PubMed · 10700271
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K S Matthews, C M Falcon, L Swint-Kruse. 2000. Relieving repression.. https://doi.org/10.1038/73274
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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.
Although structures of single-stranded (ss)DNA-binding proteins (SSBs) have been reported with and without ssDNA, the mechanism of ssDNA binding in eukarya remains speculative. Here we report a 2.5 Angstroms structure of the ssDNA-binding domain of human replication protein A (RPA) (eukaryotic SSB), for which we previously reported a structure in complex with ssDNA. A comparison of free and bound forms of RPA revealed that ssDNA binding is associated with a major reorientation between, and significant conformational changes within, the structural modules--OB-folds--which comprise the DNA-binding domain. Two OB-folds, whose tandem orientation was stabilized by the presence of DNA, adopted multiple orientations in its absence. Within the OB-folds, extended loops implicated in DNA binding significantly changed conformation in the absence of DNA. Analysis of intermolecular contacts suggested the possibility that other RPA molecules and/or other proteins could compete with DNA for the same binding site. Using this mechanism, protein-protein interactions can regulate, and/or be regulated by DNA binding. Combined with available biochemical data, this structure also suggested a dynamic model for the DNA-binding mechanism.
Recent crystallographic studies have revealed that 12 alpha-helices can pack in an anti-parallel fashion to form a hollow cylinder of nearly uniform radius. In this architecture, which we refer to as an alpha-barrel, the helices are inclined with respect to the cylindrical axis, and thus they curve and twist. As with conventional coiled-coils, the helices of the barrel associate via "knobs-into-holes" interactions; however, their packing is distinct in several important ways. First, the alpha-barrel helices untwist in comparison with the helices found in two-stranded coiled-coils and, as a consequence of this distortion, their knobs approach closely one end of the complementary holes. This effect defines a requirement for particular size and shape of the protruding residues, and it is associated with a relative axial translation of the paired helices. Second, as each helix packs laterally with two neighbours, the helices have two sequence patterns that are phased to match the two interfaces. The two types of interface are not equivalent and, as one travels around the circumference of the cylinder's interior, they alternate between one type where the knobs approach the holes straight-on, and a second type in which they are inclined. The choice of amino acid depends on the interface type, with small hydrophobic side-chains preferred for the direct contacts and larger aliphatic side-chains for the inclined contacts. Third, small residues are found preferentially on the inside of the tube, in order to make the "wedge" angle between helices compatible with a 12-member tube. Finally, hydrogen-bonding interactions of side-chains within and between helices support the assembly. Using these salient structural features, we present a sequence template that is compatible with some underlying rules for the packing of helices in the barrel, and which may have application to the design of higher-order assemblies from peptides, such as nano-tubes. We discuss the general implications of relative axial translation in coiled-coils and, in particular, the potential role that this movement could play in allosteric mechanisms.