Vity reported for NetPhos predictions cover a range from 69?6 [25], partially due to the lack of insufficient dataPLOS ONE | DOI:10.1371/journal.pone.0151961 March 22,15 /Evolutionary Dynamics of Sequence, Structure, and Phosphorylation in the p53, p63, and p73 Paralogsavailable to train phosphorylation predictors [43]. Still, these are all standard prediction methods, widely used in computational and molecular biology when experimental data is not available. By comparing approximately 300 protein sequences from the vertebrate p53 family and an additional 50 invertebrate p53 DBD domain sequences, we have investigated diverging properties from sequence to structure to regulation in the p53 family. From the invertebrate p53 DBD phylogeny, it appears that p53 DBD sequences primarily form clades based on the domain content of the full-length protein. If the p53 DBD containing proteins from Fig 1 are arranged by species in the order of taxonomy and with focus on their domain composition, a picture of the main evolutionary events of the p53 family emerges (Fig 8). As previously shown, a three domain p53 DBD containing protein is present in choanoflagellates [12]. The shared precursor of this protein and the very first metazoan p53 protein must have had at least three of the four domains found in present day vertebrate p53 family proteins. We observe proteins with all four domains in gastropods, hemichordates, and early chordates. Since these belong to Bilateria, it is clear that the bilaterian ancestor had all four domains. It should also be noted that other species not included here, such as the placozoan, Trichoplax adhaerens, have an MDM2 binding site [44]. Although Pfam does not classify this protein to have a TAD domain, the MDM2 binding site indicates that it does, or at least that it used to have a TAD domain. Thus, TAD predates the buy ��-Amanitin divergence of Bilateria and Placozoa. Further, TAD and the other non-p53 DBD domains, are frequently lost (Fig 8). In Ecdysozoa, some of these domain losses are due to actual sequence segment loss and others are due to the sequence signature being depleted. Altogether, this clearly suggests that early metazoan, and perhaps even chanoflagellates have p53 family proteins that diverged less than many of the ecdysozoan p53 family proteins that have lost most domains and frequently only consist of the p53 DBD itself. There may be other equally or more remote p53 DBD proteins in other 6-Methoxybaicalein chemical information invertebrates, like j.jebo.2013.04.005 e.g. CEP-1 in Caenorhabditis elegans [44]. Lineage-specific gene duplications are frequent in invertebrates, but a last common ancestor of all proteins in the vertebrate p53 family is shared with B. floridae (Figs 1 and 8). It is also clear that the p53 DBD is less structurally disordered in single domain invertebrate proteins. In vertebrates, the three paralogs p53, p63, and p73, are diverging at different rates: p63 is highly constrained while p53 is not. Ray-finned fish are demonstrating rapid lineagespecific diversification among all three paralogs. Although this study is mostly focused on the functional domains and their divergence, the inter-domain linkers vary in length and in disorder/order and secondary SART.S23503 structure composition. Linkers are not just flexible spacers but important for controlling the conformational ensemble [45]. The divergence in linker 1 between p53 and p63 and p73 is profound and suggests functional change. TAD is rapidly diverging amongst p53 in different vertebrates, and has already d.Vity reported for NetPhos predictions cover a range from 69?6 [25], partially due to the lack of insufficient dataPLOS ONE | DOI:10.1371/journal.pone.0151961 March 22,15 /Evolutionary Dynamics of Sequence, Structure, and Phosphorylation in the p53, p63, and p73 Paralogsavailable to train phosphorylation predictors [43]. Still, these are all standard prediction methods, widely used in computational and molecular biology when experimental data is not available. By comparing approximately 300 protein sequences from the vertebrate p53 family and an additional 50 invertebrate p53 DBD domain sequences, we have investigated diverging properties from sequence to structure to regulation in the p53 family. From the invertebrate p53 DBD phylogeny, it appears that p53 DBD sequences primarily form clades based on the domain content of the full-length protein. If the p53 DBD containing proteins from Fig 1 are arranged by species in the order of taxonomy and with focus on their domain composition, a picture of the main evolutionary events of the p53 family emerges (Fig 8). As previously shown, a three domain p53 DBD containing protein is present in choanoflagellates [12]. The shared precursor of this protein and the very first metazoan p53 protein must have had at least three of the four domains found in present day vertebrate p53 family proteins. We observe proteins with all four domains in gastropods, hemichordates, and early chordates. Since these belong to Bilateria, it is clear that the bilaterian ancestor had all four domains. It should also be noted that other species not included here, such as the placozoan, Trichoplax adhaerens, have an MDM2 binding site [44]. Although Pfam does not classify this protein to have a TAD domain, the MDM2 binding site indicates that it does, or at least that it used to have a TAD domain. Thus, TAD predates the divergence of Bilateria and Placozoa. Further, TAD and the other non-p53 DBD domains, are frequently lost (Fig 8). In Ecdysozoa, some of these domain losses are due to actual sequence segment loss and others are due to the sequence signature being depleted. Altogether, this clearly suggests that early metazoan, and perhaps even chanoflagellates have p53 family proteins that diverged less than many of the ecdysozoan p53 family proteins that have lost most domains and frequently only consist of the p53 DBD itself. There may be other equally or more remote p53 DBD proteins in other invertebrates, like j.jebo.2013.04.005 e.g. CEP-1 in Caenorhabditis elegans [44]. Lineage-specific gene duplications are frequent in invertebrates, but a last common ancestor of all proteins in the vertebrate p53 family is shared with B. floridae (Figs 1 and 8). It is also clear that the p53 DBD is less structurally disordered in single domain invertebrate proteins. In vertebrates, the three paralogs p53, p63, and p73, are diverging at different rates: p63 is highly constrained while p53 is not. Ray-finned fish are demonstrating rapid lineagespecific diversification among all three paralogs. Although this study is mostly focused on the functional domains and their divergence, the inter-domain linkers vary in length and in disorder/order and secondary SART.S23503 structure composition. Linkers are not just flexible spacers but important for controlling the conformational ensemble [45]. The divergence in linker 1 between p53 and p63 and p73 is profound and suggests functional change. TAD is rapidly diverging amongst p53 in different vertebrates, and has already d.