PubMed · 12475200
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Nathan C Rockwell, Damian J Krysan, Tomoko Komiyama, Robert S Fuller. 2002. Precursor processing by kex2/furin proteases.. https://doi.org/10.1021/cr010168i
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Post-translational control enables rapid and precise regulation of cell behavior. Despite these advantages, general strategies to build phosphorylation-based synthetic circuits are limited. Here we reasoned that engineered allostery, a technique that has been applied to design light- and chemically gated protein switches, could also be used to engineer phosphorylation-controlled protein switches (phospho-switches). Using an allosterically controllable Gal4 transcription factor as a scaffold, we show that a classic kinase Förster resonance energy transfer biosensor architecture can be used as a starting point for phospho-switch design. We optimize all features of the phospho-switch to develop an ERK-controlled transcription factor with a 20-fold phosphorylation-dependent change in transcriptional output. The resulting synthetic ERK-responsive transcription factor responds with comparable sensitivity to the c-fos promoter and reveals spatial ERK signaling patterns in mammalian developmental organoids. We further show that our switch architecture can be generalized to other input kinases and allosterically controlled targets. This work provides a general platform for a new generation of kinase-responsive tools for biosensing and synthetic biology applications.
Modification of Escherichia coli phosphofructokinase-2 (Pfk-2) with pyrene maleimide (PM) results in a rapid inactivation of the enzyme. The loss of enzyme activity correlates with the incorporation of 2 mol of PM/mol of subunit and the concomitant dissociation of the dimeric enzyme. The two modified residues were identified as Cys-238 and Cys-295. In the presence of the negative allosteric effector, MgATP, Cys-238 was the only modified cysteine residue. Kinetic characterization of the Cys-238-labelled Pfk-2 indicates that the enzyme is fully active, with the kinetic constants ( K(m), kcat) being almost identical to the ones obtained for the native enzyme. The modified enzyme is a monomer in the absence of ligands and, like the native enzyme, behaves as a tetramer in the presence of the nucleotide. However, in the presence of fructose-6-phosphate (fru-6-P) and ATP(-4), the enzyme behaves as a dimer, suggesting that the monomers undergo re-association in the presence of the substrates and that the active species is a dimer. Modification of Pfk-2 with eosin-5-maleimide (EM) results in the labelling of Cys-295. This modified enzyme is inactive and is not able to bind to the allosteric effector, remaining as a dimer in its presence. Nonetheless, Cys-295-labelled Pfk-2 is able to bind to the substrate fru-6-P in an hyperbolic fashion with a K(d) value that is 6-fold higher than the one determined for the native enzyme. These are the first residues to be implicated in the activity and/or structure of the Pfk-2.
Many proteins are built from structurally and functionally distinct domains. A major goal is to understand how conformational change transmits information between domains in order to achieve biological activity. A two-domain, bi-functional fusion protein has been designed so that the mechanical stress imposed by the folded structure of one subunit causes the other subunit to unfold, and vice versa. The construct consists of ubiquitin inserted into a surface loop of barnase. The distance between the amino and carboxyl ends of ubiquitin is much greater than the distance between the termini of the barnase loop. This topological constraint causes the two domains to engage in a thermodynamic tug-of-war in which only one can exist in its folded state at any given time. This conformational equilibrium, which is cooperative, reversible, and controllable by ligand binding, serves as a model for the coupled binding and folding mechanism widely used to mediate protein-protein interactions and cellular signaling processes. The position of the equilibrium can be adjusted by temperature or ligand binding and is monitored in vivo by cell death. This design forms the basis for a new class of cytotoxic proteins that can be activated by cell-specific effector molecules, and can thus target particular cell types for destruction.