Bacteriophage Cro repressors provide the most extreme known violation of the expectation that natural protein domains with ~40% sequence identity share the same fold: the homologous Cro proteins Xfaso 1 (all-α helical) and Pfl 6 (mixed α+β) from the transitive-homology chain connecting P22 Cro and λ Cro. The 2008 discovery paper left the evolutionary account of this fold switch open, and later work has established the plausibility of smooth transitions without reconstructing the actual pathway. The open problem is to produce a complete, phylogenetically grounded evolutionary account of the Cro α→β fold switch: a rigorous Cro-family phylogeny that places the fold change, identification of the natural mutational and insertion/deletion (indel) path and any intermediate conformational states between the all-α and α+β folds, and determination of whether strengthened dimerization (including development of the hydrophobic ball-and-socket interface) arose before, during, or after the secondary-structure switch.

Background

Cro proteins are small (~60–70 residue) transcriptional repressors from temperate bacteriophages (viruses that infect bacteria) that bind specific operator DNA sites (the repressor-binding sequences near phage lytic genes) as dimers to promote lytic growth. In the classic lambdoid phages, P22 Cro adopts an all-α helical fold and is monomeric in solution, whereas λ Cro adopts a mixed α-helix/β-sheet fold and dimerizes at low micromolar affinity. Despite only ~25% sequence identity between them, conserved gene context, parallel DNA-binding function, partial structural similarity, and remote sequence homology indicate that P22 Cro and λ Cro are true homologs (derived from a common ancestral gene), making their different folds a case of evolutionary fold switching — the remodeling of a protein's secondary and tertiary structure during evolution.

To trace how this fold change happened, Roessler, Hall, Anderson, Ingram, Roberts, Montfort and Cordes (PNAS 2008) used transitive homology: instead of directly aligning the two divergent endpoints, they identified intermediate Cro sequences from bacterial prophages (viral genomes integrated in bacterial chromosomes) such that each neighboring pair in the chain shares ~40% sequence identity. They then solved crystal structures of two stepping stones, Xfaso 1 and Pfl 6, which share 40% identity over 65 aligned residues yet adopt different folds (all-α versus α+β), with the structural crossover falling abruptly between these two proteins. Notably, only the α+β Cro proteins showed appreciable solution dimerization, and λ Cro's tight dimer interface contains a hydrophobic "ball-and-socket" contact (a bulky side chain packing into a cavity on the partner subunit), previously implicated by mutagenesis of Ala33 and Phe58, that is shallower in Pfl 6 (Met33/Ile58) — suggesting dimerization strength and fold may have evolved on partially independent schedules.

The 2008 paper explicitly left the evolutionary account unfinished: a rigorous global phylogeny (evolutionary tree) of Cro sequences was still lacking, complicated by extreme sequence diversity and the mosaic (patchwork, recombination-driven) nature of phage genomes; the actual sequence changes and any intermediate conformational states between the two folds were unknown; and the causal order of fold conversion versus dimerization strengthening was unresolved. Subsequent work by the same laboratory (designed "chameleon" sequences that fold either way, indel and chimera experiments, and the engineered intermediate hybrids XPH1/XPH2 with partly rearranged secondary structure) demonstrated that stepwise, multistep transformations are plausible, while a broader literature established that fold-switching proteins are far more common than once thought and that analogous fold transitions occurred in other families (e.g., from helix-turn-helix (a two-helix DNA-binding motif) to winged-helix (a variant with an added β-sheet "wing") folds in bacterial response regulators (two-component signaling proteins)). None of this, however, reconstructs the natural historical pathway in the Cro family itself. The broader questions of how frequently homologous fold switching occurs across protein families, and how best to design transitive-homology targeting, are out of scope for this task.

Problem Statement

Reconstruct the evolutionary pathway by which the bacteriophage Cro protein family switched folds: provide a rigorous, global phylogeny of Cro sequences that robustly places the α→β secondary-structure transition (the crossover between the all-α fold of Xfaso 1 and relatives such as P22 Cro and the α+β fold of Pfl 6 and λ Cro) on the Cro family tree despite extreme sequence divergence and the mosaic genomes of bacteriophages; identify the natural mutational and insertion/deletion events, and characterize any intermediate conformational states, sufficient to convert the all-α fold into the α+β fold (or establish that no structurally intermediate, stably folded states existed along the actual historical path, i.e., that the switch was effectively all-or-nothing) while preserving regulatory function; and determine the evolutionary order and causal relationship between the secondary-structure switch and the strengthening of dimerization — specifically whether the low-micromolar dimerization of λ Cro, including development of the hydrophobic ball-and-socket contacts at positions 33 and 58, arose before, during, or after the fold conversion, using the weaker (~1 mM) dimerization of Pfl 6 and the monomeric all-α Cros as calibration points. The phylogenetic placement, the experimentally supported sequence of molecular changes, and the ordering of interface evolution are to explain jointly how a ~65-residue domain changed its global fold through ordinary mutations without loss of function.

References

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