Variable and Conserved Regions of Secondary Structure in the β-Trefoil Fold: Structure Versus Function

Abstract

β-trefoil proteins exhibit an approximate C₃ rotational symmetry. An analysis of the secondary structure for members of this diverse superfamily of proteins indicates that it is comprised of remarkably conserved β-strands and highly-divergent turn regions. A fundamental "minimal" architecture can be identified that is devoid of heterogenous and extended turn regions, and is conserved among all family members. Conversely, the different functional families of β-trefoils can potentially be identified by their unique turn patterns (or turn "signature"). Such analyses provide clues as to the evolution of the β-trefoil family, suggesting a folding/stability role for the β-strands and a functional role for turn regions. This viewpoint can also guide de novo protein design of β-trefoil proteins having novel functionality.

Keywords: de novo design; folding nucleus; hydrophobic patterning; ligand; protein symmetry.

FIGURE 1

The primary, secondary, and tertiary structure of the Symfoil (“Symfoil-4T”) reference β-trefoil protein. Upper panel: A “ribbon” diagram of the Symfoil protein (RCSB 3O4B). The colored region (blue: β-strand; red: turn) identifies the first of three repeating “trefoil” motifs in the structure (the other two colored in gray). Middle panel: A two-dimensional representation of the overall β-trefoil architecture and indicating the strand and turn numbering and the number of residues in each type of secondary structure (referencing Symfoil). Lower panel: the primary structure of the Symfoil protein indicating the secondary structure positions (β-strands underlined and indicated by “S”, and turns indicated by “T”).

FIGURE 2

A comparison of secondary structure insertions/deletions for three symmetric designed β-trefoil proteins. The Symfoil, Threefoil, and Mitsuba-1 proteins are three independently de novo designed, purely-symmetric β-trefoil proteins. The most compact of these three is Symfoil, primarily due to ligand-binding turns T2, T6, and T10, engineered into both Threefoil and Misuba-1.

FIGURE 3

Relative insertions or deletions in secondary structure elements among the β-trefoil superfamily of proteins. The reference protein is the Symfoil protein—a de novo designed, purely-symmetric, minimalist, and functionless β-trefoil protein (see text).

FIGURE 4

Cα structural conservation (<1.5 Å rmsd) within secondary structure elements for the β-trefoil family of proteins. The reference protein is the Symfoil protein (RCSB 3O4B)—a de novo designed, purely-symmetric, minimalist, and functionless β-trefoil protein (see Figure 1).

FIGURE 5

Relative secondary structure insertions or deletions of individual β-trefoil superfamilies. (A): Ricin B-like lectin superfamily (n = 22). (B): Cytokine superfamily (n = 10). (C): 30 K Lipoprotein superfamily (n = 3).

FIGURE 6

Sequence logo plots for the β-strand secondary structure in the β-trefoil superfamily. Equivalent β-strands are grouped by the C₃ rotational symmetry of the β-trefoil for all members of the superfamily in Table 1. Thus, strands S2, S6, and S10 are grouped together in this analysis, and similarly for the other symmetry-related β-strands (therefore, n = 126 at each position). The single letter amino acid code is utilized, and the height indicates the relative prevalence of a particular amino acid at each position. The amino acids are colored according to chemical properties (see text); however, hydrophobic is indicated by black.

FIGURE 7

Examples of β-trefoil proteins having distinct C₃ symmetry at the T2/T6/T10 turn region. The turn length in reference to the Symfoil protein is -1 (3PG0), 0 (2FDB), +1 (1UPS), +5 (3PG0), +6 (4IY9), and +8 (1T9F). The view is down the C₃ axis of rotational symmetry. Such symmetric relationships in turn structure suggests divergence of this turn structure occurred prior to duplication/fusion/truncation events leading to the extant β-trefoil architecture.

FIGURE 8

Sequence logo plot of the set of symmetric core-packing residues (see Figure 6) present in de novo designed symmetric β-trefoil proteins Symfoil-4T (3O4B), Phifoil (4OW4), Threefoil (3PG0), and Misuba-1 (5XG5). The positions within a single trefoil motif are shown, but these are replicated exactly for the other two trefoil motifs in each protein. Position #4 in S1, and position #3 in S2, have the highest neighbor contacts among the set of core residues (Blaber, 2021).