UC San Diego

Interleukin (IL)-1[Beta] is one of the "master" cytokines that initiates the innate immune response. IL-1[Beta] is a tightly regulated signaling molecule, and requires a delicate balance between its on/off states to maintain homeostasis in the organisms that produce it. Local and system-wide deleterious effects are seen when the actions of IL-1[Beta] are thrown off-balance. Therefore, there are multiple mechanisms that exist to keep this cytokine in check, from transcription to degradation. In this study, we characterize the native state of the inactive precursor form of IL-1[Beta], pro-IL-1[Beta]. Little detailed structural information is known about pro-IL-1[Beta], despite the extreme importance of IL-1[Beta] as an immune system modulator. We find that pro-IL-1[Beta] is an extended, loosely packed confirmation with respect to the well-folded mature protein. The presence of the N-terminal region in pro-IL-1[Beta] prevents the C-terminal region, the eventual mature protein, from functioning by binding IL-1RI. With a combination of proteolysis and deutermium exchange mass spectrometry, we show that while pro-IL- 1[Beta] is in a different conformation that the mature protein, an area important in the kinetic refolding pathway of the mature protein is protected from both proteolysis and deuterium incorporation into the amide backbone. Thus, pro-IL-1[Beta] is primed for quick- conversion into the mature form once the appropriate combination of signals is received by the cell producing it. IL-1[Beta] is also regulated extracellularly. It has co-evolved with a competitive inhibitor, IL-1 receptor antagonist (IL-1Ra). Both IL-1[Beta] and IL-1Ra bind IL- 1RI, but only IL-1[Beta] elicits a cell-signaling response. Despite sharing only 30% sequence identity, IL- 1[Beta] and IL-1Ra share the same tertiary structure, the [Beta]-trefoil fold. Since the structures between the agonist and antagonist are conserved, we would also like to know if their folding behavior and stability pathways are conserved as well. The dominant view of protein folding is the energy landscape theory, where protein sequences are designed well enough to fold on a "minimally -frustrated" funnel-shaped landscape. While minimizing frustration, or energetic traps on the landscape on-route to the native state leads robust and faster folding, areas within a structure that are functionally important have been shown to add frustration into a folding pathway. Therefore a balance must be struck between maintaining a functional protein that folds well. Recent minimalist C- alpha, or Go-model simulations of IL-1[Beta] determined that a region in IL-1[Beta] important for function (the [Beta] -bulge) added topological frustration into folding landscape for this protein, where contacts formed early in the folding trajectory must be unmade and remade again late in the native state formation. As predicted from these theoretical simulations, removal of this area abolishes most of the frustration on the folding landscape. Since IL-1Ra lacks the [Beta]-bulge, we undertook equilibrium and kinetic folding studies to determine if indeed the folding of IL-1Ra is faster than that of IL-1[Beta]. We find that IL-1Ra, while maintaining the similar thermodynamic stability, folds faster from the I to N transition, indicating a less-frustrated landscape. Go-model simulations of IL-1Ra folding revealed a region of frustration in the vicinity of the loop between strands 11 and 12 of the protein, not in IL-1[Beta]. This area is in close proximity the receptor binding sites of both IL- 1Ra and IL-1[Beta]. Folding and biological activity studies mutations made to this area in IL-1Ra, along with two other functionally important areas were undertaken. These studies revealed new insights into the functional landscape of IL-1Ra that could not be inferred by inspection and comparison of the structures of unbound/ bound IL-1Ra and IL-1[Beta] to IL-1RI alone. Finally, the newest member of the IL-1 family of proteins, IL-33, contains a C-terminal b-trefoil structural motif. Early in vivo functional studies indicate it has a unique functional landscape as compared to all other family members. We undertook a combined folding and structural study of the [Beta]-trefoil region of IL-33, and show it is slightly destabilized and faster folding as compared to wt IL-1Ra and IL-1[Beta] in the same conditions