In the Cro protein family, an evolutionary change in secondary structure has converted an alpha-helical fold to a mixture of alpha-helix and beta-sheet. P22 Cro and lambda Cro represent the ancestral all-alpha and descendant alpha+beta folds, respectively. The major structural differences between these proteins are at the C-terminal end of the domain (residues 34-56), where two alpha-helices in P22 Cro align with two beta-strands in lambda Cro. We sought to assess the possibility that smooth evolutionary transitions could have converted the all-alpha structure to the alpha+beta structure through sequences that could adopt both folds. First, we used scanning mutagenesis to identify and compare patterns of key stabilizing residues in the C-terminal regions of both P22 Cro and lambda Cro. These patterns exhibited little similarity to each other, with structurally important residues in the two proteins most often occurring at different sequence positions. Second, "hybrid scanning" studies, involving replacement of each wild-type residue in P22 Cro with the aligned wild-type residue in lambda Cro and vice versa, revealed five or six residues in each protein that strongly destabilized the other. These results suggest that key stability determinants for each Cro fold are quite different and that the P22 Cro sequence strongly favors the all-alpha structure while the lambda Cro sequence strongly favors the alpha+beta structure. Nonetheless, we were able to design a "structurally ambivalent" sequence fragment (SASF1), which corresponded to residues 39-56 and simultaneously incorporated most key stabilizing residues for both P22 Cro and lambda Cro. NMR experiments showed SASF1 to stably fold as a beta-hairpin when incorporated into the lambda Cro sequence but as a pair of alpha-helices when incorporated into P22 Cro.
PMID: 22325767;Abstract:
Fold switching may play a role in the evolution of new protein folds and functions. He et al., in this issue of Structure, use protein design to illustrate that the same drastic change in a protein fold can occur via multiple different mutational pathways. © 2012 Elsevier Ltd. All rights reserved.
Arc repressor is a homodimeric protein with a ribbon-helix-helix fold. A single polar-to-hydrophobic substitution (N11L) at a solvent-exposed position leads to population of an alternate dimeric fold in which 3₁₀ helices replace a β-sheet. Here we find that the variant Q9V/N11L/R13V (S-VLV), with two additional polar-to-hydrophobic surface mutations in the same β-sheet, forms a highly stable, reversibly folded octamer with approximately half the α-helical content of wild-type Arc. At low protein concentration and low ionic strength, S-VLV also populates both dimeric topologies previously observed for N11L, as judged by NMR chemical shift comparisons. Thus, accumulation of simple hydrophobic mutations in Arc progressively reduces fold specificity, leading first to a sequence with two folds and then to a manifold bridge sequence with at least three different topologies. Residues 9-14 of S-VLV form a highly hydrophobic stretch that is predicted to be amyloidogenic, but we do not observe aggregates of higher order than octamer. Increases in sequence hydrophobicity can promote amyloid aggregation but also exert broader and more complex effects on fold specificity. Altered native folds, changes in fold coupled to oligomerization, toxic pre-amyloid oligomers, and amyloid fibrils may represent a near continuum of accessible alternatives in protein structure space.
PMID: 16344489;PMCID: PMC1317976;Abstract:
The Arc repressor of bacteriophage P22 is a dimeric member of the ribbon-helix-helix family of transcription factors. Residues 9-14 of each wild-type Arc subunit pair to form two antiparallel -strands and have the alternating pattern of polar and nonpolar residues expected for a β-ribbon with one solvent-exposed face and one face that forms part of the hydrophobic core. Simultaneously switching Asn-11 to Leu and Leu-12 to Asn changes the local binary sequence pattern to that of an amphipathic helix. Previous studies have shown that this double mutation results in replacement of the wild-type β-ribbon by two right-handed Biohelices. Moreover, an Arc variant bearing just the Asn-11 → Leu mutation has an ambiguous binary pattern and can form either the ribbon or the helical structures, which interchange rapidly. Here, we study Arc mutants in which position 11 is occupied by Gly, Ala, Val, Ile, Leu, Met, Phe, or Tyr. These mutants adopt the wild-type β-ribbon structure in a sequence context that stabilizes this fold, but they assume the alternative helical structure in a sequence background in which the wild-type fold is precluded by negative design. In an otherwise wild-type sequence background, the detailed chemical properties of the position 11 side chain dictate which of the two competing conformational folds is preferred. © 2005 by The National Academy of Sciences of the USA.
To study the sequence determinants governing protein fold evolution, we generated hybrid sequences from two homologous proteins with 40% identity but different folds: Pfl 6 Cro, which has a mixed α + β structure, and Xfaso 1 Cro, which has an all α-helical structure. First, we first examined eight chimeric hybrids in which the more structurally conserved N-terminal half of one protein was fused to the more structurally divergent C-terminal half of the other. None of these chimeras folded, as judged by circular dichroism spectra and thermal melts, suggesting that both halves have strong intrinsic preferences for the native global fold pattern, and/or that the interfaces between the halves are not readily interchangeable. Second, we examined 10 hybrids in which blocks of the structurally divergent C-terminal region were exchanged. These hybrids showed varying levels of thermal stability and suggested that the key residues in the Xfaso 1 C terminus specifying the all-α fold were concentrated near the end of helix 4 in Xfaso 1, which aligns to the end of strand 2 in Pfl 6. Finally, we generated hybrid substitutions for each individual residue in this critical region and measured thermal stabilities. The results suggested that R47 and V48 were the strongest factors that excluded formation of the α + β fold in the C-terminal region of Xfaso 1. In support of this idea, we found that the folding stability of one of the original eight chimeras could be rescued by back-substituting these two residues. Overall, the results show not only that the key factors for Cro fold specificity and evolution are global and multifarious, but also that some all-α Cro proteins have a C-terminal subdomain sequence within a few substitutions of switching to the α + β fold.
