Proteins that share common ancestry may differ in structure and function because of divergent evolution of their amino acid sequences. For a typical diverse protein superfamily, the properties of a few scattered members are known from experiment. A satisfying picture of functional and structural evolution in relation to sequence changes, however, may require characterization of a larger, well chosen subset. Here, we employ a ''stepping-stone'' method, based on transitive homology, to target sequences intermediate between two related proteins with known divergent properties. We apply the approach to the question of how new protein folds can evolve from preexisting folds and, in particular, to an evolutionary change in secondary structure and oligomeric state in the Cro family of bacteriophage transcription factors, initially identified by sequence-structure comparison of distant homologs from phages P22 and . We report crystal structures of two Cro proteins, Xfaso 1 and Pfl 6, with sequences intermediate between those of P22 and . The domains show 40% sequence identity but differ by switching of ␣-helix to ␤-sheet in a C-terminal region spanning Ϸ25 residues. Sedimentation analysis also suggests a correlation between helix-to-sheet conversion and strengthened dimerization.conformational switching ͉ structural evolution ͉ transitive homology ͉ x-ray crystallography T he amino acid sequences of proteins evolve faster than the structures and functions encoded by these sequences. This neutral sequence drift allows annotation of an uncharacterized protein-coding gene based on common ancestry (homology) with a characterized gene, even if the protein sequences are quite different. Conservation of structure and function may hold even for homology so distant that no clear sequence similarity has survived evolutionary divergence. Yet there are limits: the structural and functional evolution of proteins is not completely static, and the likelihood of two proteins evolving divergent properties increases with the extent of sequence change separating them. Remote homology detection methods [for example, PSI-BLAST (1), COMPASS (2), and HHpred (3)] thus yield diminishing returns for gene annotation by grouping distantly related proteins into superfamilies that encompass diverse properties and biological roles. Simultaneously, however, the excavation of distant relationships opens a rich field for experimental studies of protein evolution, with the promise of recovered annotation power as one elucidates how structure and function vary across the ''sequence space'' of a superfamily.Transitive sequence comparison is one method for detecting distant homology between highly diverged sequences (4-8). In this approach, two dissimilar sequences, A and C, are indirectly linked if a third ''intermediate'' sequence B exists with sufficient similarity to both A and C to imply homology with both proteins. The relationships between A and B and between B and C combine to support distant common ancestry between A and C. Transitivity can extend ...

Toxoplasma gondii chronic infection in rodent secondary hosts has been reported to lead to a loss of innate, hard-wired fear toward cats, its primary host. However the generality of this response across T. gondii strains and the underlying mechanism for this pathogen-mediated behavioral change remain unknown. To begin exploring these questions, we evaluated the effects of infection with two previously uninvestigated isolates from the three major North American clonal lineages of T. gondii, Type III and an attenuated strain of Type I. Using an hour-long open field activity assay optimized for this purpose, we measured mouse aversion toward predator and non-predator urines. We show that loss of innate aversion of cat urine is a general trait caused by infection with any of the three major clonal lineages of parasite. Surprisingly, we found that infection with the attenuated Type I parasite results in sustained loss of aversion at times post infection when neither parasite nor ongoing brain inflammation were detectable. This suggests that T. gondii-mediated interruption of mouse innate aversion toward cat urine may occur during early acute infection in a permanent manner, not requiring persistence of parasite cysts or continuing brain inflammation.

The immediate-early gene early growth response 3 (Egr3) is associated with schizophrenia and expressed at reduced levels in postmortem patients' brains. We have previously reported that Egr3-deficient (Egr3(-/-)) mice display reduced sensitivity to the sedating effects of clozapine compared with wild-type (WT) littermates, paralleling the heightened tolerance of schizophrenia patients to antipsychotic side effects. In this study, we have used a pharmacological dissection approach to identify a neurotransmitter receptor defect in Egr3(-/-) mice that may mediate their resistance to the locomotor suppressive effects of clozapine. We report that this response is specific to second-generation antipsychotic agents (SGAs), as first-generation medications suppress the locomotor activity of Egr3(-/-) and WT mice to a similar degree. Further, in contrast to the leading theory that sedation by clozapine results from anti-histaminergic effects, we show that H1 histamine receptors are not responsible for this effect in C57BL/6 mice. Instead, selective serotonin 2A receptor (5HT(2A)R) antagonists ketanserin and MDL-11939 replicate the effect of SGAs, repressing the activity in WT mice at a dosage that fails to suppress the activity of Egr3(-/-) mice. Radioligand binding revealed nearly 70% reduction in 5HT(2A)R expression in the prefrontal cortex of Egr3(-/-) mice compared with controls. Egr3(-/-) mice also exhibit a decreased head-twitch response to 5HT(2A)R agonist 1-(2,5-dimethoxy 4-iodophenyl)-2-amino propane (DOI). These findings provide a mechanism to explain the reduced sensitivity of Egr3(-/-) mice to the locomotor suppressive effects of SGAs, and suggest that 5HT(2A)Rs may also contribute to the sedating properties of these medications in humans. Moreover, as the deficit in cortical 5HT(2A)R in Egr3(-/-) mice aligns with numerous studies reporting decreased 5HT(2A)R levels in the brains of schizophrenia patients, and the gene encoding the 5HT(2A)R is itself a leading schizophrenia candidate gene, these findings suggest a potential mechanism by which putative dysfunction in EGR3 in humans may influence risk for schizophrenia.

Bacteriophage Cro proteins bind to target DNA as dimers but do not all dimerize with equal strength, and differ in fold in the region of the dimer interface. We report the structure of the Cro protein from Enterobacteria phage N15 at 1.05 Å resolution. The subunit fold contains five a-helices and is closely similar to the structure of P22 Cro (1.3 Å backbone room mean square difference over 52 residues), but quite different from that of l Cro, a structurally diverged member of this family with a mixed a-helix/ b-sheet fold. N15 Cro crystallizes as a biological dimer with an extensive interface (1303 Å 2 change in accessible surface area per dimer) and also dimerizes in solution with a K d of 5.1 6 1.5 mM. Its dimerization is much stronger than that of its structural homolog P22 Cro, which does not self-associate detectably in solution. Instead, the level of self-association and interfacial area for N15 Cro is similar to that of l Cro, even though these two orthologs do not share the same fold and have dimer interfaces that are qualitatively different in structure. The common Cro ancestor is thought to be an all-helical monomer similar to P22 Cro. We propose that two Cro descendants independently developed stronger dimerization by entirely different mechanisms.

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.