Visualization of Transient Protein-Protein Interactions that Promote or Inhibit Amyloid Assembly

Abstract

SummaryIn the early stages of amyloid formation, heterogeneous populations of oligomeric species are generated, the affinity, specificity, and nature of which may promote, inhibit, or define the course of assembly. Despite the importance of the intermolecular interactions that initiate amyloid assembly, our understanding of these events remains poor. Here, using amyloidogenic and nonamyloidogenic variants of β2-microglobulin, we identify the interactions that inhibit or promote fibril formation in atomic detail. The results reveal that different outcomes of assembly result from biomolecular interactions involving similar surfaces. Specifically, inhibition occurs via rigid body docking of monomers in a head-to-head orientation to form kinetically trapped dimers. By contrast, the promotion of fibrillation involves relatively weak protein association in a similar orientation, which results in conformational changes in the initially nonfibrillogenic partner. The results highlight the complexity of interactions early in amyloid assembly and reveal atomic-level information about species barriers in amyloid formation.

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

“…S9). Previous studies have shown that oligomers of this size do not accumulate during fibril formation of ΔN6 at pH 6.2 or from wild-type β 2 m at pH 2.0, and thus their accumulation is a unique consequence of fibril disassembly at pH 6.4 (16,26). Further structural characterization revealed that these species are not recognized by the antibody WO1 (which recognizes the cross-β fold of amyloid; ref.…”

“…Wild-type β 2 m, 15 N-labeled β 2 m and β 2 m R3C were expressed and purified as described (9,16). The R3C variant was covalently labeled with maleimide-Alexa488 (Invitrogen) according to manufacturer's instructions.…”

“…Because amyloid formation is under kinetic control, minor changes in the population of an amyloid precursor can lead to dramatic changes in the rate of fibril formation and in the repertoire of nonnative species formed (15,16). Conversely, the kinetic stability of amyloid fibrils can be enhanced by alterations in the protein sequence (17), or by adding small molecules (18,19) or molecular chaperones (20).…”

Energy landscapes of functional proteins are inherently risky

“…Most importantly, however, and by contrast with native β 2 m, I T and its structural analog, ΔN6, are highly aggregation prone 64,71,72 , assembling rapidly into amyloid fibrils that resemble those that form in the disease dialysis-related amyloidosis 73,74 . Moreover, this kinetically trapped non-native conformation of the protein can promote misfolding of the initially innocuous native state into an amyloidogenic conformer, analogous to conformational conversion associated with prion disease 75 . Thus, trapped partially folded proteins formed on rugged folding landscapes not only give rise to difficulties by creating folding bottlenecks and enhancing aggregate potential, but they can also wield a second blow by turning soluble proteins into aggregation precursors.…”

An Internal Disulfide Locks a Misfolded Aggregation-Prone Intermediate in Cataract-Linked Mutants of Human Gamma-D Crystallin

“…Hence, some pathways of protein aggregation depend strongly on the redox environment. Notable examples include superoxide dismutase 1 (34,35) and β2-microglobulin (36)(37)(38). We and others have demonstrated that γ-crystallins, too, form crucial disulfide-mediated interactions.…”