Accommodation of a highly symmetric core within a symmetric protein superfold

Abstract

An alternative core packing group, involving a set of five positions, has been introduced into human acidic FGF-1. This alternative group was designed so as to constrain the primary structure within the core region to the same threefold symmetry present in the tertiary structure of the protein fold (the beta-trefoil superfold). The alternative core is essentially indistinguishable from the WT core with regard to structure, stability, and folding kinetics. The results show that the beta-trefoil superfold is compatible with a threefold symmetric constraint on the core region, as might be the case if the superfold arose as a result of gene duplication/fusion events. Furthermore, this new core arrangement can form the basis of a structural "building block" that can greatly simplify the de novo design of beta-trefoil proteins by using symmetric structural complementarity. Remaining asymmetry within the core appears to be related to asymmetry in the tertiary structure associated with receptor and heparin binding functionality of the growth factor.

Figure 1.

(Top panel) Relaxed stereo ribbon diagram (side and top views) detailing the β-trefoil tertiary structure of FGF-1 and the associated threefold symmetry. The green color identifies the repeated structural domain. The cyan color within this domain identifies structurally conserved elements. (Middle panel) Relaxed stereo diagram showing an overlay of the three structural domains within FGF-1 (regions 11–52, 53–93, and 94–137). The overlay was performed using the structurally conserved regions (cyan color) in the top panel. (Bottom panel) Alignment of the 140-amino-acid primary structure of FGF-1 according to the threefold tertiary structure symmetry present in the β-trefoil superfold (Murzin et al. 1992). Light gray boxes indicate residue positions with two residues in common. The dark gray box indicates the single position where all three residues are in common. The open boxes indicate the residues comprising the core packing group.

Figure 2.

Chevron plot of the folding and unfolding kinetic data for WT (filled circles), Leu 44→Phe (filled diamonds), Leu 73→Val (filled triangles), Val 109→Leu (filled squares), and the SYM5 mutant (open circles). The Cys 117→Val and Leu 111→Val point mutations are essentially indistinguishable from WT and are omitted for clarity.

Figure 3.

Relaxed stereo diagram showing overlaid subdomains within WT FGF-1 (top panel) and SYM5 (bottom panel) for the core positions mutated in this study, and illustrating the symmetric rotamer orientations and packing environments for these positions.

Figure 4.

(Top panel) ΔΔG values for the effects on stability of point and combination mutations (negative values indicate a stabilizing mutation). (Middle panel) ΔΔG_‡-D values for point and combination mutations derived from folding kinetic data (positive values indicate the TSE has been stabilized relative to the native state). (Bottom panel) ΔΔG_‡-N values for point and combination mutations derived from unfolding kinetic data (positive values indicate the TSE has been stabilized relative to the native state). In each panel, the solid columns indicate the experimentally determined values, and the hatched columns indicate the values expected from a simple sum of the individual point mutations.

Figure 5.

Relaxed stereo diagram of WT FGF-1 illustrating the core packing arrangement. Moving from top to bottom panels, the sequential packing of positions 31, 73, 117; 25, 67, 111; 23, 65, 109; and 44, 85, 132 are shown. The color of the three symmetry-related subdomains is the same as in Figure 1 ▶ (middle panel).

Figure 6.

Relaxed stereo diagram of SYM5 illustrating the core packing arrangement (details follow that of Fig. 5 ▶).