Cells can activate the same proteins at different times or places to generate diverse effects — for example, the same enzymes can be involved in both cell growth and programmed cell death. Many cellular processes that depend on the timing and site of protein activity can be studied in living cells, by triggering localized protein activity and examining the effects. In the past few years, scientists have developed ‘photoactivation’ methods that allow protein functions to be switched on by light1,2. Writing in Nature, Wang et al.3 now describe a photoactivation strategy that is both broadly applicable and minimally perturbs normal protein functions.
Approaches for the photoactivation and photoinhibition of proteins are available, but it is often difficult to apply these without modifying some of the proteins’ activities. These methods work by manipulating amino-acid residues involved in the target protein’s mechanism of action, within the active site. However, the structure and active sites of many proteins are poorly understood, preventing such methods from being applied to many important systems.
Wang and colleagues report a method that they term computationally aided and genetically encoded proximal decaging (CAGE-prox). In CAGE-prox, a straightforward computational method is used to identify a position in a protein of interest at which the introduction of a bulky chemical group is likely to perturb the protein’s interaction with its substrate. The amino-acid residue at that position is then replaced with a tyrosine residue that has been modified4 to carry a group that can be cleaved using light. Once installed, this bulky group blocks the protein’s activity until light irradiation ‘prunes’ it back to the normal tyrosine structure (Fig. 1), whereupon activity is restored.
The computational method requires no information about a protein’s mechanism of action. CAGE-prox can therefore be applied to an amazingly wide range of target proteins. The authors elected to use a modified tyrosine residue, rather than other amino acids that could be modified with a light-cleavable group, because they found that the normal tyrosine produced after cleavage proved least likely to perturb folding or normal protein binding.
One of the most striking advantages of CAGE-prox is its ability to produce an almost-native protein analogue — irradiation produces a protein that differs from the normal one by only a single amino-acid residue, which need not be in the active site. Other approaches have used light to link a fragment of an active protein to a second protein anchored at a specific site in the cell, thereby driving the fragment to specific regions1. Alternatively, engineered protein domains that change shape when irradiated have been inserted into a target protein to alter its conformation when illuminated, or have been positioned so that they block the target’s active site only in the dark1,2. These previously reported methods alter the overall structure of the target protein, thus potentially affecting its ability to interact normally with its biological partners.
By contrast, the surfaces used by wild-type proteins to mediate interactions with other cell components are retained almost intact in the CAGE-prox proteins after irradiation. This allows the modified proteins to target their normal binding partners in cells, and to be simultaneously activated at multiple locations in the same way as the wild-type protein.
It is perhaps surprising that the small changes associated with just one light-sensitive tyrosine residue can affect the interactions of a target protein so effectively. The success of Wang and colleagues’ method depends crucially on the ability to select the best site for modification. Encouragingly, the computational modelling used in CAGE-prox identified fewer than ten possible modification sites for each of the diverse proteins studied, limiting the number of amino-acid positions that had to be tested experimentally to find the most effective one.
The researchers used CAGE-prox not only to activate diverse protein structures, but also to control the sensitivity of kinase enzymes to inhibitors, thereby allowing modified and wild-type kinases to be inhibited independently. Just as impressively, Wang et al. used their method to activate proteins that have anticancer activity, and showed that light-triggered activation of these proteins inhibits tumour growth in vivo in mice.
In other photoactivation methods, a key amino-acid residue in the active site is identified and replaced by a modified version of that residue. By contrast, in CAGE-prox, the identified residue does not have to be in the active site and is always replaced by the same modified tyrosine residue. This tyrosine is introduced using a cell-based technique called unnatural-amino-acid mutagenesis5,6. The use of this technique could be seen as a weakness of Wang and co-workers’ approach, because unnatural-amino-acid mutagenesis is not suitable for all cell types, and can require substantial optimization for each application. Furthermore, the covalent bond that is cleaved to remove the bulky side chain from the light-sensitive tyrosine residue can be broken only by using high-energy light (wavelengths of less than 400 nanometres), which is toxic to living cells. These are likely to be short-term obstacles, however, because many laboratories are actively pursuing and improving methods for altering proteins in cultured cells, and even in vivo.
With its remarkable simplicity and generality, CAGE-prox opens the door to studies of previously inaccessible cellular pathways, and of the spatio-temporal control of processes that determine cell behaviour. The range of applications that Wang et al. have already proved in principle for their technique is remarkable. No doubt, many more will soon follow.
Nature 569, 490-491 (2019)