Enthalpic Classification of Water Molecules in Target-Ligand Binding.

Water molecules play various roles in target-ligand binding. For example, they can be replaced by the ligand and leave the surface of the binding pocket or stay conserved in the interface and form bridges with the target. While experimental techniques supply target-ligand complex structures at an increasing rate, they often have limitations in the measurement of a detailed water structure. Moreover, measurements of binding thermodynamics cannot distinguish between the different roles of individual water molecules. However, such a distinction and classification of the role of individual water molecules would be key to their application in drug design at atomic resolution. In this study, we investigate a quantitative approach for the description of the role of water molecules during ligand binding. Starting from complete hydration structures of the free and ligand-bound target molecules, binding enthalpy scores are calculated for each water molecule using quantum mechanical calculations. A statistical evaluation showed that the scores can distinguish between conserved and displaced classes of water molecules. The classification system was calibrated and tested on more than 1000 individual water positions. The practical tests of the enthalpic classification included important cases of antiviral drug research on HIV-1 protease inhibitors and the Influenza A ion channel. The methodology of classification is based on open source program packages, Gromacs, Mopac, and MobyWat, freely available to the scientific community.

Efficient Refinement of Complex Structures of Flexible Histone Peptides Using Post-Docking Molecular Dynamics Protocols.

Histones are keys to many epigenetic events and their complexes have therapeutic and diagnostic importance. The determination of the structures of histone complexes is fundamental in the design of new drugs. Computational molecular docking is widely used for the prediction of target-ligand complexes. Large, linear peptides like the tail regions of histones are challenging ligands for docking due to their large conformational flexibility, extensive hydration, and weak interactions with the shallow binding pockets of their reader proteins. Thus, fast docking methods often fail to produce complex structures of such peptide ligands at a level appropriate for drug design. To address this challenge, and improve the structural quality of the docked complexes, post-docking refinement has been applied using various molecular dynamics (MD) approaches. However, a final consensus has not been reached on the desired MD refinement protocol. In this present study, MD refinement strategies were systematically explored on a set of problematic complexes of histone peptide ligands with relatively large errors in their docked geometries. Six protocols were compared that differ in their MD simulation parameters. In all cases, pre-MD hydration of the complex interface regions was applied to avoid the unwanted presence of empty cavities. The best-performing protocol achieved a median of 32% improvement over the docked structures in terms of the change in root mean squared deviations from the experimental references. The influence of structural factors and explicit hydration on the performance of post-docking MD refinements are also discussed to help with their implementation in future methods and applications.

Target-ligand binding affinity from single point enthalpy calculation and elemental composition.

Reliable target-ligand binding thermodynamics data are essential for successful drug design and molecular engineering projects. Besides experimental methods, a number of theoretical approaches have been introduced for the generation of binding thermodynamics data. However, available approaches often neglect electronic effects or explicit water molecules influencing target-ligand interactions. To handle electronic effects within a reasonable time frame, we introduce a fast calculator QMH-L using a single target-ligand complex structure pre-optimized at the molecular mechanics level. QMH-L is composed of the semi-empirical quantum mechanics calculation of binding enthalpy with predicted explicit water molecules at the complex interface, and a simple descriptor based on the elemental composition of the ligand. QMH-L estimates the target-ligand binding free energy with a root mean square error (RMSE) of 0.94 kcal mol-1. The calculations also provide binding enthalpy values and they were compared with experimental binding thermodynamics data collected from the most reliable isothermal titration calorimetry studies of systems including various protein targets and challenging, large peptide ligands with a molecular weight of up to 2-3 thousand. The single point enthalpy calculations of QMH-L require modest computational resources and are based on short runs with open source and/or free software like Gromacs, Mopac, MobyWat, and Fragmenter. QMH-L can be applied for fast, automated scoring of drug candidates during a virtual screen, enthalpic engineering of new ligands or thermodynamic explanation of complex interactions.

Construction of Histone-Protein Complex Structures by Peptide Growing.

The structures of histone complexes are master keys to epigenetics. Linear histone peptide tails often bind to shallow pockets of reader proteins via weak interactions, rendering their structure determination challenging. In the present study, a new protocol, PepGrow, is introduced. PepGrow uses docked histone fragments as seeds and grows the full peptide tails in the reader-binding pocket, producing atomic-resolution structures of histone-reader complexes. PepGrow is able to handle the flexibility of histone peptides, and it is demonstrated to be more efficient than linking pre-docked peptide fragments. The new protocol combines the advantages of popular program packages and allows fast generation of solution structures. AutoDock, a force-field-based program, is used to supply the docked peptide fragments used as structural seeds, and the building algorithm of Modeller is adopted and tested as a peptide growing engine. The performance of PepGrow is compared to ten other docking methods, and it is concluded that in situ growing of a ligand from a seed is a viable strategy for the production of complex structures of histone peptides at atomic resolution.

The Advances and Limitations of the Determination and Applications of Water Structure in Molecular Engineering.

Water is a key actor of various processes of nature and, therefore, molecular engineering has to take the structural and energetic consequences of hydration into account. While the present review focuses on the target-ligand interactions in drug design, with a focus on biomolecules, these methods and applications can be easily adapted to other fields of the molecular engineering of molecular complexes, including solid hydrates. The review starts with the problems and solutions of the determination of water structures. The experimental approaches and theoretical calculations are summarized, including conceptual classifications. The implementations and applications of water models are featured for the calculation of the binding thermodynamics and computational ligand docking. It is concluded that theoretical approaches not only reproduce or complete experimental water structures, but also provide key information on the contribution of individual water molecules and are indispensable tools in molecular engineering.

The Structural Effects of Phosphorylation of Protein Arginine Methyltransferase 5 on Its Binding to Histone H4.

The protein arginine methyltransferase 5 (PRMT5) enzyme is responsible for arginine methylation on various proteins, including histone H4. PRMT5 is a promising drug target, playing a role in the pathomechanism of several diseases, especially in the progression of certain types of cancer. It was recently proved that the phosphorylation of PRMT5 on T80 residue increases its methyltransferase activity; furthermore, elevated levels of the enzyme were measured in the case of human hepatocellular carcinoma and other types of tumours. In this study, we constructed the complexes of the unmodified human PRMT5-methylosome protein 50 (MEP50) structure and its T80-phosphorylated variant in complex with the full-length histone H4 peptide. The full-length histone H4 was built in situ into the human PRMT5-MEP50 enzyme using experimental H4 fragments. Extensive molecular dynamic simulations and structure and energy analyses were performed for the complexed and apo protein partners, as well. Our results provided an atomic level explanation for two important experimental findings: (1) the increased methyltransferase activity of the phosphorylated PRMT5 when compared to the unmodified type; (2) the PRMT5 methylates only the free form of histone H4 not bound in the nucleosome. The atomic level complex structure H4-PRMT5-MEP50 will help the design of new inhibitors and in uncovering further structure-function relationships of PRMT enzymes.

Binding Networks Identify Targetable Protein Pockets for Mechanism-Based Drug Design.

The human genome codes only a few thousand druggable proteins, mainly receptors and enzymes. While this pool of available drug targets is limited, there is an untapped potential for discovering new drug-binding mechanisms and modes. For example, enzymes with long binding cavities offer numerous prerequisite binding sites that may be visited by an inhibitor during migration from a bulk solution to the destination site. Drug design can use these prerequisite sites as new structural targets. However, identifying these ephemeral sites is challenging. Here, we introduce a new method called NetBinder for the systematic identification and classification of prerequisite binding sites at atomic resolution. NetBinder is based on atomistic simulations of the full inhibitor binding process and provides a networking framework on which to select the most important binding modes and uncover the entire binding mechanism, including previously undiscovered events. NetBinder was validated by a study of the binding mechanism of blebbistatin (a potent inhibitor) to myosin 2 (a promising target for cancer chemotherapy). Myosin 2 is a good test enzyme because, like other potential targets, it has a long internal binding cavity that provides blebbistatin with numerous potential prerequisite binding sites. The mechanism proposed by NetBinder of myosin 2 structural changes during blebbistatin binding shows excellent agreement with experimentally determined binding sites and structural changes. While NetBinder was tested on myosin 2, it may easily be adopted to other proteins with long internal cavities, such as G-protein-coupled receptors or ion channels, the most popular current drug targets. NetBinder provides a new paradigm for drug design by a network-based elucidation of binding mechanisms at an atomic resolution.

Exploration of Somatostatin Binding Mechanism to Somatostatin Receptor Subtype 4.

Somatostatin (also named as growth hormone-inhibiting hormone or somatotropin release-inhibiting factor) is a regulatory peptide important for the proper functioning of the endocrine system, local inflammatory reactions, mood and motor coordination, and behavioral responses to stress. Somatostatin exerts its effects via binding to G-protein-coupled somatostatin receptors of which the fourth subtype (SSTR4) is a particularly important receptor mediating analgesic, anti-inflammatory, and anti-depressant effects without endocrine actions. Thus, SSTR4 agonists are promising drug candidates. Although the knowledge of the atomic resolution-binding modes of SST would be essential for drug development, experimental elucidation of the structures of SSTR4 and its complexes is still awaiting. In the present study, structures of the somatostatin-SSTR4 complex were produced using an unbiased, blind docking approach. Beyond the static structures, the binding mechanism of SST was also elucidated in the explicit water molecular dynamics (MD) calculations, and key binding modes (external, intermediate, and internal) were distinguished. The most important residues on both receptor and SST sides were identified. An energetic comparison of SST binding to SSTR4 and 2 offered a residue-level explanation of receptor subtype selectivity. The calculated structures show good agreement with available experimental results and indicate that somatostatin binding is realized via prerequisite binding modes and an induced fit mechanism. The identified binding modes and the corresponding key residues provide useful information for future drug design targeting SSTR4.

Testing Serum Albumins and Cyclodextrins as Potential Binders of the Mycotoxin Metabolites Alternariol-3-Sulfate, Alternariol-9-Monomethylether and Alternariol-9-Monomethylether-3-Sulfate.

Alternaria mycotoxins, including alternariol (AOH), alternariol-9-monomethylether (AME), and their masked/modified derivatives (e.g., sulfates or glycosides), are common food contaminants. Their acute toxicity is relatively low, while chronic exposure can lead to the development of adverse health effects. Masked/modified metabolites can probably release the more toxic parent mycotoxin due to their enzymatic hydrolysis in the intestines. Previously, we demonstrated the complex formation of AOH with serum albumins and cyclodextrins; these interactions were successfully applied for the extraction of AOH from aqueous matrices (including beverages). Therefore, in this study, the interactions of AME, alternariol-3-sulfate (AS), and alternariol-9-monomethylether-3-sulfate (AMS) were investigated with albumins (human, bovine, porcine, and rat) and with cyclodextrins (sulfobutylether-ß-cyclodextrin, sugammadex, and cyclodextrin bead polymers). Our major results/conclusions are the following: (1) The stability of mycotoxin-albumin complexes showed only minor species dependent variations. (2) AS and AMS formed highly stable complexes with albumins in a wide pH range, while AME-albumin interactions preferred alkaline conditions. (3) AME formed more stable complexes with the cyclodextrins examined than AS and AMS. (4) Beta-cyclodextrin bead polymer proved to be highly suitable for the extraction of AME, AS, and AMS from aqueous solution. (5) Albumins and cyclodextrins are promising binders of the mycotoxins tested.

Determination of Ligand Binding Modes in Hydrated Viral Ion Channels to Foster Drug Design and Repositioning.

Target-based design and repositioning are mainstream strategies of drug discovery. Numerous drug design and repositioning projects have been launched to fight the ongoing COVID-19 pandemic. The resulting drug candidates have often failed due to the misprediction of their target-bound structures. The determination of water positions of such structures is particularly challenging due to the large number of possible drugs and the diversity of their hydration patterns. To answer this challenge and help correct predictions, we introduce a new protocol HydroDock, which can build hydrated drug-target complexes from scratch. HydroDock requires only the dry target and drug structures and produces their complexes with appropriately positioned water molecules. As a test application of the protocol, we built the structures of amantadine derivatives in complex with the influenza M2 transmembrane ion channel. The repositioning of amantadine derivatives from this influenza target to the SARS-CoV-2 envelope protein was also investigated. Excellent agreement was observed between experiments and the structures determined by HydroDock. The atomic resolution complex structures showed that water plays a similar role in the binding of amphipathic amantadine derivatives to transmembrane ion channels of both influenza A and SARS-CoV-2. While the hydrophobic regions of the channels capture the bulky hydrocarbon group of the ligand, the surrounding waters direct its orientation parallel with the axes of the channels via bridging interactions with the ionic ligand head. As HydroDock supplied otherwise undetermined structural details, it can be recommended to improve the reliability of future design and repositioning of antiviral drug candidates and many other ligands with an influence of water structure on their mechanism of action.

Ndr/Lats Kinases Bind Specific Mob-Family Coactivators through a Conserved and Modular Interface.

Ndr/Lats kinases bind Mob coactivator proteins to form complexes that are essential and evolutionarily conserved components of "Hippo" signaling pathways, which control cell proliferation and morphogenesis in eukaryotes. All Ndr/Lats kinases have a characteristic N-terminal regulatory (NTR) region that binds a specific Mob cofactor: Lats kinases associate with Mob1 proteins, and Ndr kinases associate with Mob2 proteins. To better understand the significance of the association of Mob protein with Ndr/Lats kinases and selective binding of Ndr and Lats to distinct Mob cofactors, we determined crystal structures of Saccharomyces cerevisiae Cbk1NTR-Mob2 and Dbf2NTR-Mob1 and experimentally assessed determinants of Mob cofactor binding and specificity. This allowed a significant improvement in the previously determined structure of Cbk1 kinase bound to Mob2, presently the only crystallographic model of a full length Ndr/Lats kinase complexed with a Mob cofactor. Our analysis indicates that the Ndr/LatsNTR-Mob interface provides a distinctive kinase regulation mechanism, in which the Mob cofactor organizes the Ndr/Lats NTR to interact with the AGC kinase C-terminal hydrophobic motif (HM), which is involved in allosteric regulation. The Mob-organized NTR appears to mediate association of the HM with an allosteric site on the N-terminal kinase lobe. We also found that Cbk1 and Dbf2 associated specifically with Mob2 and Mob1, respectively. Alteration of residues in the Cbk1 NTR allows association of the noncognate Mob cofactor, indicating that cofactor specificity is restricted by discrete sites rather than being broadly distributed. Overall, our analysis provides a new picture of the functional role of Mob association and indicates that the Ndr/LatsNTR-Mob interface is largely a common structural platform that mediates kinase-cofactor binding.

Effects of Chrysin and Its Major Conjugated Metabolites Chrysin-7-Sulfate and Chrysin-7-Glucuronide on Cytochrome P450 Enzymes and on OATP, P-gp, BCRP, and MRP2 Transporters.

Chrysin is an abundant flavonoid in nature, and it is also contained by several dietary supplements. Chrysin is highly biotransformed in the body, during which conjugated metabolites chrysin-7-sulfate and chrysin-7-glucuronide are formed. These conjugates appear at considerably higher concentrations in the circulation than the parent compound. Based on previous studies, chrysin can interact with biotransformation enzymes and transporters; however, the interactions of its metabolites have been barely examined. In this in vitro study, the effects of chrysin, chrysin-7-sulfate, and chrysin-7-glucuronide on cytochrome P450 enzymes (2C9, 2C19, 3A4, and 2D6) as well as on organic anion-transporting polypeptides (OATPs; 1A2, 1B1, 1B3, and 2B1) and ATP binding cassette [P-glycoprotein, multidrug resistance-associated protein 2, and breast cancer resistance protein (BCRP)] transporters were investigated. Our observations revealed that chrysin conjugates are strong inhibitors of certain biotransformation enzymes (e.g., CYP2C9) and transporters (e.g., OATP1B1, OATP1B3, OATP2B1, and BCRP) examined. Therefore, the simultaneous administration of chrysin-containing dietary supplements with medications needs to be carefully considered due to the possible development of pharmacokinetic interactions. SIGNIFICANCE STATEMENT: Chrysin-7-sulfate and chrysin-7-glucuronide are the major metabolites of flavonoid chrysin. In this study, we examined the effects of chrysin and its conjugates on cytochrome P450 enzymes and on organic anion-transporting polypeptides and ATP binding cassette transporters (P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2). Our results demonstrate that chrysin and/or its conjugates can significantly inhibit some of these proteins. Since chrysin is also contained by dietary supplements, high intake of chrysin may interrupt the transport and/or the biotransformation of drugs.

Molecular Structure, Binding Affinity, and Biological Activity in the Epigenome.

Development of valid structure-activity relationships (SARs) is a key to the elucidation of pathomechanisms of epigenetic diseases and the development of efficient, new drugs. The present review is based on selected methodologies and applications supplying molecular structure, binding affinity and biological activity data for the development of new SARs. An emphasis is placed on emerging trends and permanent challenges of new discoveries of SARs in the context of proteins as epigenetic drug targets. The review gives a brief overview and classification of the molecular background of epigenetic changes, and surveys both experimental and theoretical approaches in the field. Besides the results of sophisticated, cutting edge techniques such as cryo-electron microscopy, protein crystallography, and isothermal titration calorimetry, examples of frequently used assays and fast screening techniques are also selected. The review features how different experimental methods and theoretical approaches complement each other and result in valid SARs of the epigenome.

Synthesis of Spin-Labelled Bergamottin: A Potent CYP3A4 Inhibitor with Antiproliferative Activity.

Bergamottin (BM, 1), a component of grapefruit juice, acts as an inhibitor of some isoforms of the cytochrome P450 (CYP) enzyme, particularly CYP3A4. Herein, a new bergamottin containing a nitroxide moiety (SL-bergamottin, SL-BM, 10) was synthesized; chemically characterized, evaluated as a potential inhibitor of the CYP2C19, CYP3A4, and CYP2C9 enzymes; and compared to BM and known inhibitors such as ketoconazole (KET) (3A4), warfarin (WAR) (2C9), and ticlopidine (TIC) (2C19). The antitumor activity of the new SL-bergamottin was also investigated. Among the compounds studied, BM showed the strongest inhibition of the CYP2C9 and 2C19 enzymes. SL-BM is a more potent inhibitor of CYP3A4 than the parent compound; this finding was also supported by docking studies, suggesting that the binding positions of BM and SL-BM to the active site of CYP3A4 are very similar, but that SL-BM had a better ∆Gbind value than that of BM. The nitroxide moiety markedly increased the antitumor activity of BM toward HeLa cells and marginally increased its toxicity toward a normal cell line. In conclusion, modification of the geranyl sidechain of BM can result in new CYP3A4 enzyme inhibitors with strong antitumor effects.

Towards Unraveling the Histone Code by Fragment Blind Docking.

Histones serve as protein spools for winding the DNA in the nucleosome. High variability of their post-translational modifications result in a unique code system often responsible for the pathomechanisms of epigenetics-based diseases. Decoding is performed by reader proteins via complex formation with the N-terminal peptide tails of histones. Determination of structures of histone-reader complexes would be a key to unravel the histone code and the design of new drugs. However, the large number of possible histone complex variations imposes a true challenge for experimental structure determination techniques. Calculation of such complexes is difficult due to considerable size and flexibility of peptides and the shallow binding surfaces of the readers. Moreover, location of the binding sites is often unknown, which requires a blind docking search over the entire surface of the target protein. To accelerate the work in this field, a new approach is presented for prediction of the structure of histone H3 peptide tails docked to their targets. Using a fragmenting protocol and a systematic blind docking method, a collection of well-positioned fragments of the H3 peptide is produced. After linking the fragments, reconstitution of anchoring regions of the target-bound H3 peptide conformations was possible. As a first attempt of combination of blind and fragment docking approaches, our new method is named fragment blind docking (FBD).

A Fragmenting Protocol with Explicit Hydration for Calculation of Binding Enthalpies of Target-Ligand Complexes at a Quantum Mechanical Level.

Optimization of the enthalpy component of binding thermodynamics of drug candidates is a successful pathway of rational molecular design. However, the large size and missing hydration structure of target-ligand complexes often hinder such optimizations with quantum mechanical (QM) methods. At the same time, QM calculations are often necessitated for proper handling of electronic effects. To overcome the above problems, and help the QM design of new drugs, a protocol is introduced for atomic level determination of hydration structure and extraction of structures of target-ligand complex interfaces. The protocol is a combination of a previously published program MobyWat, an engine for assigning explicit water positions, and Fragmenter, a new tool for optimal fragmentation of protein targets. The protocol fostered a series of fast calculations of ligand binding enthalpies at the semi-empirical QM level. Ligands of diverse chemistry ranging from small aromatic compounds up to a large peptide helix of a molecular weight of 3000 targeting a leukemia protein were selected for systematic investigations. Comparison of various combinations of implicit and explicit water models demonstrated that the presence of accurately predicted explicit water molecules in the complex interface considerably improved the agreement with experimental results. A single scaling factor was derived for conversion of QM reaction heats into binding enthalpy values. The factor links molecular structure with binding thermodynamics via QM calculations. The new protocol and scaling factor will help automated optimization of binding enthalpy in future molecular design projects.

Novel Drug-Like Somatostatin Receptor 4 Agonists are Potential Analgesics for Neuropathic Pain.

Somatostatin released from the capsaicin-sensitive sensory nerves mediates analgesic and anti-inflammatory effects via the somatostatin sst4 receptor without endocrine actions. Therefore, sst4 is considered to be a novel target for drug development in pain including chronic neuropathy, which is an emerging unmet medical need. Here, we examined the in silico binding, the sst4-linked G-protein activation on stable receptor expressing cells (1 nM to 10 µM), and the effects of our novel pyrrolo-pyrimidine molecules in mouse inflammatory and neuropathic pain models. All four of the tested compounds (C1-C4) bind to the same binding site of the sst4 receptor with similar interaction energy to high-affinity reference sst4 agonists, and they all induce G-protein activation. C1 is the more efficacious (γ-GTP-binding: 218.2% ± 36.5%) and most potent (EC50: 37 nM) ligand. In vivo testing of the actions of orally administered C1 and C2 (500 µg/kg) showed that only C1 decreased the resiniferatoxin-induced acute neurogenic inflammatory thermal allodynia and mechanical hyperalgesia significantly. Meanwhile, both of them remarkably reduced partial sciatic nerve ligation-induced chronic neuropathic mechanical hyperalgesia after a single oral administration of the 500 µg/kg dose. These orally active novel sst4 agonists exert potent anti-hyperalgesic effect in a chronic neuropathy model, and therefore, they can open promising drug developmental perspectives.

Inhibitory Effects of Quercetin and Its Human and Microbial Metabolites on Xanthine Oxidase Enzyme.

Quercetin is an abundant flavonoid in nature and is used in several dietary supplements. Although quercetin is extensively metabolized by human enzymes and the colonic microflora, we have only few data regarding the pharmacokinetic interactions of its metabolites. Therefore, we investigated the interaction of human and microbial metabolites of quercetin with the xanthine oxidase enzyme. Inhibitory effects of five conjugates and 23 microbial metabolites were examined with 6-mercaptopurine and xanthine substrates (both at 5 µM), employing allopurinol as a positive control. Quercetin-3'-sulfate, isorhamnetin, tamarixetin, and pyrogallol proved to be strong inhibitors of xanthine oxidase. Sulfate and methyl conjugates were similarly strong inhibitors of both 6-mercaptopurine and xanthine oxidations (IC50 = 0.2-0.7 µM); however, pyrogallol inhibited xanthine oxidation (IC50 = 1.8 µM) with higher potency vs. 6-MP oxidation (IC50 = 10.1 µM). Sulfate and methyl conjugates were approximately ten-fold stronger inhibitors (IC50 = 0.2-0.6 µM) of 6-mercaptopurine oxidation than allopurinol (IC50 = 7.0 µM), and induced more potent inhibition compared to quercetin (IC50 = 1.4 µM). These observations highlight that some quercetin metabolites can exert similar or even a stronger inhibitory effect on xanthine oxidase than the parent compound, which may lead to the development of quercetin-drug interactions (e.g., with 6-mercaptopurin or azathioprine).

The Structure of an NDR/LATS Kinase-Mob Complex Reveals a Novel Kinase-Coactivator System and Substrate Docking Mechanism.

Eukaryotic cells commonly use protein kinases in signaling systems that relay information and control a wide range of processes. These enzymes have a fundamentally similar structure, but achieve functional diversity through variable regions that determine how the catalytic core is activated and recruited to phosphorylation targets. "Hippo" pathways are ancient protein kinase signaling systems that control cell proliferation and morphogenesis; the NDR/LATS family protein kinases, which associate with "Mob" coactivator proteins, are central but incompletely understood components of these pathways. Here we describe the crystal structure of budding yeast Cbk1-Mob2, to our knowledge the first of an NDR/LATS kinase-Mob complex. It shows a novel coactivator-organized activation region that may be unique to NDR/LATS kinases, in which a key regulatory motif apparently shifts from an inactive binding mode to an active one upon phosphorylation. We also provide a structural basis for a substrate docking mechanism previously unknown in AGC family kinases, and show that docking interaction provides robustness to Cbk1's regulation of its two known in vivo substrates. Co-evolution of docking motifs and phosphorylation consensus sites strongly indicates that a protein is an in vivo regulatory target of this hippo pathway, and predicts a new group of high-confidence Cbk1 substrates that function at sites of cytokinesis and cell growth. Moreover, docking peptides arise in unstructured regions of proteins that are probably already kinase substrates, suggesting a broad sequential model for adaptive acquisition of kinase docking in rapidly evolving intrinsically disordered polypeptides.

Structural assembly of the signaling competent ERK2-RSK1 heterodimeric protein kinase complex.

Mitogen-activated protein kinases (MAPKs) bind and activate their downstream kinase substrates, MAPK-activated protein kinases (MAPKAPKs). Notably, extracellular signal regulated kinase 2 (ERK2) phosphorylates ribosomal S6 kinase 1 (RSK1), which promotes cellular growth. Here, we determined the crystal structure of an RSK1 construct in complex with its activator kinase. The structure captures the kinase-kinase complex in a precatalytic state where the activation loop of the downstream kinase (RSK1) faces the enzyme's (ERK2) catalytic site. Molecular dynamics simulation was used to show how this heterodimer could shift into a signaling-competent state. This structural analysis combined with biochemical and cellular studies on MAPK→MAPKAPK signaling showed that the interaction between the MAPK binding linear motif (residing in a disordered kinase domain extension) and the ERK2 "docking" groove plays the major role in making an encounter complex. This interaction holds kinase domains proximal as they "readjust," whereas generic kinase domain surface contacts bring them into a catalytically competent state.