title: Substitution of proline32 by α-methylproline preorganizes β2-microglobulin for oligomerization but not for aggregation into amyloids.
abstract: Conversion of soluble folded proteins into insoluble amyloids generally proceeds in three distinct mechanistic stages: (1) initial protein misfolding into aggregation-competent conformers, (2) subsequent formation of oligomeric species and, finally, (3) self-assembly into extended amyloid fibrils. In the work reported herein, we interrogated the amyloidogenesis mechanism of human β2-microglobulin (β2m), which is thought to be triggered by a pivotal cis-trans isomerization of a proline residue at position 32 in the polypeptide, with nonstandard amino acids. Using chemical protein synthesis we prepared a β2m analogue in which Pro32 was replaced by the conformationally constrained amino acid α-methylproline (MePro). The strong propensity of MePro to adopt a trans prolyl bond led to enhanced population of a non-native [trans-MePro32]β2m protein conformer, which readily formed oligomers at neutral pH. In the presence of the antibiotic rifamycin SV, which inhibits amyloid growth of wild-type β2m, [MePro32]β2m was nearly quantitatively converted into different spherical oligomeric species. Self-assembly into amyloid fibrils was not observed in the absence of seeding, however, even at low pH (<3), where wild-type β2m spontaneously forms amyloids. Nevertheless, we found that aggregation-preorganized [MePro32]β2m can act in a prion-like fashion, templating misfolded conformations in a natively folded protein. Overall, these results provide detailed insight into the role of cis-trans isomerization of Pro32 and ensuing structural rearrangements that lead to initial β2m misfolding and aggregation. They corroborate the view that conformational protein dynamics enabled by reversible Pro32 cis-trans interconversion rather than simple population of the trans conformer is critical for both nucleation and subsequent growth of β2m amyloid structures.
authors: Laboratory of Organic Chemistry and ‡Laboratory of Physical Chemistry, ETH Zurich , Zurich CH-8093, Switzerland.
authors: Vladimir
authors: V
authors: Torbeev
authors: Marc-Olivier
authors: MO
authors: Ebert
authors: Jozica
authors: J
authors: Dolenc
authors: Donald
authors: D
authors: Hilvert
mesh_terms: D019943:Amino Acid Substitution; D000682:Amyloid / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D056004:Molecular Dynamics Simulation; D011392:Proline / Q000031:analogs & derivatives* / Q000737:chemistry; D055503:Protein Multimerization / Q000187:drug effects; D055550:Protein Stability; D017433:Protein Structure, Secondary; D012294:Rifamycins / Q000494:pharmacology; D013237:Stereoisomerism; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000235:genetics
chemical_list: D000682:Amyloid; D012294:Rifamycins; D001613:beta 2-Microglobulin; C051624:2-methylproline; D011392:Proline; C023808:rifamycin SV