Protein kinases expedite the cell signaling processes by facilitating the transfer of a phosphate from the nucleotide triphosphate to the target protein. Kinase-mediated phosphorylation is critical for a wide spectrum of cell signaling processes such as transcription, translation, cell proliferation, receptor downregulation, metabolism, and apoptosis (Cheng et al., 2011). The dysregulation of kinases alters cell function and may lead to diseases such as schizophrenia (Bentea et al., 2019), Alzheimer’s disease (Ohno, 2014), cancer (Sarkar et al., 2023), and cardiovascular diseases (Chen et al., 2022a). Kinases undergo dynamic conformational changes to elicit their function during cell signaling (Dixon et al., 2004). All kinases have a conserved residue sequence that forms the nucleotide-binding domain to bind to adenosine triphosphate (ATP) or guanosine triphosphate (GTP) (Cheng et al., 2011; Taylor and Kornev, 2011). The binding of the nucleotides is facilitated by phosphorylation of the activation loop (Adams, 2003). Therapies targeting kinases focus on the nucleotide-binding sites (active/orthosteric sites) that regulate the kinase function. Therapies targeting orthosteric sites to regulate kinase activity compete with the native ligand (ATP or GTP) to bind to the active site and need to be highly selective for the active site. Due to the conservation of the nucleotide-binding domain across all protein kinases, specifically targeting the active/orthosteric sites of one type of kinase is challenging.

Kinase activity is also regulated by autoinhibitory domains (Soderling, 1990; Li et al., 2021). Autoinhibitory domains suppress the interaction between the kinases and the effector molecule (nucleotides). The autoinhibitory domains are the sites distal to the active sites that affect the activity of the kinase at the active site through the induction of conformational and dynamic changes in the protein structure (Nussinov and Tsai, 2013; Cui and Karplus, 2008; Guo and Zhou, 2016). Allosteric regulation does not involve direct competition with the native ligand. Instead, it perturbs active site structure or dynamics. The kinases, especially in diseased conditions, may develop resistance to orthosterically regulated therapies by incorporating mutations at the active site, thus enabling kinases to elicit catalytic activity while disallowing the binding of the orthosteric regulators. Allosteric control of kinase function allows the bypass of this resistance (Cui and Karplus, 2008).

Finding allosteric sites that effectively regulate active sites is critical for establishing allosteric control over a particular kinase. X-ray crystallography (Keedy, 2019) and nuclear magnetic resonance (NMR) spectroscopy (Grutsch et al., 2016) techniques are widely used to observe the structures of a protein. The changes occurring in the structure of protein molecules during the allosteric binding of effector molecules can be mapped by comparing structures at different points in time. However, these techniques are expensive and time-consuming. Modern computational approaches offer an inexpensive and fast assessment of protein allosteric ‘wiring.’ However, the computational approaches only offer hypothetical predictions and still need to be tested experimentally. Experimental validation does not require the structural characterization of the protein; instead, a direct test of the predictions in activity assays demonstrates the functioning of the established allosteric control (Vishweshwaraiah et al., 2021b). Here, we discuss computational and experimental approaches to map allosteric communications, and tools used to control protein function through allosteric regulation.

Allosteric regulation of protein kinases

Kinases are considered to be biological switches due to their ability to change structural conformation upon activation (Taylor et al., 2012b). Since kinases make up 2% of the protein-coding genome (Manning et al., 2002) in humans and are implicated in multiple diseases, this family of proteins is an important therapeutic target. Protein kinases phosphorylate serines, threonines, or tyrosines by transferring the γ-phosphate of ATP to the hydroxyl group causing structural changes in the protein, eliciting its function as an enzyme (Cohen, 2002). Kinases have a widespread role in cellular signaling, thereby making them desirable targets for establishing control over cellular processes (Vishweshwaraiah et al., 2021b; Chen et al., 2022b). In eukaryotic cells, across all different types of protein kinases, there are close to 250 residues worthy of conserved sequences (Taylor et al., 2012b). One conserved region is attributed to be the nucleotide-binding domain (NBD), which is a cleft that forms the binding pocket for ATP and is located between the N-terminal lobe and the C-terminal lobe of the protein. The flexibility of the protein is maintained by two ensembles of hydrophobic residues or ‘spines’ that connect the α-helix of the C lobe to the β-sheet of the N lobe (Johnson and Lewis, 2001). These spines are called the ‘regulatory spine (R spine)’ due to their ability to regulate the assembly and disassembly of the active kinase, and the ‘catalytic spine (C spine)’ since these residues balance the catalytic activity of the kinases upon ATP binding. The changes in structure of the kinases due to their complex structure–activity relationship are likely to be resolved due to the presence of the spines (Taylor et al., 2012a). The dynamic structure of kinases has proved to be of great importance for the functionality of the protein and the NMR structures confirming these dynamic structural changes have underscored the presence and importance of allostery of protein kinases (Masterson et al., 2012; Figure 1).

Figure 1

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Several approaches have been developed using the preexisting tools to control protein function for the allosteric regulation of protein kinases. Kinase activity can be controlled by ligands (Zorba et al., 2019), made use of monobodies (Sha et al., 2017) (a type of synthetic binding protein) to control allostery in Aurora A (AurA) (Zhou et al., 1998). AurA is a protein kinase responsible for the maturation of the centrosome and duplication through its effect on the microtubule spindle formation in mitosis (Glover et al., 1995). AurA serves to be a major oncogene since its overexpression has been associated with a variety of cancers. The authors used monobodies (Koide et al., 2012) to bind to the allosteric sites of AurA and produce complete inhibition or complete activation without having to actively compete with ATP to bind to the NBD. The effect of the binding of monobodies to AurA and the activity of the protein were mapped using X-ray crystallography. The binding of the monobody showed a 20-fold inhibition of the kinases, proving its effectiveness as an allosteric inhibitor and demonstrating promising therapeutic potential. Wojcik et al., 2016 designed high-affinity monobodies to bind to the kinase Bcr-Abl formed by the fusion of breakpoint cluster region (Bcr) and Abelson tyrosine kinase 1 (Abl1). Bcr-Abl activity leads to the development of chronic myelogenous leukemia (Deininger et al., 2000). Studies have shown an interaction between Src homology domain (SH2) and tyrosine kinase, critical to kinase activity and mutations arising at these interfaces (I164E), and leads to inhibition of the kinase activity and oncogenesis induced by Bcr-Abl (Grebien et al., 2011). The authors developed monobodies for these critical residues to control the kinase activity of Bcr-Abl allosterically. The monobodies were developed using combinatorial synthesis and the binding affinity was tested using chemical shift perturbation assays that identify binding sites and predict the binding efficiency of ligands. Inhibition of interaction of the SH2 domain and the kinase domain due to monobodies decreased the activity of Bcr-Abl (validated by western blot and immunohistochemistry experiments). Monobodies can be used as a tool for controlling kinase activity, providing an alternative approach to chemogenetic and optogenetic control of protein function as monobodies do not require engineering of the target kinase (Figure 2).

Figure 2

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Figure 2—source data 1

Source data for Figure 2.

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Dagliyan et al., 2017 developed a rapamycin-binding domain that destabilized the NBD, thereby disrupting the kinase activity in the P21-activated kinase (PAK1). PAK1 is known to play a role in angiogenesis, neurogenesis, and cell motility (Hofmann et al., 2004). The dysregulation of this protein leads to the development of speech impairment, seizures, and macrocephaly. The authors developed a protein domain that is activated upon dopamine binding to the PAK1 domain. PAK1 is overexpressed in breast cancer and hence is identified as an oncogene (Radu et al., 2014). The authors tested this rapamycin-PAK construct (RapR-PAK1) in breast cancer cells to observe changes in the kinase activity due to the effect of rapamycin. The construct was successfully able to control the kinase activity of PAK, demonstrating the applicability of ligands to control protein activity allosterically by either inhibiting activity or inducing it.

Karginov et al., 2011 used a similar strategy for regulating kinase activity, but instead of using a ligand-based approach, the authors used light as the input signal for controlling activity. The authors built a photoactivatable analog of rapamycin to regulate the dimerization of proteins. The photo-inducible small molecules are generated by the installation of ‘caging groups’ (Riggsbee and Deiters, 2010; Mayer and Heckel, 2006) that keep the molecule inactive until removed. Caging groups sequester the site on the ligand necessary for activation. Dissociation of the caging groups from the activation site through irradiation by light leads to activation of the small molecules. The authors used ‘caged-rapamycin (pRap)’ to regulate both the kinase activity and protein dimerization.

Zhou et al., 2017 developed an optically inducible, single-chain protein that could undergo conformational changes upon exposure to light. The authors induced modifications to DronpaN145, a photo-inducible green fluorescent protein to form pdDronpa1 (Zhou et al., 2012) that could dimerize at the kinase active site in the presence of 500 nm cyan and stymie kinase activity. Upon removal of light, the kinase activity was restored. The authors used dual-specificity mitogen-activated protein kinase 1 (MEK1) to test the pdDronpa1 construct. The authors were able to successfully control the kinase activity of MEK1, providing a unique and versatile approach toward optogenetics.

Vishweshwaraiah et al., 2021a developed a protein-based nanocomputing agent (Dokholyan, 2021) containing two inputs that had opposing effects on the kinase activity, forming a logic OR gate to regulate allosteric communications. The authors inserted a chemogenetic (uniRapR; Dagliyan et al., 2013) and an optogenetic domain (LOV2; Lee et al., 2008) within FAK. The uniRapR construct was inserted in the allosteric site of the kinase domain, predicted using Ohm (Wang et al., 2020), such that it could activate the protein upon interaction with rapamycin. This activation pathway of the protein upon interaction with rapamycin was predicted by discrete molecular dynamics (DMD) simulations. The LOV2 construct was inserted in loop 2 of the FERM domain (allosteric site on FERM domain was predicted using Ohm), leading to an inhibition of kinase activity by inactivation of the protein (Lietha et al., 2007). Inactivation of the protein was predicted using DMD simulations in blue light on/off conditions. The authors observed a decrease in kinase activity upon exposure to blue light and an increase in kinase activity upon exposure to rapamycin upon live cell imaging (changes in phenotype were used to determine kinase activity). This successful study provides a basis for the development of rationally designed nano-computing agents and can be used in diagnostics, drug delivery, regulation of cell signaling, and metabolic flux. In order to demonstrate that the nanocomputing agents are effective in other proteins as well, Chen et al. developed an Src-uniRapR-LOV2 construct that provided a dual control on the function of Src kinase. The uniRapR construct was able to activate the Src kinase while the LOV2 construct rendered the protein inactive upon irradiation by blue light. The on/off protein switch was tested by visualization of the focal adhesion. With rapamycin as the first signal, followed by blue light as the second (Chen et al., 2023). Along with computational methods such as multiple sequence alignments (MSA), molecular dynamics (MD), and DMD simulations, molecular docking and in silico modeling (Yuan et al., 2022). paired with mass spectroscopy, have also been used to define protein structures and map allosteric sites in proteins. Through microscopic techniques, such as cryo-EM, two-dimensional structures of proteins can be acquired and morphed into three-dimensional models using artificial intelligence (Chirigati, 2021). In addition to small molecules, biologics such as antibodies or various input stimuli (such as pH, temperature, or pressure) can be made use of to regulate protein function by exploitation of allosteric communication. Protein allostery proves to be an essential tool in controlling protein function in translational research.

Optogenetic control

Photoreceptors are molecules that can change their conformation upon irradiation by light. The light-sensitive domains of the photoreceptors can be used as sensor domains to control protein function (Padmanabhan et al., 2022). The phytochrome B (PhyB) (Ni et al., 1999) is used extensively as a red light photoreceptor since many optical systems do not have far-infrared light range lasers, and red light photoreceptors have limitations to their usage (Ni et al., 1999). Light oxygen voltage (LOV2) and cryptochromes are the two main types of blue light photoreceptors used to control protein function. The (LOV2) domain of phototropin 1 (Halavaty and Moffat, 2007) obtained from Avena sativa has been shown to produce dramatic conformational changes upon exposure to blue light and is used to control protein function. Zimmerman et al., 2016 developed LOV2 photoswitches and validated them by inserting them in various protein domains and determining the activity of the protein upon exposure to blue light. Dagliyan et al., 2016 developed a photo-inhibitable Src kinase domain by inserting LOV2 in the L1 loop connecting β-strands of Src kinase causing inhibition of the kinase upon activation of LOV2. However, insertion of the LOV2 domain into the protein without causing structural perturbations in the native protein structure is difficult due to limitations arising from the lack of availability of caging surfaces leading to non-specific activation of the photoreceptors. Caging surfaces or photocages are photosensitive groups that prevent the protein from reacting to small molecules or other proteins that might produce a biological function of the protein. The caging groups ensure activation of proteins only upon irradiation of the proteins by light of suitable wavelength (Bojtár et al., 2020). To overcome this limitation, He et al., 2021 developed a circular permutant of LOV2 (cpLOV2). The cpLOV2 is constructed by introduction of a glycine and serine-rich linker in the surface-exposed loop. The construct remains compatible with the existing LOV2-based engineered proteins with the advantage of having more caging surfaces thus improving its applicability (Figure 2).

Owing to the difficulty of optogenetic tools to control allosteric activity, arising due to the limitations in achieving subcellular control of activity (Wang et al., 2019, Shaaya et al., 2020) developed an optogenetic tool, LightR, that enables subcellular control of protein and provides a tight temporal regulation of protein activity. LightR comprises of two tandemly connected Vivid photoreceptor (Nihongaki et al., 2014) domains that form an antiparallel homodimer upon irradiation by blue light causing its activation. The use of LightR in controlling allosteric activity was validated in Src kinase, where irradiation with blue light caused activation of Src kinase. LightR provides better spatiotemporal resolution than LOV2 due to the subcellular control of proteins (Figure 3).

Figure 3

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Author details

1. Shivani Sujay Godbole

   Department of Pharmacology, Penn State College of Medicine, Hershey, United States

   Contribution

   Conceptualization, Writing – original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0177-2286

2. Nikolay V Dokholyan

   1. Department of Pharmacology, Penn State College of Medicine, Hershey, United States
   2. Department of Biomedical Engineering, Penn State University, University Park, Hershey, United States
   3. Department of Engineering Science and Mechanics, Penn State University, University Park, Hershey, United States
   4. Department of Biochemistry & Molecular Biology, Penn State College of Medicine, Hershey, United States
   5. Department of Chemistry, Penn State University, University Park, Hershey, United States

   Contribution

   Conceptualization, Writing – original draft

   For correspondence

   dokh@psu.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8225-4025

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