Ancestral resurrection reveals evolutionary mechanisms of kinase plasticity

Howard, Conor J; Hanson-Smith, Victor; Kennedy, Kristopher J.; Miller, Chad J.; Lou, Hua; Johnson, Alexander D.; Turk, Benjamin E.; Holt, Liam J.

doi:10.7554/elife.04126

Cited by 60 publications

(73 citation statements)

References 79 publications

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“…That is, the allosteric sites may have evolved in a species-dependent manner based on individual needs, leading to larger sequence variations at those sites and higher ambiguity in the APR analysis. Overall, our data agree with previous studies showing that the pool of possible ancestors at the nodes of a phylogenetic tree reflect ancestral mechanistic function experimentally [74][75][76].…”

Section: Discussionsupporting

confidence: 92%

Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity

Grinshpon

Shrestha

Titus-McQuillan

et al. 2019

Preprint

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Apoptotic caspases evolved with metazoans more than 950 million years ago (MYA), and a series of gene duplications resulted in two subfamilies consisting of initiator and effector caspases. The effector caspase genes (caspases-3, -6, and -7) were subsequently fixed into the Chordata phylum more than 650 MYA when the gene for a common ancestor (CA) duplicated, and the three effector caspases have persisted throughout mammalian evolution. All caspases require an aspartate residue at the P1 position of substrates, so each caspase evolved discrete cellular roles through changes in substrate recognition at the P4 position combined with allosteric regulation. We examined the evolution of substrate specificity in caspase-6, which prefers valine at the P4 residue, compared to caspases-3 and -7, which prefer aspartate, by reconstructing the CA of effector caspases (AncCP-Ef1) and the CA of caspase-6 (AncCP-6An). We show that AncCP-Ef1 is a promiscuous enzyme with little distinction between Asp, Val, or Leu at P4. The specificity of caspase-6 was defined early in its evolution, where AncCP-6An demonstrates preference for Val over Asp at P4. Structures of AncCP-Ef1 and of AncCP-6An show a network of charged amino acids near the S4 pocket that, when combined with repositioning a flexible active site loop, resulted in a more hydrophobic binding pocket in AncCP-6An. The ancestral protein reconstructions show that the caspasehemoglobinase fold has been conserved for over 650 million years and that only three substitutions in the scaffold are necessary to shift substrate selection toward Val over Asp.

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Section: Discussionsupporting

confidence: 92%

Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity

Grinshpon

Shrestha

Titus-McQuillan

et al. 2019

Preprint

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“…6A and B). These analyses showed that Ssp2 KAD /Smk1 most strongly selects a P at ϩ1, but like some other CMGC kinases, it appeared to tolerate a G or A at that position as well (20)(21)(22). Similar to other MAPKs, Ssp2 KAD /Smk1 preferred a P and, to a lesser extent, other aliphatic residues at position Ϫ2.…”

Section: Resultsmentioning

confidence: 80%

Ssp2 Binding Activates the Smk1 Mitogen-Activated Protein Kinase

Tio

Omerza

Phillips

et al. 2017

Molecular and Cellular Biology

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Smk1 is a meiosis-specific mitogen-activated protein kinase (MAPK) in Saccharomyces cerevisiae that couples spore morphogenesis to the completion of chromosome segregation. Similar to other MAPKs, Smk1 is controlled by phosphorylation of a threonine (T) and a tyrosine (Y) in its activation loop. However, it is not activated by a dual-specificity MAPK kinase. Instead, T207 in Smk1's activation loop is phosphorylated by the cyclin-dependent kinase (CDK)-activating kinase (Cak1), and Y209 is autophosphorylated in an intramolecular reaction that requires the meiosis-specific protein Ssp2. In this study, we show that Smk1 is catalytically inert unless it is bound by Ssp2. While Ssp2 binding activates Smk1 by a mechanism that is independent of activation loop phosphorylation, binding also triggers autophosphorylation of Y209 in Smk1, which, along with Cak1-mediated phosphorylation of T207, further activates the kinase. Autophosphorylation of Smk1 on Y209 also appears to modify the specificity of the MAPK by suppressing Y kinase and enhancing S/T kinase activity. We also found that the phosphoconsensus motif preference of Ssp2/Smk1 is more extensive than that of other characterized MAPKs. This study therefore defines a novel mechanism of MAPK activation requiring binding of an activator and also shows that MAPKs can be diversified to recognize unique phosphorylation motifs.

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“…Although there have been several attempts to modify HLD substrate specificity by engineering their active sites, halide‐binding sites, and access tunnels, only two studies achieved a shift in HLD substrate specificity . Modification of substrate specificity by ASR has been reported for ancestral α‐glucosidases, which preferentially convert maltose‐like sugars, whereas their present‐day counterparts degrade isomaltose‐like sugars; ancestral aldehyde dehydrogenase ALDH1/2 converted small substrates, whereas the present‐day enzymes accommodate bulky aldehydes; and ancestral kinases phosphorylated protein substrates containing proline or arginine at the first position . Therefore, ASR clearly represents a useful strategy for the modification of substrate specificity.…”

Section: Discussionmentioning

confidence: 99%

Ancestral Haloalkane Dehalogenases Show Robustness and Unique Substrate Specificity

et al. 2017

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Ancestral sequence reconstruction (ASR) represents a powerful approach for empirical testing structure-function relationships of diverse proteins. We employed ASR to predict sequences of five ancestral haloalkane dehalogenases (HLDs) from the HLD-II subfamily. Genes encoding the inferred ancestral sequences were synthesized and expressed in Escherichia coli, and the resurrected ancestral enzymes (AncHLD1-5) were experimentally characterized. Strikingly, the ancestral HLDs exhibited significantly enhanced thermodynamic stability compared to extant enzymes (ΔT up to 24 °C), as well as higher specific activities with preference for short multi-substituted halogenated substrates. Moreover, multivariate statistical analysis revealed a shift in the substrate specificity profiles of AncHLD1 and AncHLD2. This is extremely difficult to achieve by rational protein engineering. The study highlights that ASR is an efficient approach for the development of novel biocatalysts and robust templates for directed evolution.

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Ancestral resurrection reveals evolutionary mechanisms of kinase plasticity

Cited by 60 publications

References 79 publications

Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity

Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity

Ssp2 Binding Activates the Smk1 Mitogen-Activated Protein Kinase

Ancestral Haloalkane Dehalogenases Show Robustness and Unique Substrate Specificity

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