Hydrophobic Motif Research Articles

A conserved juxtamembrane motif in plant NFR5 receptors is essential for root nodule symbiosis

Establishment of root nodule symbiosis is initiated by the perception of bacterial Nod factor ligands by the plant LysM receptor kinases NFR1 and NFR5. Receptor signaling initiating the symbiotic pathway depends on the kinase activity of NFR1, while the signaling mechanism of the catalytically inactive NFR5 pseudokinase is unknown. Here, we present the crystal structure of the signaling-competent Lotus japonicus NFR5 intracellular domain, comprising the juxtamembrane region and pseudokinase domain. The juxtamembrane region is structurally well defined and forms two α-helices, αA and αA', which contain an exposed hydrophobic motif. We demonstrate that this "juxtamembrane motif" promotes NFR5-NFR5 and NFR1-NFR5 interactions and is essential for symbiotic signaling. Conservation analysis reveals that the juxtamembrane motif is present throughout NFR5-type receptors and is required for symbiosis signaling from barley RLK10, suggesting a conserved and broader function for this motif in plant-microbe symbioses.

Capturing Autoinhibited PDK1 Reveals the Linker's Regulatory Role, Informing Innovative Inhibitor Design.

PDK1 is crucial for PI3K/AKT/mTOR and Ras/MAPK cancer signaling. It phosphorylates AKT in a PIP3-dependent but S6K, SGK, and RSK kinases in a PIP3-independent manner. Unlike its substrates, its autoinhibited monomeric state has been unclear, likely due to its low population time, and phosphorylation in the absence of PIP3 has been puzzling too. Here, guided by experimental data, we constructed models and performed all-atom molecular dynamics simulations. In the autoinhibited PDK1 conformation that resembles autoinhibited AKT, binding of the linker between the kinase and PH domains to the PIF-binding pocket promotes the formation of the Glu130-Lys111 salt bridge and weakens the association of the kinase domain with the PH domain, shifting the population from the autoinhibited state to states accessible to the membrane and its kinase substrates. The interaction of the substrates' hydrophobic motif and the PDK1 PIF-binding pocket facilitates the release of the autoinhibition even in the absence of PIP3. Phosphorylation of the serine-rich motif within the linker further attenuates the association of the PH domain with the kinase domain. These suggest that while the monomeric autoinhibited state is relatively stable, it can readily shift to its active, catalysis-prone state to phosphorylate its diverse substrates. Our findings reveal the PDK1 activation mechanism and discover the regulatory role of PDK1's linker, which lead to two innovative linker-based inhibitor strategies: (i) locking the autoinhibited PDK1 through optimization of the interactions of AKT inhibitors with the PH domain of PDK1 and (ii) analogs (small molecules or peptidomimetics) that mimic the linker interactions with the PIF-binding pocket.

Fatty acid derivatization and cyclization of the immunomodulatory peptide RP-182 targeting CD206high macrophages improves anti-tumor activity.

As tumor-associated macrophages (TAMs) exercise a plethora of pro-tumor and immune evasive functions, novel strategies targeting TAMs to inhibit tumor progression have emerged within the current arena of cancer immunotherapy. Activation of the mannose receptor 1 (Mrc1; CD206) is a recent approach that recognizes immune suppressive CD206high M2-like TAMs as a drug target. Ligation of CD206 both induces reprogramming of CD206high TAMs towards a pro-inflammatory phenotype and selectively triggers apoptosis in these cells. CD206-activating therapeutics are currently limited to the linear, 10mer peptide RP-182, 1, which is not a drug candidate. Here we sought to identify a better suitable candidate for future clinical development by synthesizing and evaluating a series of RP-182 analogues. Surprisingly, fatty acid derivative 1a (RP-182-PEG3-K(palmitic acid)) not only showed improved stability but also increased affinity to the CD206 receptor through enhanced interaction with a hydrophobic binding motif of CD206. Peptide 1a showed superior in vitro activity in cell-based assays of macrophage activation which was restricted to CD206high M2-polarized macrophages. Improvement of responses was disproportionally skewed towards improved induction of phagocytosis including cancer cell phagocytosis. 1a reprogrammed the immune landscape in genetically engineered murine KPC pancreatic tumors towards increased innate immune surveillance and improved tumor control, and effectively suppressed tumor growth of murine B16 melanoma allografts.

The membrane insertion of the pro-apoptotic protein Bax is a Tom22-dependent multi-step process: a study in nanodiscs

Membrane insertion of the pro-apoptotic protein Bax was investigated by setting up cell-free synthesis of full-length Bax in the presence of pre-formed nanodiscs. While Bax was spontaneously poorly inserted in nanodiscs, co-synthesis with the mitochondrial receptor Tom22 stimulated Bax membrane insertion. The initial interaction of Bax with the lipid bilayer exposed the hydrophobic GALLL motif in Hα1 leading to Bax precipitation through hydrophobic interactions. The same motif was recognized by Tom22, triggering conformational changes leading to the extrusion and the ensuing membrane insertion of the C-terminal hydrophobic Hα9. Tom22 was also required for Bax-membrane insertion after Bax was activated either by BH3-activators or by its release from Bcl-xL by WEHI-539. The effect of Tom22 was impaired by D154Y substitution in Bax-Hα7 and T174P substitution in Bax-Hα9, which are found in several tumors. Conversely, a R9E substitution promoted a spontaneous insertion of Bax in nanodiscs, in the absence of Tom22. Both Tom22-activated Bax and BaxR9E alone permeabilized liposomes to dextran-10kDa and formed ~5-nm-diameter pores in nanodiscs. The concerted regulation of Bax membrane insertion by Tom22 and BH3-activators is discussed.

Molecular insights into the regulatory landscape of PKC-related kinase-2 (PRK2/PKN2) using targeted small compounds

The protein kinase C-related kinases (PRKs, also termed PKNs) are important in cell migration, cancer, hepatitis C infection, and nutrient sensing. They belong to a group of protein kinases called AGC kinases that share common features like a C-terminal extension to the catalytic domain comprising a hydrophobic motif. PRKs are regulated by N-terminal domains, a pseudosubstrate sequence, Rho-binding domains and a C2 domain involved in inhibition and dimerization, while Rho and lipids are activators. We investigated the allosteric regulation of PRK2 and its interaction with its upstream kinase PDK1 using a chemical biology approach. We confirmed the PIF-mediated docking interaction of PRK2 with PDK1 and showed that this interaction can be modulated allosterically. We showed that the polypeptide PIFtide and a small compound binding to the PIF-pocket of PRK2 were allosteric activators, by displacing the pseudosubstrate PKL region from the active site. In addition, a small compound binding to the PIF-pocket allosterically inhibited the catalytic activity of PRK2. Together, we confirmed the docking interaction and allostery between PRK2 and PDK1 and described an allosteric communication between the PIF-pocket and the active site of PRK2, both modulating the conformation of the ATP-binding site and the pseudosubstrate PKL-binding site. Our study highlights the allosteric modulation of the activity and the conformation of PRK2 in addition to the existence of at least two different complexes between PRK2 and its upstream kinase PDK1. Finally, the study highlights the potential for developing allosteric drugs to modulate PRK2 kinase conformations and catalytic activity.

Molecular basis for different substrate-binding sites and chaperone functions of the BRICHOS domain.

Proteins can misfold into fibrillar or amorphous aggregates and molecular chaperones act as crucial guardians against these undesirable processes. The BRICHOS chaperone domain, found in several otherwise unrelated proproteins that contain amyloidogenic regions, effectively inhibits amyloid formation and toxicity but can in some cases also prevent non-fibrillar, amorphous protein aggregation. Here, we elucidate the molecular basis behind the multifaceted chaperone activities of the BRICHOS domain from the Bri2 proprotein. High-confidence AlphaFold2 and RoseTTAFold predictions suggest that the intramolecular amyloidogenic region (Bri23) is part of the hydrophobic core of the proprotein, where it occupies the proposed amyloid binding site, explaining the markedly reduced ability of the proprotein to prevent an exogenous amyloidogenic peptide from aggregating. However, the BRICHOS-Bri23 complex maintains its ability to form large polydisperse oligomers that prevent amorphous protein aggregation. A cryo-EM-derived model of the Bri2 BRICHOS oligomer is compatible with surface-exposed hydrophobic motifs that get exposed and come together during oligomerization, explaining its effects against amorphous aggregation. These findings provide a molecular basis for the BRICHOS chaperone domain function, where distinct surfaces are employed against different forms of protein aggregation.

The polarity protein Yurt associates with the plasma membrane via basic and hydrophobic motifs embedded in its FERM domain.

The subcellular distribution of the polarity protein Yurt (Yrt) is subjected to a spatio-temporal regulation in Drosophila melanogaster embryonic epithelia. After cellularization, Yrt binds to the lateral membrane of ectodermal cells and maintains this localization throughout embryogenesis. During terminal differentiation of the epidermis, Yrt accumulates at septate junctions and is also recruited to the apical domain. Although the mechanisms through which Yrt associates with septate junctions and the apical domain have been deciphered, how Yrt binds to the lateral membrane remains as an outstanding puzzle. Here, we show that the FERM domain of Yrt is necessary and sufficient for membrane localization. Our data also establish that the FERM domain of Yrt directly binds negatively charged phospholipids. Moreover, we demonstrate that positively charged amino acid motifs embedded within the FERM domain mediates Yrt membrane association. Finally, we provide evidence suggesting that Yrt membrane association is functionally important. Overall, our study highlights the molecular basis of how Yrt associates with the lateral membrane during the developmental time window where it is required for segregation of lateral and apical domains.

A novel bivalent interaction mode underlies a non-catalytic mechanism for Pin1-mediated protein kinase C regulation.

Regulated hydrolysis of the phosphoinositide phosphatidylinositol(4,5)-bis-phosphate to diacylglycerol and inositol-1,4,5-P3 defines a major eukaryotic pathway for translation of extracellular cues to intracellular signaling circuits. Members of the lipid-activated protein kinase C isoenzyme family (PKCs) play central roles in this signaling circuit. One of the regulatory mechanisms employed to downregulate stimulated PKC activity is via a proteasome-dependent degradation pathway that is potentiated by peptidyl-prolyl isomerase Pin1. Here, we show that contrary to prevailing models, Pin1 does not regulate conventional PKC isoforms α and βII via a canonical cis-trans isomerization of the peptidyl-prolyl bond. Rather, Pin1 acts as a PKC binding partner that controls PKC activity via sequestration of the C-terminal tail of the kinase. The high-resolution structure of full-length Pin1 complexed to the C-terminal tail of PKCβII reveals that a novel bivalent interaction mode underlies the non-catalytic mode of Pin1 action. Specifically, Pin1 adopts a conformation in which it uses the WW and PPIase domains to engage two conserved phosphorylated PKC motifs, the turn motif and hydrophobic motif, respectively. Hydrophobic motif is a non-canonical Pin1-interacting element. The structural information combined with the results of extensive binding studies and experiments in cultured cells suggest that non-catalytic mechanisms represent unappreciated modes of Pin1-mediated regulation of AGC kinases and other key enzymes/substrates.

The Chaperone-Active State of HdeB at pH 4 Arises from Its Conformational Rearrangement and Enhanced Stability Instead of Surface Hydrophobicity.

HdeA and HdeB are dimeric ATP-independent acid-stress chaperones, which protect the periplasmic proteins of enteric bacteria at pH 2.0 and 4.0, respectively, during their passage through the acidic environment of the mammalian stomach. Despite being structurally similar, they exhibit distinct functional pH optima and conformational prerequisite for their chaperone action. HdeA undergoes a dimer-to-monomer transition at pH 2.0, whereas HdeB remains dimeric at pH 4.0. The monomerization of HdeA exposes its hydrophobic motifs, which facilitates its interaction with the partially folded substrates. How HdeB functions despite maintaining its dimeric conformation has been poorly elucidated in the literature. Herein, we characterized the conformational states and stability of HdeB at its physiologically relevant pH and compared the data with those of HdeA. At pH 4.0, HdeB exhibited distinct spectroscopic signatures and higher stability against heat and guanidine-HCl-induced denaturation than at pH 7.5. We affirm that the pH 4.0 conformer of HdeB was distinct from that at pH 7.5 and that these two conformational states were hierarchically unrelated. Salt-bridge mutations that perturbed HdeB's intersubunit interactions resulted in the loss of its stability and function at pH 4.0. In contrast, mutations affecting intrasubunit interactions enhanced its function, albeit with a reduction in stability. These findings suggest that, unlike HdeA, HdeB acts as a noncanonical chaperone, where pH-dependent stability and conformational rearrangement at pH 4.0 play a core role in its chaperone function rather than its surface hydrophobicity. Such rearrangement establishes a stability-function trade-off that allows HdeB to function while maintaining its stable dimeric state.

N-terminal signals in the SNX-BAR paralogs Vps5 and Vin1 guide endosomal coat complex formation.

Endosomal coats incorporate membrane-binding subunits such as sorting nexin (SNX) proteins. The Saccharomyces cerevisiae SNX-BAR paralogs Vin1 and Vps5 are respective subunits of the endosomal VINE and retromer complexes whose dimerizing BAR domains are required for complex assembly and membrane association. However, a degree of promiscuity is predicted for yeast BAR-BAR pairings, and recent work has implicated the unstructured N-terminal domains of Vin1 and Vps5 in coat formation. Here, we map N-terminal signals in both SNX-BAR paralogs that contribute to the assembly and function of two distinct endosomal coats in vivo. Whereas Vin1 leverages a polybasic region and adjacent hydrophobic motif to bind Vrl1 and form VINE, the N-terminus of Vps5 interacts with the retromer subunit Vps29 at two sites, including a conserved hydrophobic pocket in Vps29 that engages other accessory proteins in humans. We also examined the sole isoform of Vps5 from the milk yeast Kluyveromyces lactis and found that ancestral yeasts may have used a nested N-terminal signal to form both VINE and retromer. Our results suggest that the specific assembly of Vps5-family SNX-BAR coats depends on inputs from unique N-terminal sequence features in addition to BAR domain coupling, expanding our understanding of endosomal coat biology.

MLL4 binds TET3

Human mixed lineage leukemia 4 (MLL4), also known as KMT2D, regulates cell type specific transcriptional programs through enhancer activation. Along with the catalytic methyltransferase domain, MLL4 contains seven less characterized plant homeodomain (PHD) fingers. Here, we report that the sixth PHD finger of MLL4 (MLL4PHD6) binds to the hydrophobic motif of ten-eleven translocation 3 (TET3), a dioxygenase that converts methylated cytosine into oxidized derivatives. The solution NMR structure of the TET3-MLL4PHD6 complex and binding assays show that, like histone H4 tail, TET3 occupies the hydrophobic site of MLL4PHD6, and that this interaction is conserved in the seventh PHD finger of homologous MLL3 (MLL3PHD7). Analysis of genomic localization of endogenous MLL4 and ectopically expressed TET3 in mouse embryonic stem cells reveals a high degree overlap on active enhancers and suggests a potential functional relationship of MLL4 and TET3.

Exploring the Landscape of Aptamers: From Cross-Reactive to Selective to Specific, High-Affinity Receptors for Cocaine.

We reported over 20 years ago MNS-4.1, the first DNA aptamer with a micromolar affinity for cocaine. MNS-4.1 is based on a structural motif that is very common in any random pool of oligonucleotides, and it is actually a nonspecific hydrophobic receptor with wide cross-reactivity with alkaloids and steroids. Despite such weaknesses preventing broad applications, this aptamer became widely used in proof-of-concept demonstrations of new formats of biosensors. We now report a series of progressively improved DNA aptamers recognizing cocaine, with the final optimized receptors having low nanomolar affinity and over a thousand-fold selectivity over the initial cross-reactants. In the process of optimization, we tested different methods to eliminate cross-reactivities and improve affinity, eventually achieving properties that are comparable to those of the reported monoclonal antibody candidates for the therapy of overdose. Multiple aptamers that we now report share structural motifs with the previously reported receptor for serotonin. Further mutagenesis studies revealed a palindromic, highly adaptable, broadly cross-reactive hydrophobic motif that could be rebuilt through mutagenesis, expansion of linker regions, and selections into receptors with exceptional affinities and varying specificities.

In-silico investigation of RPS6KB1 associated cancer inhibitor: a drug repurposing study

The ribosomal protein S6 kinase beta-1 (RPS6KB1), also known as p70S6 kinase, plays a crucial role in various disease-related conditions such as diabetes, obesity, and cancer. Its activity is regulated by phosphorylation events, including phosphorylation of Threonine 389 in the hydrophobic motif by the mammalian target of rapamycin complex 1 (mTORC1) and phosphorylation of Threonine 229 in the activation loop by PDK1 (phosphoinositide-dependent kinase 1). However, other phenomena connected to RPS6KB1 remain unknown. In this study, we employed virtual screening and molecular docking techniques on the molecules in the ZINC library to identify potential inhibitors. Molecular dynamics (MD) simulations and MMGBSA calculations were carried out on promising compounds to determine their binding affinity and stability. We also evaluated the drug-likeness properties of the selected compounds. A comparative study between the native RPS6KB1 structure, co-crystal ligands, and the shortlisted molecules from the ZINC dataset was carried out. The identified molecules possess significant potential for future in vitro and in vivo studies, paving the way for developing effective cancer treatments. Communicated by Ramaswamy H. Sarma

High-Throughput Screening of pH-Dependent β-sheet Self-Assembling Peptide.

pH-dependent peptide biomaterials hold tremendous potential for cell delivery and tissue engineering. However, identification of responsive self-assembling sequences with specified secondary structure remains a challenge. In this work, An experimental procedure based on the one-bead one-compound (OBOC) combinatorial library is developed to rapidly screen self-assembling β-sheet peptides at neutral aqueous solution (pH 7.5) and disassemble at weak acidic condition (pH 6.5). Using the hydrophobic fluorescent molecule thioflavin T (ThT) as a probe, resin beads displaying self-assembling peptides show fluorescence under pH 7.5 due to the insertion of ThT into the hydrophobic domain, and are further cultured in pH 6.5 solution. The beads with extinguished fluorescence are selected. Three heptapeptides are identified that can self-assemble into nanofibers or nanoparticles at pH 7.5 and disassemble at pH 6.5. P1 (LVEFRHY) shows a rapid acid response and morphology transformation with pH modulation. Changes in the charges of histidine and hydrophobic phenyl motif of phenylalanine may play important roles in the formation of pH-responsive β-sheet nanofiber. This high-throughput screening method provides an efficient way to identify pH-dependent β-sheet self-assembling peptide and gain insights into structural design of such nanomaterials.

A peptide selectively recognizes Gram-negative bacteria and forms a bacterial extracellular trap (BET) through interfacial self-assembly.

An innate immune system intricately leverages unique mechanisms to inhibit colonization of external invasive Bacteria, for example human defensin-6, through responsive encapsulation of bacteria. Infection and accompanying antibiotic resistance stemming from Gram-negative bacteria aggregation represent an emerging public health crisis, which calls for research into novel anti-bacterial therapeutics. Herein, inspired by naturally found host-defense peptides, we design a defensin-like peptide ligand, bacteria extracellular trap (BET) peptide, with modular design composed of targeting, assembly, and hydrophobic motifs with an aggregation-induced emission feature. The ligand specifically recognizes Gram-negative bacteria via targeting cell wall conserved lipopolysaccharides (LPS) and transforms from nanoparticles to nanofibrous networks in situ to trap bacteria and induce aggregation. Importantly, treatment of the BET peptide was found to have an antibacterial effect on the Pseudomonas aeruginosa strain, which is comparable to neomycin. Animal studies further demonstrate its ability to trigger aggregation of bacteria in vivo. This biomimetic self-assembling BET peptide provides a novel approach to fight against pathogenic Gram-negative bacteria.

Gibberellic-acid-dependent expression of α-amylase in wheat aleurone cells is mediated by target of rapamycin (TOR) signaling

Target of rapamycin (TOR) signaling is an essential nutrient-dependent pathway controlling cell growth in all eukaryotes. TOR signaling is well characterized in yeast and animals but remains poorly investigated in plants. The hormonal action of gibberellic acid (GA) is a crucial factor for wheat germination by inducing the synthesis of α-amylase in wheat aleurone cells. Here we showed that GA promotes the activation of Triticum aestivum TOR (TaTOR) signaling as evidenced by increased phosphorylation of T. aestivum S6K1 (TaS6K1) on its conserved hydrophobic motif together with proteasomal degradation of growth-inhibitory factor Rht-1. GA-dependent activation of TaTOR signaling led to α-amylase synthesis and Rht-1 proteasomal degradation because both GA-dependent events were sensitive to TaTOR inhibition. Using antibodies specific to TaTOR, we successfully identified the presence of endogenous TaTOR protein in terminally differentiated wheat aleurone layers. Additionally, by examining the rapamycin-sensitive phosphorylation of S6K1 as a reliable indicator of endogenous TOR kinase activity, we demonstrated that the activity of TaTOR in aleurone layers is enhanced by GA. Importantly, this stimulation is not associated with the regulation of either TaTOR transcription or the accumulation of TaTOR protein. In yeast and pull-down assays, a robust interaction between TaS6K1 and the N terminus of Rht-1 (amino acids 1–234) was observed, a finding further supported by co-immunoprecipitation of endogenous Rht-1 and TaS6K1. Furthermore, the administration of mTOR inhibitors significantly attenuated GA-induced degradation of endogenous Rht-1 and prolonged the persistence of the complex formed by these two proteins. We propose that TaTOR-TaS6K1 signaling contributes to GA-dependent wheat germination by mediating α-amylase synthesis and controlling proteasomal degradation of Rht-1 in wheat aleurone cells.

Development of Tris‐(triazolyl)‐s–triazine Linker‐Based Cationic Amphiphiles: Stability and Gene Delivery Applications

AbstractThe ability to readily be designed, manufactured, and described has made cationic lipids one of the highly adaptable vehicles for the transfer of DNA, RNA, and numerous additional therapeutic compounds. In this article, we synthesized a variety of Tris‐(triazolyl)‐s‐triazine linker‐based cationic lipids with different types of anchoring groups, and assessed the transfection efficacy. Specifically, two identical tails like either two alpha‐tocopherol domains or cholesterol domains or aliphatic chains (saturated), and on the other hand, two different tails, alpha‐tocopherol domain and cholesterol domain or alpha‐tocopherol domain and aliphatic chain (saturated) were employed as hydrophobic motifs to synthesize the cationic lipids. The stability, size, and surface charge of liposomes were performed by dynamic light scattering technique. Cytotoxicity study on cell survival showed that the synthesized lipids are less hazardous to the cells despite the fact that cationic lipids have large molecular structure. The lipid having di alpha‐tocopherol domains demonstrated phenomenal β‐galactosidase protein expression and enhanced green fluorescence expression in the hepatocellular carcinoma cell line. Findings from the physico‐chemical and biological evaluations the developed lipids are compatible in various forms while maintaining low cytotoxicity and efficient carriers for nucleic acid delivery.

Molecular Basis for SPINDOC-Spindlin1 Engagement and Its Role in Transcriptional Attenuation

Spindlin1 is a histone reader with three Tudor-like domains and its transcriptional co-activator activity could be attenuated by SPINDOC. The first two Tudors are involved in histone methylation readout, while the function of Tudor 3 is largely unknown. Here our structural and binding studies revealed an engagement mode of SPINDOC-Spindlin1, in which a hydrophobic motif of SPINDOC, DOCpep3, stably interacts with Spindlin1 Tudor 3, and two neighboring K/R-rich motifs, DOCpep1 and DOCpep2, bind to the acidic surface of Spindlin1 Tudor 2. Although DOCpep3-Spindlin1 engagement is compatible with histone readout, an extended SPINDOC fragment containing the K/R-rich region attenuates histone or TCF4 binding by Spindlin1 due to introduced competition. This inhibitory effect is more pronounced for weaker binding targets but not for strong ones such as H3 “K4me3-K9me3” bivalent mark. Further ChIP-seq and RT-qPCR indicated that SPINDOC could promote genomic relocation of Spindlin1, thus modulate downstream gene transcription. Collectively, we revealed multivalent engagement between SPINDOC and Spindlin1, in which a hydrophobic motif acts as the primary binding site for stable SPINDOC-Spindlin1 association, while K/R-rich region modulates the target selectivity of Spindlin1 via competitive inhibition, therefore attenuating the transcriptional co-activator activity of Spindlin1.

An essential Trypanosoma brucei protein kinase: a functional analysis of regulation and the identification of inhibitors

IntroductionThe protein serine/threonine kinase AEK1 is essential in the pathogenic stage of Trypanosoma brucei, the causative agent of African trypanosomiasis. AEK1 is a member of the AGC protein kinase family, although it is not closely related to a specific human AGC kinase. Our previous chemical genetic studies showed that targeted inhibition of AEK1 in parasites expressing analog-sensitive AEK1 blocked parasite growth and enhanced survival of infected mice.MethodsTo further validate AEK1 as a drug target, we used the chemical genetic system to determine the effect of a 24 hour loss of AEK1 activity on cell viability at the clonal level. A panel of 429 protein kinase inhibitors were screened against the wild-type protein for binding, using time-resolved fluorescence energy transfer (TR-FRET). The role of phosphorylation sites and motifs was probed by determining whether expression of proteins harboring mutations in these sequences could rescue AEK1 conditional knockout parasites. To determine the effect that mutations in the phosphosites have on the kinase activity of cellular AEK1 we compared the in vitro kinase activity of mutant and wild-type proteins immunoprecipitated from parasite lysates using the exogenous substrate MBP. Finally, the tagged AEK1 protein was localized by deconvolution microscopy.ResultsAfter a 24 hour exposure to an AEK1 inhibitory analog in the chemical genetic system, less than five percent of the remaining live cells can clonally expand, further validating AEK1 as a drug target. In the AEK1 inhibitor screening assay, we identified 17 hit compounds. Complementation studies showed that of the two known phosphorylation sites in the activation loop; mutation of one abolished function while mutation of the other had no discernable effect. Mutation of the other two AEK1 phosphosites gave intermediate phenotypes. Mutations in either the hydrophobic motif at the C-terminus of the protein or in the region of AEK1 predicted to bind the hydrophobic motif were also required for function. All parasites with defective AEK1 showed reduced proliferation and defects in cytokinesis, although the tested mutations differed in terms of the extent of cell death. Kinase activity of immunoprecipitated AEK1 phosphosite mutants largely paralleled the effects seen in complementation studies, although the mutation of the phosphosite adjacent to the hydrophobic motif had a greater impact on activity than predicted by the complementation studies. AEK1 was localized to cytoplasmic puncta distinct from glycosomes and acidocalcisomes.DiscussionThe rapid loss of viability of cells inhibited for AEK1 supports the idea that a short course of treatment that target AEK1 may be sufficient for treatment of people or animals infected with T. brucei. Key regulatory elements between AEK1 and its closest mammalian homolog appear to be largely conserved despite the vast evolutionary distance between mammals and T. brucei. The presence of AEK1 in cytoplasmic puncta raises the possibility that its localization may also play a role in functional activity.

From Local Hydration Motifs in Aqueous Acetic Acid Solutions to Macroscopic Mixing Thermodynamics: A Quantitative Connection from THz-Calorimetry.

We report the results of THz measurements (30-440 cm-1) of aqueous acetic acid solutions over the full mixing range (XAcAc = 0-1). We recorded spectroscopic observables as a function of the acetic acid concentration in the frequency range of the intermolecular stretch at 150 cm-1 and of the librational modes at 350-440 cm-1. This allowed us to unravel changes in hydrophobic and hydrophilic hydration motifs, respectively. By means of a novel THz-calorimetry approach, we quantitatively correlated these changes in local hydration motifs to excess mixing entropy and enthalpy. We find that ΔHmix is determined by both hydrophobic and hydrophilic solvation contributions. In contrast, ΔSmix is governed by hydrophobic cavity formation. Our results further suggest that acetic acid-water mixtures are systems at the edge of phase separation due to endothermic contributions from both hydrophilic and hydrophobic solvation in a large portion of the miscibility range. Our work establishes a quantitative relationship between the balance of local hydrophobic and hydrophilic solvation motifs and the macroscopic mixing thermodynamic properties.