Relationship between Sequence Determinants of Stability for Two Natural Homologous Proteins with Different Folds

Abstract

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.

A polymetamorphic protein

“…5,18,51 Sequences that can encode two stable folds have been referred to as 'bridges', 'switches', 'metamorphic proteins' and 'transformer proteins' among other terms. 2,15,52-54 S-VLV is in effect a three-way bridge or three-fold switch, 53 capable in principle of acting as a hub for mutational traffic between at least three stable folding patterns related by nontrivial changes in secondary structure.…”

Re‐engineering a β‐lactamase using prototype peptides from a library of local structural motifs

“…The I-sites library collects protein sequences of [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] residues that correlate strongly with local structural motifs and are not limited by family boundaries. 27,28 Each structural motif is associated to several sequence clusters, which are in turn associated to a paradigm sequence.…”

“…(a) proteins tolerate a surprisingly large number of mutations [2][3][4][5][6][7][8] ; (b) there are several examples of similar protein folds encoded by completely different sequences 9 ; (c) conversely, proteins with highly similar sequence but different folds have been created [10][11][12][13][14][15] ; (d) novel folds can be generated by nonhomologous domain or subdomain recombination [16][17][18] ; (e) identical sequences of 8-11 residues can adopt different structure in different proteins 17,19,20 ; (f) well documented examples of alternative native folds for the same sequence are known 10,17,21 ; and (g) specific long-range tertiary interactions might not be the main determinant of protein conformation. 22 Yet, protein functions and folds evolve by sequence changes, and therefore there should be a way to reconcile all the above properties in a single folding theory.…”

A folding space odyssey

“…5. The identification of highly similar homologous proteins with different structures, such as Pfl 6 and Xfaso 1, opens up the feasibility of determining structure switching mechanisms through mutagenesis studies (6).…”