Dimerizers allowing inducible control of protein-protein interactions are powerful tools for manipulating biological processes. Here we describe genetically encoded light-inducible protein interaction modules based on Arabidopsis thaliana cryptochrome 2 and CIB1 that require no exogenous ligands and dimerize on blue light exposure with sub-second time resolution and subcellular spatial resolution. We demonstrate the general utility of this system by inducing protein translocation, transcription, and Cre-mediated DNA recombination using light.

Arabidopsis thaliana cryptochrome 2 (AtCRY2), a light-sensitive photosensory protein, was previously adapted for use controling protein-protein interactions through light-dependent binding to a partner protein, CIB1. While the existing CRY2/CIB dimerization system has been used extensively for optogenetic applications, some limitations exist. Here, we set out to optimize function of the CRY2/CIB system, to identify versions of CRY2/CIB that are smaller, show reduced dark interaction, and maintain longer or shorter signaling states in response to a pulse of light. We describe minimal functional CRY2 and CIB1 domains maintaining light-dependent interaction and new signaling mutations affecting AtCRY2 photocycle kinetics. The latter work implicates a α13-α14 turn motif within plant CRYs where perturbations alter signaling state lifetime. Using a long-lived L348F photocycle mutant, we engineered a second generation photoactivatable Cre recombinase, PA-Cre2.0, that shows five-fold improved dynamic range allowing robust recombination following exposure to a single, brief pulse of light.

However, whether the cationic component of the interaction is necessary for binding in the aromatic cage has not been addressed. In this article, the interaction of trimethyllysine with tryptophan is compared with that of its neutral analog, tert-butylnorleucine (2-amino-7,7-dimethyloctanoic acid), within the context of a ␤-hairpin peptide model system. These two side chains have near-identical size, shape, and polarizabilities but differ in their charges. Comparison of the two peptides reveals that the neutral side chain has no preference for interacting with tryptophan, unlike trimethyllysine, which interacts strongly in a defined geometry. In vitro binding studies of the histone 3A peptide containing trimethyllysine or tert-butylnorleucine to HP1 chromodomain indicate that the cationic moiety is critical for binding in the aromatic cage. This difference in binding affinities demonstrates the necessity of the cation-interaction to binding with the chromodomain and its role in providing specificity. This article presents an excellent example of synergy between model systems and in vitro studies that allows for the investigation of the key forces that control biomolecular recognition.cation-pi interactions ͉ histone code ͉ lysine methylation ͉ posttranslational modifications ͉ protein-protein interactions W ith rapid advancements in genomics, epigenetics has become the next major challenge in understanding how genetic information is controlled (1). It is becoming clear that posttranslational modifications of proteins are a key component in controlling gene expression. These modifications include a number of subtle structural changes, including Lys and Arg methylation, Lys acylation, and Ser/Thr/Tyr phosphorylation, which act as chemical switches to induce or repress proteinprotein interactions. Among all histone modifications, lysine methylation is especially important for chromatin function because of its stability and direct contribution to heritable patterns of gene expression (for review, see ref.2). To understand how such modest structural modifications can control biomolecular recognition events, it is critical to understand the underlying noncovalent interactions involved.Methylation of Lys induces a protein-protein interaction through the binding of methyl lysine (KMe n , n ϭ 1-3) in an aromatic cage. This interaction first was described for the binding of methylated histone 3 (H3) tail to the HP1 chromodomain ( Fig. 1) (3, 4). HP1 and methylated H3 interact specifically whether lysine 9 is mono-, di-, or trimethylated. However, the binding is most effective when lysine is trimethylated (5). In addition, more recent findings have shown that phosphorylation of serine 10 prevents interaction of HP1 with methylated H3 (for review, see ref. 6). Therefore, a binary switch mechanism has been proposed for the recognition of methyllysine-containing peptides by chromodomains. Interestingly, binding of a methylated lysine in an aromatic cage is not exclusive to chromodomains. Plant homeobox domain (PHD) fingers an...

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The methylation of lysine in histone tails is a common posttranslational modification that functions in histone-regulated chromatin condensation, with binding of methylated lysine occurring in aromatic pockets on chromodomain proteins. We have synthesized a highly stable 12-residue beta-hairpin peptide that exploits the histone-related cation-pi interaction between a methylated lysine residue and a tryptophan residue. Thermodynamic analysis reveals significant entropic stabilization of the peptide due to methylation of the lysine residue. Chemical denaturation of the peptide demonstrates two-state behavior. In comparison to other reported, highly stable designed beta-hairpins, this peptide is the most thermally stable beta-hairpin reported to date. This study provides insight into the role of Lys methylation in histone proteins and more generally in mediating protein-protein interactions.