Conserved and nonconserved features of the folding pathway of hisactophilin, a β‐trefoil protein

Abstract

Based on previous studies of interleukin-1␤ (IL-1␤) and both acidic and basic fibroblast growth factors (FGFs), it has been suggested that the folding of ␤-trefoil proteins is intrinsically slow and may occur via the formation of essential intermediates. Using optical and NMR-detected quenched-flow hydrogen/deuterium exchange methods, we have measured the folding kinetics of hisactophilin, another ␤-trefoil protein that has <10% sequence identity and unrelated function to IL-1␤ and FGFs. We find that hisactophilin can fold rapidly and with apparently two-state kinetics, except under the most stabilizing conditions investigated where there is evidence for formation of a folding intermediate. The hisactophilin intermediate has significant structural similarities to the IL-1␤ intermediate that has been observed experimentally and predicted theoretically using a simple, topology-based folding model; however, it appears to be different from the folding intermediate observed experimentally for acidic FGF. For hisactophilin and acidic FGF, intermediates are much less prominent during folding than for IL-1␤. Considering the structures of the different ␤-trefoil proteins, it appears that differences in nonconserved loops and hydrophobic interactions may play an important role in differential stabilization of the intermediates for these proteins.

Nonnative interactions regulate folding and switching of myristoylated protein

“…The modeling is consistent with experimentally observed large decreases in hisactophilin folding rate with decreasing pH (19,24).…”

“…For hisactophilin, we observed two-state folding with a relatively high energy barrier (5.9 kT) (Fig. 1C) consistent with experimentally observed relatively slow folding (24). While the native topology-based model captures many features of the folding energy landscape, it neglects the roughness of the landscape due to nonnative interactions, which surely exist to some extent.…”

“…The function of hisactophilin is to reversibly recruit actin filaments to membranes during chemotaxis and osmotic stress. Previous studies revealed that increasing hisactophilin charge with decreasing pH favors actin and membrane binding but decreases protein stability and folding kinetics (23,24). The myristoyl increases hisactophilin stability with an apparent pK a of 6.95 as it switches between a "sequestered" state at high pH, where the myristoyl is buried in the protein core (Fig.…”

Experimental support for the foldability–function tradeoff hypothesis: Segregation of the folding nucleus and functional regions in fibroblast growth factor‐1

“…As highlighted by Meiering and coworkers, 47 the folding of FGF-1 was thought to differ from the folding pathways of both hisactophilin and IL-1b. The basis for this conclusion was a report that probed the folding of FGF-1 using NMR H/D exchange studies and identified the association of the N-and C-termini as the first step in the folding pathway.…”

“…51 Thus, our analysis of the folding of FGF-1 is inconsistent with the published report of Yu and coworkers, 48 but is in general agreement with the folding studies of other b-trefoil proteins in which the central strands of the protein are observed to fold faster than those on the periphery. 47,52,53 Only a key subset of structural elements (turns #2-#5 and turn #7, spanning $50% of the overall protein) appears necessary to confer efficient foldability to FGF-1. The entire protein is not (and apparently does not need to be) optimized for foldability; the regions not contributing to formation of the folding transition state instead segregate to regions of HSPG and receptor-binding functionalities.…”