta2m to organize into native-like dimers prior to forming amyloid fibrils. Here, we report the results from selective covalent labeling reactions combined with mass spectrometry that provide insight into the amino acid residues that mediate dimer formation in the wild-type protein. Using three complementary covalent labeling reagents, we find that the dimer interface is formed by the antiparallel stacking of ABED beta-sheets from two beta2m monomers. In addition, our data clearly indicate that a dimer interface involving the interactions of D-D strands from separate protein units as seen in the recent crystal structures of two mutant beta2m oligomers is unlikely.
Project description:Many human neurodegenerative diseases are associated with amyloid fibril formation. The human 99-residue beta(2)-microglobulin (beta2m) is one of the most intensively studied amyloid-forming proteins. Recent studies show that the C-terminal fragments 72-99, 83-89, and 91-96 form by themselves amyloid fibrils in vitro and play a significant role in fibrillization of the full-length beta2m protein under acidic pH conditions. In this work, we have studied the equilibrium structures of the 17-residue fragment 83-99 in solution, and investigated its dimerization process by multiple molecular dynamics simulations. We find that an intertwined dimer, with the positions of the beta-strands consistent with the results for the monomer, is a possible structure for two beta2m(83-89) peptides. Based on our molecular-dynamics-generated dimeric structure, a protofibril model is proposed for the full-length beta2m protein.
Project description:The main pathogenic process underlying dialysis-related amyloidosis is the accumulation of ?-2-microglobulin (?2m) as amyloid fibrils in the musculoskeletal system, and some evidence suggests that Cu(II) may play a role in ?2m amyloid formation. Cu(II)-induced ?2m fibril formation is preceded by the formation of discrete, oligomeric intermediates, including dimers, tetramers, and hexamers. In this work, we use selective covalent labeling reactions combined with mass spectrometry to investigate the amino acids responsible for mediating tetramer formation in wild-type ?2m. By comparing the labeling patterns of the monomer, dimer, and tetramer, we find evidence that the tetramer interface is formed by the interaction of D strands from one dimer unit and G strands from another dimer unit. These covalent labeling data along with molecular dynamics calculations allow the construction of a tetramer model that indicates how the protein might proceed to form even higher-order oligomers.
Project description:Beta-2-microglobulin (beta2m) deposits as amyloid fibrils in the musculoskeletal system of patients undergoing long-term dialysis treatment as a result of kidney failure. Previous work has shown that Cu(II) binding causes beta2m to organize into nativelike dimers and tetramers that precede amyloid formation. Cu(II) is then released from higher-order oligomers before mature Cu(II)-free amyloid fibrils are formed. While some of the Cu(II)-induced structural changes that enable beta2m self-assembly are starting to be revealed, the details of how the Cu(II) binding site evolves from the monomer to the dimers and tetramers are not known. Here, we report results from three mass spectrometry (MS)-based methods that provide insight into the changing Cu-beta2m interactions. We find that monomeric beta2m binds Cu(II) via the N-terminal amine, the amide of Gln2, His31, and Asp59. In the dimer and tetramer, Asp59 is no longer bound to Cu(II), but the other residues still comprise a well-defined albeit weaker binding site that is better able to release Cu(II). Consistent with this is the observation that a fraction of the tetrameric species no longer binds Cu(II) at this weakened binding site, which agrees with a previous report that suggested the tetramer as the first Cu(II)-free oligomer. Our results also provide some insight into structural changes caused by Cu(II) binding that facilitate oligomer formation. Specifically, binding by Asp59 in the monomer requires significant movement of this residue, and we propose that this repositioning is important for establishing a pair of dimer-stabilizing salt bridges between this residue and Lys19. We also find evidence that Cu(II) binding in the N-terminal region of the monomer repels Arg3, which likely allows this residue to form a pair of dimer-stabilizing salt bridges with Glu16. Overall, our measurements suggest that the previously proposed conformational switch caused by Cu(II) binding includes not only a cis-trans isomerization at Pro32 but also the repositioning of residues that are critical for the formation of new electrostatic interactions.
Project description:In humans suffering from dialysis-related amyloidosis, the protein beta2-microglobulin (beta2M) is deposited as an amyloid; however, an amyloid of beta2M is unknown in mice. beta2M sequences from human and mouse are 70% identical, but there is a seven-residue peptide in which six residues differ. This peptide from human beta2M forms amyloid in vitro, whereas the mouse peptide does not. Substitution of the human peptide for its counterpart in the mouse sequence results in the formation of amyloid in vitro. These results show that a seven-residue segment of human beta2M is sufficient to convert beta2M to the amyloid state, and that specific residue interactions are crucial to the conversion. These observations are consistent with a proposed Zipper-spine model for beta2M amyloid, in which the spine of the fibril consists of an anhydrous beta-sheet.
Project description:Amyloid fibrils formed from unrelated proteins often share morphological similarities, suggesting common biophysical mechanisms for amyloidogenesis. Biochemical studies of human beta-2 microglobulin (beta2M) have shown that its transition from a water-soluble protein to insoluble aggregates can be triggered by low pH. Additionally, biophysical measurements of beta2M using NMR have identified residues of the protein that participate in the formation of amyloid fibrils. The crystal structure of monomeric human beta2M determined at pH 5.7 shows that one of its edge beta-strands (strand D) adopts a conformation that differs from other structures of the same protein obtained at higher pH. This alternate beta-strand arrangement lacks a beta-bulge, which may facilitate protein aggregation through intermolecular beta-sheet association. To explore whether the pH change may yield the observed conformational difference, molecular dynamics simulations of beta2M were performed. The effects of pH were modeled by specifying the protonation states of Asp, Glu, and His, as well as the C terminus of the main chain. The bulged conformation of strand D is preferred at medium pH (pH 5-7), whereas at low pH (pH < 4) the straight conformation is observed. Therefore, low pH may stabilize the straight conformation of edge strand D and thus increase the amyloidogenicity of beta2M.
Project description:Beta-2 microglobulin (beta2m) is a small globular protein implicated in amyloid fiber formation in renal patients on long-term hemodialysis therapy. In vitro, under physiological conditions, beta2m is not aggregation prone. However, in the presence of stoichiometric Cu(2+), beta2m readily self-associates ultimately leading to heterogeneously sized aggregates. As this process occurs under near physiological solution conditions where the fold is >or=20 kJ/mol stabilized over the unfolded state, local conformational rearrangements are critical to understanding the oligomerization of beta2m. The isomerization of a conserved cis proline at residue 32 is a recognized step in this process that can be initiated by Cu(2+) binding. To better understand the structural basis of metal-induced oligomerization of beta2m, we set out to determine the role of individual imidazole side chains in mediating metal binding affinity, native state stability, and oligomerization in the framework of P32A beta2m. We find that P32A in the presence of Cu(2+) forms a tetramer in an apparently cooperative manner. One interface of this tetramer appears to reside along an edge strand as H51 is a key residue in mediating oligomerization. Furthermore, H31 is the main Cu(2+) binding residue in P32A and has an important role in stabilizing the protein in its holo form. Importantly, Cu(2+) binding affinity in P32A is much greater than in WT. Here, we show that this strong binding affinity need not be directly coupled to oligomerization. We interpret our results in terms of the known structures of beta2m(apo) and a reversible hexameric state of beta2m(holo).
Project description:Beta-2 microglobulin (beta2m) is a globular protein that self-associates into fibrillar amyloid deposits in patients undergoing hemodialysis therapy. Formation of these beta-sheet-rich assemblies is a fundamental property of polypeptides that can be triggered by diverse conditions. For beta2m, oligomerization into pre-amyloidogenic states occurs in specific response to coordination by Cu2+. Here we report the basis for this self-association at atomic resolution. Metal is not a direct participant in the molecular interface. Rather, binding results in distal alterations enabling the formation of two new surfaces. These interact to form a closed hexameric species. The origins of this include isomerization of a buried and conserved cis-proline previously implicated in the beta2m aggregation pathway. The consequences of this isomerization are evident and reveal a molecular basis for the conversion of this robust monomeric protein into an amyloid-competent state.
Project description:Beta2-microglobulin (beta2m), the light chain of class I major histocompatibility complex, is responsible for the dialysis-related amyloidosis and, in patients undergoing long term dialysis, the full-length and chemically unmodified beta2m converts into amyloid fibrils. The protein, belonging to the immunoglobulin superfamily, in common to other members of this family, experiences during its folding a long-lived intermediate associated to the trans-to-cis isomerization of Pro-32 that has been addressed as the precursor of the amyloid fibril formation. In this respect, previous studies on the W60G beta2m mutant, showing that the lack of Trp-60 prevents fibril formation in mild aggregating condition, prompted us to reinvestigate the refolding kinetics of wild type and W60G beta2m at atomic resolution by real-time NMR. The analysis, conducted at ambient temperature by the band selective flip angle short transient real-time two-dimensional NMR techniques and probing the beta2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type beta2m, involving more than a single intermediate species, and shedding new light into the fibrillogenic pathway. Moreover, a significant difference in the kinetic scheme previously characterized by optical spectroscopic methods was discovered for the W60G beta2m mutant.
Project description:Three-dimensional structures of beta(2)-microglobulin (beta2m) from chicken and various mammals have been described previously, but aside from genomic sequences, very little is known about the three-dimensional structures of beta2m in species other than warm-blooded vertebrates. Here, we present the first three-dimensional structure of beta2m from bony fish grass carp (Ctid-beta2m), resolved at 2.1 A. The key structural differences between this new structure and previously published structures are two new hydrogen bonds at positions Ile(37) and Glu(38) in strand C and Lys(66) in strand E, and a hydrophobic pocket around the center of the protein found in Ctid-beta2m. Importantly, Ctid-beta2m has a short D strand and a long loop between stands C and D, rather than the flexible region found in other beta2m structures that serves as a putative binding region for the major histocompatibility complex heavy chain. Comparing the Ctid-beta2m structure with those of bovine and human beta2ms, the Calpha root mean square deviation of the latter are 1.3 A and 1.8 A, respectively. Compared with the constant domains of Lamprey T cell receptor-like receptor (Lamp-TCRLC) and Amphioxus V and C domain-bearing protein (Amphi-VCPC), Ctid-beta2m exhibits very different topology. The three-dimensional structures of domains predicted from Amphi-VCPC/Lamp-TCRLC are distinctly lacking in strand A of beta2ms. There are 18 amino acids at the N terminus of Amphi-VCPC that may have evolved into strand A of beta2ms. A mutation in the BC loops of Amphi-VCPC may have led to the novel topology found in beta2m. Based on these results, Ctid-beta2m may well reflect evolutionary characteristics of ancestral C set molecules.
Project description:?-2-Microglobulin (?2m) forms amyloid fibrils in the joints of patients undergoing dialysis treatment as a result of kidney failure. One of the ways in which ?2m can be induced to form amyloid fibrils in vitro is via incubation with stoichiometric amounts of Cu(II). To better understand the structural changes caused by Cu(II) binding that allow ?2m to form amyloid fibrils, we compared the effect of Ni(II) and Zn(II) binding, which are two similarly sized divalent metal ions that do not induce ?2m amyloid formation. Using hydrogen/deuterium exchange mass spectrometry (HDX/MS) and covalent labeling MS, we find that Ni(II) has little effect on ?2m structure, despite binding in the same region of the protein as Cu(II). This observation indicates that subtle differences in the organization of residues around Cu(II) cause distant changes that are necessary for oligomerization and eventual amyloid formation. One key difference that we find is that only Cu(II), not Ni(II) or Zn(II), is able to cause the cis-trans isomerization of Pro32 that is an important conformational switch that initiates ?2m amyloid formation. By comparing HDX/MS data from the three metal-?2m complexes, we also discover that increased dynamics in the ?-sheet formed by the A, B, D, and E ? strands of the protein and repositioning of residues in the D-E loop are necessary aspects of ?2m forming an amyloid-competent dimer. Altogether, our results reveal new structural insights into the unique effect of Cu(II) in the metal-induced amyloid formation of ?2m.