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Agrobacterium tumefaciens pathogenesis of plants is initiated with signal reception and culminates with transforming the genomic DNA of its host. The histidine sensor kinase VirA receives and reacts to discrete signaling molecules for the full induction of the genes necessary for this process. Though many of the components of this process have been identified, the precise mechanism of how VirA coordinates the response to host signals, namely phenols and sugars, is unknown. Recent advances of molecular modeling have allowed us to test structure/function predictions and contextualize previous experiments with VirA. In particular, the deep mind software AlphaFold has generated a structural model for the entire protein, allowing us to construct a model that addresses the mechanism of VirA signal reception. Here, we deepen our analysis of the region of VirA that is critical for phenol reception, model and probe potential phenol-binding sites of VirA, and refine its mechanism to strengthen our understanding of A. tumefaciens signal perception. [ABSTRACT FROM AUTHOR]

The emergence of multicellularity is strongly correlated with the expansion of tyrosine kinases, a conserved family of signaling enzymes that regulates pathways essential for cell-to-cell communication. Although tyrosine kinases have been classified from several model organisms, a molecular-level understanding of tyrosine kinase evolution across all holozoans is currently lacking. Using a hierarchical sequence constraint-based classification of diverse holozoan tyrosine kinases, we construct a new phylogenetic tree that identifies two ancient clades of cytoplasmic and receptor tyrosine kinases separated by the presence of an extended insert segment in the kinase domain connecting the D and E-helices. Present in nearly all receptor tyrosine kinases, this fast-evolving insertion imparts diverse functionalities, such as post-translational modification sites and regulatory interactions. Eph and EGFR receptor tyrosine kinases are two exceptions which lack this insert, each forming an independent lineage characterized by unique functional features. We also identify common constraints shared across multiple tyrosine kinase families which warrant the designation of three new subgroups: Src module (SrcM), insulin receptor kinase-like (IRKL), and fibroblast, platelet-derived, vascular, and growth factor receptors (FPVR). Subgroup-specific constraints reflect shared autoinhibitory interactions involved in kinase conformational regulation. Conservation analyses describe how diverse tyrosine kinase signaling functions arose through the addition of family-specific motifs upon subgroup-specific features and coevolving protein domains. We propose the oldest tyrosine kinases, IRKL, SrcM, and Csk, originated from unicellular premetazoans and were coopted for complex multicellular functions. The increased frequency of oncogenic variants in more recent tyrosine kinases suggests that lineage-specific functionalities are selectively altered in human cancers. [ABSTRACT FROM AUTHOR]

The pleckstrin homology (PH) domain is well known for its phospholipid targeting function. The PH-TEC homology (PHTH) domain within the TEC family of tyrosine kinases is also a crucial component of the autoinhibitory apparatus. The autoinhibitory surface on the PHTH domain has been previously defined, and biochemical investigations have shown that PHTH-mediated inhibition is mutually exclusive with phosphatidylinositol binding. Here we use hydrogen/deuterium exchange mass spectrometry, nuclear magnetic resonance (NMR), and evolutionary sequence comparisons to map where and how the PHTH domain affects the Bruton’s tyrosine kinase (BTK) domain. The data map a PHTH-binding site on the activation loop face of the kinase C lobe, suggesting that the PHTH domain masks the activation loop and the substrate-docking site. Moreover, localized NMR spectral changes are observed for non–surface-exposed residues in the active site and on the distal side of the kinase domain. These data suggest that the association of PHTH induces allosteric conformational shifts in regions of the kinase domain that are critical for catalysis. Through statistical comparisons of diverse tyrosine kinase sequences, we identify residues unique to BTK that coincide with the experimentally determined PHTH-binding surface on the kinase domain. Our data provide a more complete picture of the autoinhibitory conformation adopted by full-length TEC kinases, creating opportunities to target the regulatory domains to control the function of these kinases in a biological setting. [ABSTRACT FROM AUTHOR]

Protein tyrosine kinases (PTKs) are a group of closely related enzymes that have evolutionarily diverged from serine/threonine kinases (STKs) to regulate pathways associated with multi-cellularity. Evolutionary divergence of PTKs from STKs has occurred through accumulation of mutations in the active site as well as in the commonly conserved hydrophobic core. While the functional significance of active site variations is well understood, relatively little is known about how hydrophobic core variations contribute to PTK evolutionary divergence. Here, using a combination of statistical sequence comparisons, molecular dynamics simulations, mutational analysis and in vitro thermostability and kinase assays, we investigate the structural and functional significance of key PTK-specific variations in the kinase core. We find that the nature of residues and interactions in the hydrophobic core of PTKs is strikingly different from other protein kinases, and PTK-specific variations in the core contribute to functional divergence by altering the stability and dynamics of the kinase domain. In particular, a functionally critical STK-conserved histidine that stabilizes the regulatory spine in STKs is selectively mutated to an alanine, serine or glutamate in PTKs, and this loss-of-function mutation is accommodated, in part, through compensatory PTK-specific interactions in the core. In particular, a PTK-conserved phenylalanine in the I-helix appears to structurally and functionally compensate for the loss of STK-histidine by interacting with the regulatory spine, which has far-reaching effects on enzyme activity, inhibitor sensing, and stability. We propose that hydrophobic core variations provide a selective advantage during PTK evolution by increasing the conformational flexibility, and therefore the allosteric potential of the kinase domain. Our studies also suggest that Tyrosine Kinase Like kinases such as RAF are intermediates in PTK evolutionary divergence inasmuch as they share features of both PTKs and STKs in the core. Finally, our studies provide an evolutionary framework for identifying and characterizing disease and drug resistance mutations in the kinase core. [ABSTRACT FROM AUTHOR]

Pseudokinases are prevalent across species and contribute diverse, noncatalytic signaling functions. Hunting pseudokinases: Pseudokinases generally have a similar structure to that of kinases but without the catalytic function. Rather, they serve alternative, some as yet unknown, functions across various signaling niches in many cell and tissue types. Kwon et al. performed an evolutionary analysis of pseudokinases across species and classified both known and previously unknown pseudokinases into groups and families. Their analysis reveals that pseudokinases are more prevalent than was previously appreciated and shows how pseudokinase structures may have evolved to confer advantageous functions in bacteria, plants, and animals. The classification is part of a new online, mineable resource called ProKinO. Protein phosphorylation by eukaryotic protein kinases (ePKs) is a fundamental mechanism of cell signaling in all organisms. In model vertebrates, ~10% of ePKs are classified as pseudokinases, which have amino acid changes within the catalytic machinery of the kinase domain that distinguish them from their canonical kinase counterparts. However, pseudokinases still regulate various signaling pathways, usually doing so in the absence of their own catalytic output. To investigate the prevalence, evolutionary relationships, and biological diversity of these pseudoenzymes, we performed a comprehensive analysis of putative pseudokinase sequences in available eukaryotic, bacterial, and archaeal proteomes. We found that pseudokinases are present across all domains of life, and we classified nearly 30,000 eukaryotic, 1500 bacterial, and 20 archaeal pseudokinase sequences into 86 pseudokinase families, including ~30 families that were previously unknown. We uncovered a rich variety of pseudokinases with notable expansions not only in animals but also in plants, fungi, and bacteria, where pseudokinases have previously received cursory attention. These expansions are accompanied by domain shuffling, which suggests roles for pseudokinases in plant innate immunity, plant-fungal interactions, and bacterial signaling. Mechanistically, the ancestral kinase fold has diverged in many distinct ways through the enrichment of unique sequence motifs to generate new families of pseudokinases in which the kinase domain is repurposed for noncanonical nucleotide binding or to stabilize unique, inactive kinase conformations. We further provide a collection of annotated pseudokinase sequences in the Protein Kinase Ontology (ProKinO) as a new mineable resource for the signaling community. [ABSTRACT FROM AUTHOR]

A correction to the article "Hydrophobic Core Variations Provide a Structural Framework for Tyrosine Kinase Evolution and Functional Specialization" is presented.