Interleukin (IL) 11 activates multiple intracellular signaling pathways by forming a complex with its cell surface α-receptor, IL-11Rα, and the β-subunit receptor, gp130. Dysregulated IL-11 signaling has been implicated in several diseases, including some cancers and fibrosis. Mutations in IL-11Rα that reduce signaling are also associated with hereditary cranial malformations. Here we present the first crystal structure of the extracellular domains of human IL-11Rα and a structure of human IL-11 that reveals previously unresolved detail. Disease-associated mutations in IL-11Rα are generally distal to putative ligand-binding sites. Molecular dynamics simulations showed that specific mutations destabilize IL-11Rα and may have indirect effects on the cytokine-binding region. We show that IL-11 and IL-11Rα form a 1:1 complex with nanomolar affinity and present a model of the complex. Our results suggest that the thermodynamic and structural mechanisms of complex formation between IL-11 and IL-11Rα differ substantially from those previously reported for similar cytokines. This work reveals key determinants of the engagement of IL-11 by IL-11Rα that may be exploited in the development of strategies to modulate formation of the IL-11–IL-11Rα complex.

The misfolding, aggregation, and accumulation of proteins as amyloid fibrils is a defining characteristic of several debilitating diseases. Human apolipoprotein C-II (apoC-II) amyloid fibrils are representative of the fibrils formed by a number of plasma apolipoproteins implicated in amyloid-related disease. Previous structural analyses identified a buried charge pair between residues K30 and D69 within apoC-II amyloid fibrils. We have investigated the effects of amino acid substitutions of these residues on apoC-II fibril formation. Two point mutations of apoC-II, D69K and K30D, as well as a reversed ion-pair mutant containing both mutations (KDDK) were generated. Fibril formation by the double mutant, apoC-II KDDK, and apoC-II D69K was enhanced compared to that of wild-type (WT) apoC-II, while apoC-II K30D lacked the ability to form fibrils under standard conditions. Structural analyses showed that WT apoC-II, apoC-II D69K, and apoC-II KDDK fibrils have similar secondary structures and morphologies. Size distribution analyses revealed that apoC-II D69K fibrils have a broader range of fibril sizes while apoC-II KDDK fibrils showed an increased frequency of closed fibrillar loops. ApoC-II D69K fibrils exhibited reduced thioflavin T binding capacity compared to that of fibrils formed by WT apoC-II and apoC-II KDDK. These results indicate that specific charge and charge-pair mutations within apoC-II significantly alter the ability to form fibrils and that position 69 within apoC-II plays a key role in the rate-limiting step of apoC-II fibril formation.

Plasma apolipoproteins form amphipathic α helices in lipid environments but in the lipid-free state show a high propensity to form β structure and self-associate into amyloid fibrils. The widespread occurrence of apolipoproteins in amyloid plaques suggests disease-related roles, specifically in atherosclerosis. To reconcile the dual abilities of apolipoproteins to form either α helices or cross-β sheet structures, we examined fibrils formed by human apolipoprotein C-II (apoC-II). A structural model for apoC-II fibrils shows a cross-β core with parallel β strands, including a buried K30-D69 charge pair. We investigated the effect of abolishing this charge pair in mutant D69K apoC-II. Fluorescence studies indicated more rapid fibril formation and less solvent accessibility of tryptophan (W26) in D69K apoC-II fibrils than in wild-type (WT) fibrils. X-ray diffraction data of aligned D69K apoC-II fibrils yielded a typical cross-β structure with increased β sheet spacing compared to that of WT fibrils. Hydrogen/deuterium (H/D) exchange patterns were similar for D69K apoC-II fibrils compared to WT fibrils, albeit with an overall reduction in the level of slow H/D exchange, particularly around residues 29-32. Molecular dynamics simulations indicated reduced β strand content for a model D69K apoC-II tetramer compared to the WT tetramer and confirmed an expansion of the cross-β spacing that contributed to the formation of a stable charge pair between K69 and E27. The results highlight the importance of charge-pair interactions within the apoC-II fibril core, which together with numerous salt bridges in the flexible connecting loop play a major role in the ability of lipid-free apoC-II to form stable cross-β fibrils.

Small heat-shock proteins (sHSPs) are ubiquitously expressed molecular chaperones that inhibit amyloid fibril formation; however, their mechanisms of action remain poorly understood. sHSPs comprise a conserved α-crystallin domain flanked by variable N- and C-terminal regions. To investigate the functional contributions of these three regions, we compared the chaperone activities of various constructs of human αB-crystallin (HSPB5) and heat-shock 27-kDa protein (Hsp27, HSPB1) during amyloid formation by α-synuclein and apolipoprotein C-II. Using an array of approaches, including thioflavin T fluorescence assays and sedimentation analysis, we found that the N-terminal region of Hsp27 and the terminal regions of αB-crystallin are important for delaying amyloid fibril nucleation and for disaggregating mature apolipoprotein C-II fibrils. We further show that the terminal regions are required for stable fibril binding by both sHSPs and for mediating lateral fibril–fibril association, which sequesters preformed fibrils into large aggregates and is believed to have a cytoprotective function. We conclude that although the isolated α-crystallin domain retains some chaperone activity against amyloid formation, the flanking domains contribute additional and important chaperone activities, both in delaying amyloid formation and in mediating interactions of sHSPs with amyloid aggregates. Both these chaperone activities have significant implications for the pathogenesis and progression of diseases associated with amyloid deposition, such as Parkinson's and Alzheimer's diseases.

Apolipoproteins form amphipathic helical structures that bind lipid surfaces. Paradoxically, lipid-free apolipoproteins display a strong propensity to form cross-β structure and self-associate into disease-related amyloid fibrils. Studies of apolipoprotein C-II (apoC-II) amyloid fibrils suggest that a K30-D69 ion pair accounts for the dual abilities to form helix and cross-β structure. Consistent with this is the observation that a K30D mutation prevents fibril formation under standard fibril forming conditions. However, we found that fibril formation by K30D apoC-II proceeded readily at low pH and a higher salt or protein concentration. Structural analysis demonstrated that K30D apoC-II fibrils at pH 7 have a structure similar to that of the wild-type fibrils but are less stable. Molecular dynamics simulations of the wild-type apoC-II fibril model at pH 7 and 3 showed that the loss of charge on D69 at pH 3 leads to greater separation between residues K30 and D69 within the fibril with a corresponding reduction in β-strand content around residue 30. In contrast, in simulations of the K30D mutant model at pH 7 and 3, residues D30 and D69 moved closer at pH 3, accompanied by an increase in β-strand content around residue 30. The simulations also demonstrated a strong dominance of inter- over intramolecular contacts between ionic residues of apoC-II and suggested a cooperative mechanism for forming favorable interactions between the individual strands under different conditions. These observations demonstrate the important role of the buried K30-D69 ion pair in the stability and solution properties of apoC-II amyloid fibrils.