Metamorphic proteins mediate evolutionary transitions of structure

Abstract

The primary sequence of proteins usually dictates a single tertiary and quaternary structure. However, certain proteins undergo reversible backbone rearrangements. Such metamorphic proteins provide a means of facilitating the evolution of new folds and architectures. However, because natural folds emerged at the early stages of evolution, the potential role of metamorphic intermediates in mediating evolutionary transitions of structure remains largely unexplored. We evolved a set of new proteins based on approximately 100 amino acid fragments derived from tachylectin-2--a monomeric, 236 amino acids, five-bladed beta-propeller. Their structures reveal a unique pentameric assembly and novel beta-propeller structures. Although identical in sequence, the oligomeric subunits adopt two, or even three, different structures that together enable the pentameric assembly of two propellers connected via a small linker. Most of the subunits adopt a wild-type-like structure within individual five-bladed propellers. However, the bridging subunits exhibit domain swaps and asymmetric strand exchanges that allow them to complete the two propellers and connect them. Thus, the modular and metamorphic nature of these subunits enabled dramatic changes in tertiary and quaternary structure, while maintaining the lectin function. These oligomers therefore comprise putative intermediates via which beta-propellers can evolve from smaller elements. Our data also suggest that the ability of one sequence to equilibrate between different structures can be evolutionary optimized, thus facilitating the emergence of new structures.

Fig. 1.

Biophysical features of the pentameric lectin Lib1-B7. Similar patterns observed with Lib2-D2 are described in Fig. S1. (A) Mass spectrometry of a variant from second round of evolution (Lib1-B7-10, Upper Spectrum) and variant Lib1-B7-18 (Lower Spectrum) indicate that pentamers comprised the main oligomeric state throughout the evolution of Lib1-B7 (complementary data are provided as Fig. S2). (B) Elution patterns Lib1-B7 and its evolved variant from a Superdex 200/30 gel filtration column. Lib1-B7 was eluted in several peaks presumably corresponding to high molecular weight aggregates (8.05 ml), pentamers (15.35 ml), and smaller assemblies (≥16.9 ml). In contrast, Lib1-B7-18 was eluted as one peak at 15.5 ml corresponding to pentamers. (C) Guanidinium hydrochloride (GdnHCl) denaturation curves in the absence or presence of the sugar ligand (GlcNAc, 5 mM). Curves systematically deviated from the two-state model, and only apparent D₅₀ values could be derived: 3.26 and 3.71 M for Lib1-B7 without and with ligand, respectively, and 3.32 and 3.34 M for Lib1-B7-18, respectively. (D) Refolding to the native state. The starting point (Lib1-B7) and its evolved variant (Lib1-B7-18) were unfolded in various concentrations of GdnHCl and refolding was induced by dilution into buffer. The fraction of refolded protein was determined by measuring the residual mucin-binding activity.

Fig. 2.

Structures of the evolved pentameric lectins. (A) Topology diagram depicting the strand rearrangements in wild-type tachylectin-2 versus the oligomeric lectins. Magnification of the first blade with N and C termini of wild-type tachylectin-2 is provided to indicate the Velcro closure that completes the first blade. The subunits of the oligomeric lectins are colored differentially for clarity and are named A to E. (B) The evolved pentamers (shown in two 180° rotated views) comprise two five-bladed propellers connected via a short linker. The individual propellers are essentially identical to each other and to wild-type tachylectin-2 (Cα RMSD values of 0.35 Å between the individual propellers of Lib1-B7-18, and 0.87 and 0.43 Å between these two propellers and wild-type tachilectin-2; 0.44 Å, 0.52, and 0.49 Å, are the respective values for Lib2-D2-15). The five sequence-identical subunits composing the pentamers are colored as follows: intrapropeller subunits in shades of blue, and bridging subunits in purple and light blue. The bound GlcNAc ligands (five in Lib1-B7-18, and four in Lib2-D2-15) are shown in spheres; details of the binding sites are provided as Fig. S5.

Fig. 3.

Structural metamorphism in the evolved oligomeric lectins. (A) Sequence alignment of the metamorphic region in subunit C of Lib1-B7-18 with the two metamorphic regions observed in subunits C and E of Lib2-D2-15. This region is a stretch of 23 amino acids that in wild-type tachylectin-2 comprises the third and the fourth strands of a blade and provides part of the sugar-binding site. Regions with wild-type-like structures are colored in blue and the alternative structure is colored according to Fig. 2B. Residues mutated during rounds of evolution are marked in red. (B) Structures of the intrapropeller subunits represented by subunit A in each variant and the interpropeller bridging subunits (subunits C and E in Lib2-D2-15, and subunit C in Lib1-B7-18). A short segment within the bridging subunit C in Lib2-D2-15 (within the linker connecting the two propellers), and a short loop in subunit E, appear to be disordered and are presented as dashed lines.