Activation Loop Research Articles

KATANA - water activation facility at JSI TRIGA, Part I: Final design and activity calculations

Water as a primary coolant will play an important role in the performance of fusion reactors, as it causes an ionising radiation field throughout the facility after its irradiation and activation and requires improved shielding for instruments and personnel. To support ITER, the KATANA irradiation facility, which utilises a closed-water activation loop, was built, licenced and commissioned in early 2024 at the TRIGA research reactor at the Jožef Stefan Institute in Slovenia.A comprehensive overview of the KATANA irradiation facility, which serves as a well-defined and stable high-energy γ and neutron source, is given, including the final design, experimental set-up, detector systems and overall timeline. The physical characteristics of KATANA were assessed by analyses of neutron transport simulations leading to activity calculations in connection with a conventional analytical approach, without using CFD calculations. The KATANA facility demonstrated the desired operational characteristics in terms of high and stable water flow rates, leading to high activity values of the observed isotopes 16N, 17N and 19O, which is essential for minimising experimental uncertainties.The preliminary calculations provided in-depth operational knowledge and important data for the KATANA facility, forming a basis for more advanced future experiments.

Active learning assisted piezoelectric materials synthesis on the basis of composite decision-making

The synthesis and development of novel materials for soft electronics, health monitoring, etc., have become a research hotspot. Traditional laboratory synthesis is significantly time-and-resource-consuming. Machine learning therefore becomes an ideal approach for expediting experimental process, constructing a virtual and automated closed-loop material synthesis and evaluation approach. In this work, we combined piezoelectric materials’ synthesis with machine learning to achieve automatic design optimization. 300 samples with different material recipes were used to train the initial active learning model. Thereafter, more samples were fabricated based on the recommended feasible recipes for each learning loop and then proceeded to the next round of learning. Through 10 active learning loops, 105 piezoelectric samples were stagewise fabricated. Moreover, a reverse design model based on Bayesian optimization is demonstrated, and Spearman's rank correlation coefficient and p values revealed the rules for the synthesis of piezoelectric materials. Finally, according to the setup model, we fabricate optimized piezoelectric materials, and demonstrate the application in cycling monitoring. We anticipate this work establishes an essential approach to accelerate the development of new materials.

Cocrystallization of the Src-Family Kinase Hck with the ATP-Site Inhibitor A-419259 Stabilizes an Extended Activation Loop Conformation.

Hematopoietic cell kinase (Hck) is a member of the Src kinase family and is a promising drug target in myeloid leukemias. Here, we report the crystal structure of human Hck in complex with the pyrrolopyrimidine inhibitor A-419259, determined at a resolution of 1.8 Å. This structure reveals the complete Hck active site in the presence of A-419259, including the αC-helix, the DFG motif, and the activation loop. A-419259 binds at the ATP-site of Hck and induces an overall closed conformation of the kinase with the regulatory SH3 and SH2 domains bound intramolecularly to their respective internal ligands. A-419259 stabilizes the DFG-in/αC-helix-out conformation observed previously with Hck and the pyrazolopyrimidine inhibitor PP1 (PDB: 1QCF). However, the activation loop conformations are distinct, with PP1 inducing a folded loop structure with the tyrosine autophosphorylation site (Tyr416) pointing into the ATP binding site, while A-419259 stabilizes an extended loop conformation with Tyr416 facing out into the solvent. Autophosphorylation also induces activation loop extension and significantly reduces the Hck sensitivity to PP1 but not A-419259. In cancer cells where Hck is constitutively active, the extended autophosphorylation loop may render Hck more sensitive to inhibitors like A-419259 which prefer this kinase conformation. More generally, these results provide additional insight into targeted kinase inhibitor design and how conformational preferences of inhibitors may impact selectivity and potency.

SHIN-2 exerts potent activity against VanA-type vancomycin-resistant Enterococcus faecium in vitro by stabilizing the active site loop of serine hydroxymethyltransferase

Novel classes of antibiotics are needed to improve the resilience of the healthcare system to antimicrobial resistance (AMR), including vancomycin resistance. vanA gene cluster is a cause of vancomycin resistance. This gene cluster is transferred and spreads vancomycin resistance from Enterococcus spp. to Staphylococcus aureus. Therefore, novel antibacterial agents are required to combat AMR, including vanA-type vancomycin resistance. Serine hydroxymethyltransferase (SHMT) is a key target of antibacterial agents. However, the specific binding mechanisms of SHMT inhibitors remain unclear. Detailed structural information will contribute to understanding these mechanisms. In this study, we found that (+)–SHIN–2, the first in vivo active inhibitor of human SHMT, is strongly bound to the Enterococcus faecium SHMT (efmSHMT). Comparison of the crystal structures of apo- and (+)–SHIN–2-boud efmSHMT revealed that (+)–SHIN–2 stabilized the active site loop of efmSHMT via hydrogen bonds, which are critical for efmSHMT inhibition. Additionally, (+)–SHIN–2 formed hydrogen bonds with serine, forming the Schiff's base with pyridoxal 5′-phosphate, which is a co-factor of SHMT. Furthermore, (+)–SHIN–2 exerted biostatic effects on vancomycin-susceptible and vanA-type vancomycin-resistant E. faecium in vitro, indicating that SHMT inhibitors do not induce cross-resistance to vanA-type vancomycin. Overall, these findings can aid in the design of novel SHMT inhibitors to combat AMR, including vancomycin resistance.

1H, 15N and 13C resonance assignments of the S2A and H64A double mutant of human carbonic anhydrase II.

Protein-water interactions profoundly influence protein structure and dynamics. Consequently, the function of many biomacromolecules is directly related to the presence and exchange of water molecules. While structural water molecules can be readily identified through X-ray crystallography, the dynamics within functional protein-water networks remain largely elusive. Therefore, to understand the role of biological water in protein dynamics and function, we have introduced S2A and H64A mutations in human Carbonic Anhydrase II (hCAII), a model system to study protein-water interactions. The mutations of serine to alanine at position 2 and histidine to alanine at position 64 cause an increase in hydrophobicity in the N-terminus and active site loop thereby restricting water entry and disrupting the water network in the Zn2+-binding pocket. To pave the way for a detailed investigation into the structural, functional, and mechanistic aspects of the Ser2Ala/His64Ala double mutant of hCAII, we present here almost complete sequence-specific resonance assignments for 1H, 15N, and 13C. These assignments serve as the basis for comprehensive studies on the dynamics of the protein-water network within the Zn2+-binding pocket and its role in catalysis.

Effects of coronal rain on decayless kink oscillations of coronal loops

Decayless kink oscillations are ubiquitously observed in active region coronal loops with an almost constant amplitude for several cycles. Decayless kink oscillations of coronal loops triggered by coronal rain have been analyzed, but the impact of coronal rain formation in an already oscillating loop is unclear. As kink oscillations can help diagnose the local plasma conditions, it is important to understand how these are affected by coronal rain phenomena. In this study, we present the analysis of an event of coronal rain that occurred on 25 April 2014 and was simultaneously observed by Slit-Jaw Imager (SJI) on board Interface Region Imaging Spectrograph (IRIS) and Atmospheric Imaging Assembly (AIA) on board the Solar Dynamic Observatory (SDO). We investigated the oscillation properties of the coronal loop in AIA images before and after the appearance of coronal rain as observed by SJI. We find signatures of decayless oscillations before and after coronal rain at similar positions to those found during coronal rain. The individual cases show a greater amplitude and period during coronal rain. The mean period is increased by 1.3 times during coronal rain, while the average amplitude is increased by 2 times during rain, in agreement with the expected density increase from coronal rain. The existence of the oscillations in the same loop at the time of no coronal rain indicates the presence of a footpoint driver. The properties of the observed oscillations during coronal rain can result from the combined contribution of coronal rain and a footpoint driver. The oscillation amplitude associated with coronal rain is approximated to be 0.14 Mm. The properties of decayless oscillations are considerably affected by coronal rain, and without prior knowledge of coronal rain in the loop, a significant discrepancy can arise from coronal seismology with respect to the true values.

Human-in-the-loop: Using classifier decision boundary maps to improve pseudo labels

For classification tasks, several strategies aim to tackle the problem of not having sufficient labeled data, usually by automatic labeling or by fully passing this task to a user. Automatic labeling is simple to apply but can fail handling complex situations where human insights may be required to decide the correct labels. Conversely, manual labeling leverages the expertise of specialists but may waste precious effort which could be handled by automatic methods. More specifically, automatic solutions could be improved by combining an active learning loop with manual labeling assisted by visual depictions of a classifier’s behavior. We propose to include the human in the labeling loop by using manual labeling in feature spaces produced by a deep feature annotation (DeepFA) technique. To assist manual labeling, we provide users with visual insights on the classifier’s decision boundaries. Finally, we use the manual and automatically computed labels jointly to retrain the classifier in an active learning (AL) loop scheme. Experiments using a toy and a real-world application dataset show that our proposed combination of manual labeling supported by visualization of decision boundaries and automatic labeling can yield a significant increase in classifier performance with a quite limited user effort.

Developing New Peptides and Peptide-Drug Conjugates for Targeting the FGFR2 Receptor-Expressing Tumor Cells and 3D Spheroids.

In this work, we utilized a biomimetic approach for targeting KATO (III) tumor cells and 3D tumoroids. Specifically, the binding interactions of the bioactive short peptide sequences ACSAG (A-pep) and LPHVLTPEAGAT (L-pep) with the fibroblast growth factor receptor (FGFR2) kinase domain was investigated for the first time. Both peptides have been shown to be derived from natural resources previously. We then created a new fusion trimer peptide ACSAG-LPHVLTPEAGAT-GASCA (Trimer-pep) and investigated its binding interactions with the FGFR2 kinase domain in order to target the fibroblast growth factor receptor 2 (FGFR2), which is many overexpressed in tumor cells. Molecular docking and molecular dynamics simulation studies revealed critical interactions with the activation loop, hinge and glycine-rich loop regions of the FGFR2 kinase domain. To develop these peptides for drug delivery, DOX (Doxorubicin) conjugates of the peptides were created. Furthermore, the binding of the peptides with the kinase domain was further confirmed through surface plasmon resonance studies. Cell studies with gastric cancer cells (KATO III) revealed that the conjugates and the peptides induced higher cytotoxicity in the tumor cells compared to normal cells. Following confirmation of cytotoxicity against tumor cells, the ability of the conjugates and the peptides to penetrate 3D spheroids was investigated by evaluating their permeation in co-cultured spheroids grown with KATO (III) and colon tumor-associated fibroblasts (CAFs). Results demonstrated that Trimer-pep conjugated with DOX showed the highest permeation, while the ACSAG conjugate also demonstrated reasonable permeation of the drug. These results indicate that these peptides may be further explored and potentially utilized to create drug conjugates for targeting tumor cells expressing FGFR2 for developing therapeutics.

HDX-MS Analysis of Catalytic Activation of IKK2 in the IκB Kinase Complex.

The IκB Kinase (IKK) complex, containing catalytic IKK2 and noncatalytic NEMO subunits, plays essential roles in the induction of transcription factors of the NF-κB family. Catalytic activation of IKK2 via phosphorylation of its activation loop is promoted upon noncovalent association of linear or K63-linked polyubiquitin chains to NEMO within the IKK complex. The mechanisms of this activation remain speculative. To investigate interaction dynamics within the IKK complex during activation of IKK2, we conducted hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) on NEMO and IKK2 proteins in their free and complex-bound states. Altered proton exchange profiles were observed in both IKK2 and NEMO upon complex formation, and changes were consistent with the involvement of distinct regions throughout the entire length of both proteins, including previously uncharacterized segments, in direct or allosteric interactions. Association with linear tetraubiquitin (Ub4) affected multiple regions of the IKK2:NEMO complex, in addition to previously identified interaction sites on NEMO. Intriguingly, observed enhanced solvent accessibility of the IKK2 activation loop within the IKK2:NEMO:Ub4 complex, coupled with contrasting protection of surrounding segments of the catalytic subunit, suggests an allosteric role for NEMO:Ub4 in priming IKK2 for phosphorylation-dependent catalytic activation.

Water and chloride as allosteric inhibitors in WNK kinase osmosensing.

Osmotic stress and chloride regulate the autophosphorylation and activity of the WNK1 and WNK3 kinase domains. The kinase domain of unphosphorylated WNK1 (uWNK1) is an asymmetric dimer possessing water molecules conserved in multiple uWNK1 crystal structures. Conserved waters are present in two networks, referred to here as conserved water networks 1 and 2 (CWN1and CWN2). Here we show that PEG400 applied to crystals of dimeric uWNK1 induces de-dimerization. Both the WNK1 the water networks and the chloride binding site and are disrupted by PEG400. CWN1 is surrounded by a cluster of pan-WNK-conserved charged residues. Here we mutagenized these charges in WNK3, a highly active WNK isoform kinase domain, and WNK1, the isoform best studied crystallographically. Mutation of E314 in the Activation Loop of WNK3 (WNK3/E314Q and WNK3/E314A, and the homologous WNK1/E388A) enhanced the rate of autophosphorylation, and reduced chloride sensitivity. Other WNK3 Cluster mutants reduced the rate of autophosphorylation activity coupled with greater chloride sensitivity than wild-type. The water and chloride regulation thus appear linked. The lower activity of some mutants may reflect effects on catalysis. Crystallography showed that activating mutants introduced conformational changes in similar parts of the structure to those induced by PEG400. WNK activating mutations and crystallography support a role for CWN1 in WNK inhibition consistent with water functioning as an allosteric ligand.

Conformational Modulation of a Mobile Loop Controls Catalysis in the (βα)8-Barrel Enzyme of Histidine Biosynthesis HisF.

The overall significance of loop motions for enzymatic activity is generally accepted. However, it has largely remained unclear whether and how such motions can control different steps of catalysis. We have studied this problem on the example of the mobile active site β1α1-loop (loop1) of the (βα)8-barrel enzyme HisF, which is the cyclase subunit of imidazole glycerol phosphate synthase. Loop1 variants containing single mutations of conserved amino acids showed drastically reduced rates for the turnover of the substrates N'-[(5'-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PrFAR) and ammonia to the products imidazole glycerol phosphate (ImGP) and 5-aminoimidazole-4-carboxamide-ribotide (AICAR). A comprehensive mechanistic analysis including stopped-flow kinetics, X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations detected three conformations of loop1 (open, detached, closed) whose populations differed between wild-type HisF and functionally affected loop1 variants. Transient stopped-flow kinetic experiments demonstrated that wt-HisF binds PrFAR by an induced-fit mechanism whereas catalytically impaired loop1 variants bind PrFAR by a simple two-state mechanism. Our findings suggest that PrFAR-induced formation of the closed conformation of loop1 brings active site residues in a productive orientation for chemical turnover, which we show to be the rate-limiting step of HisF catalysis. After the cyclase reaction, the closed loop conformation is destabilized, which favors the formation of detached and open conformations and hence facilitates the release of the products ImGP and AICAR. Our data demonstrate how different conformations of active site loops contribute to different catalytic steps, a finding that is presumably of broad relevance for the reaction mechanisms of (βα)8-barrel enzymes and beyond.

Intermediate-based virtual screening of c-Kit kinase inhibitors as potential anti-tumor agents via ab inito folding, molecular dynamics simulation, and molecular docking

Uncontrolled activation of c-Kit is closely related to the pathogenesis and progression of leukemia, gastrointestinal cancer, and other malignant diseases. Although there are several inhibitors available, due to the limitation of selectivity and the unfavorable side effects, designing and discovering highly selective inhibitors targeting c-Kit kinase, especially the gain of function mutation (for example c-Kit D816V), is still necessary. To identify novel c-Kit inhibitors, a metastable state-based virtual screening approach, which was successfully implemented in other kinase inhibitors, was employed in the current study. The results from our current study demonstrated the residues adjacent to the DFG motif within the activation loop could fold into short α-helices aside from the random coil, which was commonly found in the crystal structure. By expanding the conformation pool of the activation loop via PyRosetta-based ab initio folding protocol, we constructed a series of structural models of the c-Kit kinase intermediate between the inactive and active states. After evaluation of the thermal stability of the metastable state with molecular dynamics simulation, one structural model showed higher stability of α-helix, and the activation loop was retained. Considering the wild-type and D816V mutated KIT kinase shared similar metastable states during the kinase activation process, we developed a hypothesis that the identified intermediate might hold the potential to identify inhibitors targeting D816V mutations from the compound database. As expected, the intermediate structure showed higher selectivity to KIT D816V selective inhibitors, such as bezuclastinib, avapritinib, BLU-263, and elenestinib, than imatinib or masitinib. The virtual screening of the available KIT kinase inhibitor database further identified vorolanib, semaxanib, henatinib, and pexmetinib may possess potential inhibitory effects against wild type, as well as the mutated c-Kit kinase. The results from our current study not only proposed a novel structural model that could be used for the identification of selective c-Kit D816V inhibitors but also identified several potential inhibitors from available kinase inhibitors, which might shed new light on the design of new therapeutic approaches for c-Kit mutation-driven malignant diseases.

Effect of active loop extrusion on the two-contact correlations in the interphase chromosome.

The population-averaged contact maps generated by the chromosome conformation capture technique provide important information about the average frequency of contact between pairs of chromatin loci as a function of the genetic distance between them. However, these datasets do not tell us anything about the joint statistics of simultaneous contacts between genomic loci in individual cells. This kind of statistical information can be extracted using the single-cell Hi-C method, which is capable of detecting a large fraction of simultaneous contacts within a single cell, as well as through modern methods of fluorescent labeling and super-resolution imaging. Motivated by the prospect of the imminent availability of relevant experimental data, in this work, we theoretically model the joint statistics of pairs of contacts located along a line perpendicular to the main diagonal of the single-cell contact map. The analysis is performed within the framework of an ideal polymer model with quenched disorder of random loops, which, as previous studies have shown, allows us to take into account the influence of the loop extrusion process on the conformational properties of interphase chromatin.

Investigating the Regulation of Ribosomal Protein S6 Kinase 1 by CoAlation.

Ribosomal protein S6 kinases belong to a family of highly conserved enzymes in eukaryotes that regulate cell growth, proliferation, survival, and the stress response. It is well established that the activation and downstream signalling of p70S6Ks involve multiple phosphorylation events by key regulators of cell growth, survival, and energy metabolism. Here, we report for the first time the covalent modification of p70S6K1 by coenzyme A (CoA) in response to oxidative stress, which regulates its kinase activity. The site of CoA binding (CoAlation) was mapped by mass spectrometry to cysteine 217 (Cys217), located in the kinase activation loop and only one amino acid away from the tripeptide DFG motif, which facilitates ATP-binding. The CoAlation of recombinant p70S6K1 was demonstrated in vitro and was shown to inhibit its kinase activity. Our molecular docking and dynamics analysis revealed the most likely mode for CoA binding to p70S6K1. This mechanism involves the non-covalent binding of the CoA ADP moiety to the p70S6K1 nucleotide-binding pocket, positioning the CoA thiol group in close proximity to form a covalent bond with the surface-exposed Cys217 residue. These findings support a "dual anchor" mechanism for protein kinase inhibition by CoAlation in cellular response to oxidative stress. Furthermore, the inhibition of S6K1 by CoAlation may open new avenues for developing novel inhibitors.

The interaction of the azetidine thiazole side chain with the active site loop (ASL) 3 drives the evolution of IMP metallo-β-lactamase against tebipenem.

Imipenemase (IMP) metallo-β-lactamases (MBLs) hydrolyze almost all available β-lactams including carbapenems and are not inhibited by any commercially available β-lactamase inhibitor. Tebipenem (TP) pivoxil is the first orally available carbapenem and possesses a unique bicyclic azetidine thiazole moiety located at the R2 position. TP has potent in vitro activity against Enterobacterales producing extended-spectrum and/or AmpC β-lactamases. Thus far, the activity of TP against IMP-producing strains is understudied. To address this knowledge gap, we explored the structure activity relationships of IMP MBLs by investigating whether IMP-6, IMP-10, IMP-25, and IMP-78 [MBLs with expanded hydrolytic activity against meropenem (MEM)] would demonstrate enhanced activity against TP. Most of the Escherichia coli DH10B strains expressing IMP-1 variants displayed a ≥twofold MIC difference between TP and MEM, while those expressing VIM or NDM variants demonstrated comparable MICs. Catalytic efficiency (kcat/KM) values for the TP hydrolysis by IMP-1, IMP-6, IMP-10, IMP-25, and IMP-78 were significantly lower than those obtained for MEM. Molecular dynamic simulations reveal that V67F and S262G substitutions (found in IMP-78) reposition active site loop 3, ASL-3, to better accommodate the bicyclic azetidine thiazole side chain, allowing microbiological/catalytic activity to approach that of comparison MBLs used in this study. These findings suggest that modifying the R2 side chain of carbapenems can significantly impact hydrolytic stability. Furthermore, changes in conformational dynamics due to single amino acid substitutions should be used to inform drug design of novel carbapenems.

Structural and kinetic characterization of DUSP5 with a Di-phosphorylated tripeptide substrate from the ERK activation loop

Introduction: Dual specific phosphatases (DUSPs) are mitogen-activated protein kinase (MAPK) regulators, which also serve as drug targets for treating various vascular diseases. Previously, we have presented mechanistic characterizations of DUSP5 and its interaction with pERK, proposing a dual active site.Methods: Herein, we characterize the interactions between the DUSP5 phosphatase domain and the pT-E-pY activation loop of ERK2, with specific active site assignments. We also report the full NMR chemical shift assignments of DUSP5 that now enable chemical shift perturbation and dynamics studies.Results and Discussion: Both phosphates of the pT-E-pY tripeptide are dephosphorylated, based on 31P NMR; but, steady state kinetic studies of the tripeptide both as a substrate and as an inhibitor indicate a preference for binding and dephosphorylation of the phospho-tyrosine before the phospho-threonine. Catalytic efficiency (kcat/Km) is 3.7 M−1S−1 for T-E-pY vs 1.3 M−1S−1 for pT-E-Y, although the diphosphorylated peptide (pT-E-pY) is a better substrate than both, with kcat/Km = 18.2 M−1S−1 . Steady state inhibition studies with the pNPP substrate yields Kis values for the peptide inhibitors of: 15.82 mM (pT-E-Y), 4.932 mM (T-E-pY), 1.672 mM (pT-E-pY). Steady state inhibition studies with pNPP substrate and with vanadate or phosphate inhibitors indicated competitive inhibition with Kis values of 0.0006122 mM (sodium vanadate) and 17.32 mM (sodium phosphate), similar to other Protein Tyrosine Phosphatases with an active site cysteine nucleophile that go through a five-coordinate high energy transition state or intermediate. Molecular dynamics (MD) studies confirm preferential binding of the diphosphorylated peptide, but with preference for binding the pY over the pT reside in the catalytic site proximal to the Cys263 nucleophile. Based on MD, the monophosphorylated peptide binds tighter if phosphorylated on the Tyr vs the Thr. And, if the starting pose of the docked diphosphorylated peptide has pT in the catalytic site, it will adjust to have the pY in the catalytic site, suggesting a dynamic shifting of the peptide orientation. 2D 1H-15N HSQC chemical shift perturbation studies confirm that DUSP5 with tripeptide bound is in a dynamic state, with extensive exchange broadening observed—especially of catalytic site residues. The availability of NMR chemical shift assignments enables additional future studies of DUSP5 binding to the ERK2 diphosphorylated activation loop.Summary: These studies indicate a preference for pY before pT binding, but with ability to bind and dephosphorylate both residues, and with a dynamic active site pocket that accommodates multiple tripeptide orientations.

Evolutionary sequence and structural basis for the distinct conformational landscapes of Tyr and Ser/Thr kinases

Protein kinases are molecular machines with rich sequence variation that distinguishes the two main evolutionary branches – tyrosine kinases (TKs) from serine/threonine kinases (STKs). Using a sequence co-variation Potts statistical energy model we previously concluded that TK catalytic domains are more likely than STKs to adopt an inactive conformation with the activation loop in an autoinhibitory folded conformation, due to intrinsic sequence effects. Here we investigate the structural basis for this phenomenon by integrating the sequence-based model with structure-based molecular dynamics (MD) to determine the effects of mutations on the free energy difference between active and inactive conformations, using a thermodynamic cycle involving many (n = 108) protein-mutation free energy perturbation (FEP) simulations in the active and inactive conformations. The sequence and structure-based results are consistent and support the hypothesis that the inactive conformation DFG-out Activation Loop Folded, is a functional regulatory state that has been stabilized in TKs relative to STKs over the course of their evolution via the accumulation of residue substitutions in the activation loop and catalytic loop that facilitate distinct substrate binding modes in trans and additional modes of regulation in cis for TKs.

Allosteric activation of the co-receptor BAK1 by the EFR receptor kinase initiates immune signaling.

Transmembrane signaling by plant receptor kinases (RKs) has long been thought to involve reciprocal trans-phosphorylation of their intracellular kinase domains. The fact that many of these are pseudokinase domains, however, suggests that additional mechanisms must govern RK signaling activation. Non-catalytic signaling mechanisms of protein kinase domains have been described in metazoans, but information is scarce for plants. Recently, a non-catalytic function was reported for the leucine-rich repeat (LRR)-RK subfamily XIIa member EFR (elongation factor Tu receptor) and phosphorylation-dependent conformational changes were proposed to regulate signaling of RKs with non-RD kinase domains. Here, using EFR as a model, we describe a non-catalytic activation mechanism for LRR-RKs with non-RD kinase domains. EFR is an active kinase, but a kinase-dead variant retains the ability to enhance catalytic activity of its co-receptor kinase BAK1/SERK3 (brassinosteroid insensitive 1-associated kinase 1/somatic embryogenesis receptor kinase 3). Applying hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis and designing homology-based intragenic suppressor mutations, we provide evidence that the EFR kinase domain must adopt its active conformation in order to activate BAK1 allosterically, likely by supporting αC-helix positioning in BAK1. Our results suggest a conformational toggle model for signaling, in which BAK1 first phosphorylates EFR in the activation loop to stabilize its active conformation, allowing EFR in turn to allosterically activate BAK1.

Cimicifugoside H-2 as an Inhibitor of IKK1/Alpha: A Molecular Docking and Dynamic Simulation Study.

One of the most challenging issues scientists face is finding a suitable non-invasive treatment for cancer, as it is widespread around the world. The efficacy of phytochemicals that target oncogenic pathways appears to be quite promising and has gained attention over the past few years. We investigated the effect of docking phytochemicals isolated from the rhizomes of the Cimicifuga foetida plant on different domains of the IκB kinase alpha (IKK1/alpha) protein. The Cimicifugoside H-2 phytochemical registered a high docking score on the activation loop of IKK1/alpha amongst the other phytochemicals compared to the positive control. The interaction of the protein with Cimicifugoside H-2 was mostly stabilized by hydrogen bonds and hydrophobic interactions. A dynamic simulation was then performed with the Cimicifugoside H-2 phytochemical on the activation loop of IKK1/alpha, revealing that Cimicifugoside H-2 is a possible inhibitor of this protein. The pharmacokinetic properties of the drug were also examined to assess the safety of administering the drug. Therefore, in this in silico study, we discovered that the Cimicifugoside H-2 phytochemical inhibits the actively mutated conformation of IKK1/alpha, potentially suppressing the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) pathway.

Multiple Functional Protein-Protein Interaction Interfaces Allosterically Regulate ATP-Binding in Cyclin-Dependent Kinase-1.

The ATP-dependent phosphorylation activity of cyclin-dependent kinase 1 (CDK1), an essential enzyme for cell cycle progression, is regulated by interactions with Cyclin-B, substrate, and Cks proteins. We have recently shown that active site acetylation in CDK1 abrogated binding to Cyclin-B which posits an intriguing long-range communication between the catalytic site and the protein-protein interaction (PPI) interface. Now, we demonstrate a general allosteric link between the CDK1 active site and all three of its PPI interfaces through atomistic molecular dynamics (MD) simulations. Specifically, we examined ATP binding free energies to CDK1 in native nonacetylated (K33wt) and acetylated (K33Ac) forms as well as the acetyl-mimic K33Q and the acetyl-null K33R mutant forms, which are accessible in vitro. In agreement with experiments, ATP binding is stronger in K33wt relative to the other three perturbed states. Free energy decomposition reveals, in addition to expected local changes, significant and selective nonlocal entropic responses to ATP binding/perturbation of K33 from the -helix, activation loop (A-loop), and - H segments in CDK1 which interface with Cyclin-B, substrate, and Cks proteins, respectively. Statistical analysis reveals that while entropic responses of protein segments to active site perturbations are on average correlated with their dynamical changes, such correlations are lost in about 9%-48% of the dataset depending on the segment. Besides proving the bi-directional communication between the active site and the CDK1:Cyclin-B interface, our study uncovers a hitherto unknown mode of ATP binding regulation by multiple PPI interfaces in CDK1.