Evolution of functional diversity in the holozoan tyrosine kinome

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 co-evolving protein domains. We propose the oldest tyrosine kinases, IRKL, SrcM, and Csk, originated from unicellular pre-metazoans and were co-opted 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.

The conformational stability of pro-apoptotic BAX is dictated by discrete residues of the protein core

BAX is a pro-apoptotic member of the BCL-2 family, which regulates the balance between cellular life and death. During homeostasis, BAX predominantly resides in the cytosol as a latent monomer but, in response to stress, transforms into an oligomeric protein that permeabilizes the mitochondria, leading to apoptosis. Because renegade BAX activation poses a grave risk to the cell, the architecture of BAX must ensure monomeric stability yet enable conformational change upon stress signaling. The specific structural features that afford both stability and dynamic flexibility remain ill-defined and represent a critical control point of BAX regulation. We identify a nexus of interactions involving four residues of the BAX core α5 helix that are individually essential to maintaining the structure and latency of monomeric BAX and are collectively required for dimeric assembly. The dual yet distinct roles of these residues reveals the intricacy of BAX conformational regulation and opportunities for therapeutic modulation.

Evolution of functional diversity in the holozoan tyrosine kinome

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. The two exceptions which lack this insert, Eph and EGFR receptor tyrosine kinases, each form 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, Vascular, and Platelet-derived 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 co-conserved protein domains. We propose the oldest tyrosine kinases, IRKL, SrcM and Csk, originated from unicellular pre-metazoans and were co-opted 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.

FimH as a scaffold for regulated molecular recognition

Background Recognition proteins are critical in many biotechnology applications and would be even more useful if their binding could be regulated. The current gold standard for recognition molecules, antibodies, lacks convenient regulation. Alternative scaffolds can be used to build recognition proteins with new functionalities, including regulated recognition molecules. Here we test the use of the bacterial adhesin FimH as a scaffold for regulated molecular recognition. FimH binds to its native small molecule target mannose in a conformation-dependent manner that can be regulated by two types of noncompetitive regulation: allosteric and parasteric. Results We demonstrate that conformational regulation of FimH can be maintained even after reengineering the binding site to recognize the non-mannosylated targets nickel or Penta-His antibody, resulting in an up to 7-fold difference in KD between the two conformations. Moreover, both the allosteric and parasteric regulatory mechanisms native to FimH can be used to regulate binding to its new target. In one mutant, addition of the native ligand mannose parasterically improves the mutant’s affinity for Penta-His 4-fold, even as their epitopes overlap. In another mutant, the allosteric antibody mab21 reduces the mutant’s affinity for Penta-His 7-fold. The advantage of noncompetitive regulation is further illustrated by the ability of this allosteric regulator to induce 98% detachment of Penta-His, even with modest differences in affinity. Conclusions This illustrates the potential of FimH, with its deeply studied conformation-dependent binding, as a scaffold for conformationally regulated binding via multiple mechanisms.

Cryo-EM structures of SERCA2b reveal the mechanism of regulation by the luminal extension tail

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps Ca2+ from the cytosol into the ER and maintains the cellular calcium homeostasis. Herein, we present cryo–electron microscopy (cryo-EM) structures of human SERCA2b in E1∙2Ca2+–adenylyl methylenediphosphonate (AMPPCP) and E2-BeF3− states at 2.9- and 2.8-Å resolutions, respectively. The structures revealed that the luminal extension tail (LE) characteristic of SERCA2b runs parallel to the lipid-water boundary near the luminal ends of transmembrane (TM) helices TM10 and TM7 and approaches the luminal loop flanked by TM7 and TM8. While the LE served to stabilize the cytosolic and TM domain arrangement of SERCA2b, deletion of the LE rendered the overall conformation resemble that of SERCA1a and SERCA2a and allowed multiple conformations. Thus, the LE appears to play a critical role in conformational regulation in SERCA2b, which likely explains the different kinetic properties of SERCA2b from those of other isoforms lacking the LE.

On the Velocity of Enzymatic Reactions in Michaelis–Menten-Like Schemes (Ensemble and Single-Molecule Versions)

In searching non-standard ways of conformational regulation, various Michaelis–Menten-like schemes attract relentless attention, resulting in sometimes too sophisticated considerations. With the example of monomeric enzymes possessing an only binding site, we define the minimal schemes capable of bearing peculiar regulatory properties like “cooperativity” or substrate inhibition. The simplest ways of calculating the enzymatic reaction velocity are exemplified, either in the ensemble or single-molecule case.