Conformational changes in protein kinase A along its activation cycle are rooted in the folding energetics of cyclic-nucleotide binding domains.

Cyclic-nucleotide binding (CNB) domains are structurally and evolutionarily conserved signaling modules that regulate proteins with diverse folds and functions. Despite a wealth of structural information, the mechanisms by which CNB domains couple cyclic-nucleotide binding to conformational changes involved in signal transduction remain unknown. Here we combined single-molecule and computational approaches to investigate the conformation and folding energetics of the two CNB domains of the regulatory subunit of protein kinase A (PKA). We found that the CNB domains exhibit different conformational and folding signatures in the apo state, when bound to cAMP, or when bound to the PKA catalytic subunit, underscoring their ability to adapt to different binding partners. Moreover, we show while the two CNB domains have near-identical structures, their thermodynamic coupling signatures are divergent, leading to distinct cAMP responses and differential mutational effects. Specifically, we demonstrate mutation W260A exerts local and allosteric effects that impact multiple steps of the PKA activation cycle. Taken together, these results highlight the complex interplay between folding energetics, conformational dynamics, and thermodynamic signatures that underlies structurally conserved signaling modules in response to ligand binding and mutational effects.

Edmond Fischer's kinase legacy: History of the protein kinase inhibitor and protein kinase A.

Although Fischer's extraordinary career came to focus mostly on the protein phosphatases, after his co-discovery of Phosphorylase Kinase with Ed Krebs he was clearly intrigued not only by cAMP-dependent protein kinase (PKA), but also by the heat-stable, high-affinity protein kinase inhibitor (PKI). PKI is an intrinsically disordered protein that contains at its N-terminus a pseudo-substrate motif that binds synergistically and with high-affinity to the PKA catalytic (C) subunit. The sequencing and characterization of this inhibitor peptide (IP20) were validated by the structure of the PKA C-subunit solved first as a binary complex with IP20 and then as a ternary complex with ATP and two magnesium ions. A second motif, nuclear export signal (NES), was later discovered in PKI. Both motifs correspond to amphipathic helices that convey high-affinity binding. The dynamic features of full-length PKI, recently captured by NMR, confirmed that the IP20 motif becomes dynamically and sequentially ordered only in the presence of the C-subunit. The type I PKA regulatory (R) subunits also contain a pseudo-substrate ATPMg2-dependent high-affinity inhibitor sequence. PKI and PKA, especially the Cß subunit, are highly expressed in the brain, and PKI expression is also cell cycle-dependent. In addition, PKI is now linked to several cancers. The full biological importance of PKI and PKA signaling in the brain, and their importance in cancer thus remains to be elucidated.

Design, Heterologous Expression, and Application of an Immobilized Protein Kinase.

Protein kinase A (PKA) is a biologically important enzyme for cell regulation, often referred to as the "central kinase". An immobilized PKA that retains substrate specificity and activity would be a useful tool for laboratory scientists, enabling targeted phosphorylation without interference from downstream kinase contamination or kinase autophosphorylation in sensitive assays. Moreover, it might also provide the benefits of robustness and reusability that are often associated with immobilized enzyme preparations. In this work, we describe the creation of a recombinant PKA fusion protein that incorporates the HaloTag covalent immobilization system. We demonstrate that protein fusion design, including affinity tag placement, is critical for optimal heterologous expression in Escherichia coli. Furthermore, we demonstrate various applications of our immobilized PKA, including the phosphorylation of recombinant PKA substrates, such as vasodilator-stimulated phosphoprotein, and endogenous PKA substrates in a cell lysate. This immobilized PKA also possesses robust activity and reusability over multiple trials. This work holds promise as a generalizable strategy for the production and application of immobilized protein kinases.

The optimal docking strength for reversibly tethered kinases.

Many kinases use reversible docking interactions to augment the specificity of their catalytic domains. Such docking interactions are often structurally independent of the catalytic domain, which allow for a flexible combination of modules in evolution and in bioengineering. The affinity of docking interactions spans several orders of magnitude. This led us to ask how the affinity of the docking interaction affects enzymatic activity and how to pick the optimal interaction module to complement a given substrate. Here, we develop equations that predict the optimal binding strength of a kinase docking interaction and validate it using numerical simulations and steady-state phosphorylation kinetics for tethered protein kinase A. We show that a kinase-substrate pair has an optimum docking strength that depends on their enzymatic constants, the tether architecture, the substrate concentration, and the kinetics of the docking interactions. We show that a reversible tether enhances phosphorylation rates most when 1) the docking strength is intermediate, 2) the substrate is nonoptimal, 3) the substrate concentration is low, 4) the docking interaction has rapid exchange kinetics, and 5) the tether optimizes the effective concentration of the intramolecular reaction. This work serves as a framework for interpreting mutations in kinase docking interactions and as a design guide for engineering enzyme scaffolds.

Non-Canonical Allostery in Cyclic Nucleotide Dependent Kinases.

The cAMP- and cGMP-dependent protein kinases (PKA and PKG) are canonically activated by the corresponding cyclic nucleotides. However, both systems are also sensitive to a wide range of non-canonical allosteric effectors, such as reactive oxygen species, which induce the formation of regulatory inter- and intra-molecular disulfide bridges, and disease-related mutations (DRMs). Here, we present a combined analysis of representative non-canonical allosteric effectors for PKA and PKG, and we identify common molecular mechanisms underlying non-canonical allostery in these kinases, from shifts in dynamical regulatory equilibria to modulation of inter-protomer interactions. In addition, mutations may also drive oligomerization beyond dimerization, and possibly phase transitions, causing loss of kinase inhibitory function and amplifying the allosteric effects of DRMs. Hence non-canonical allosteric stimuli often result in constitutive kinase activation underlying either physiological control of downstream signaling pathways or pathological outcomes, from aortic aneurisms to cancer predisposition. Overall, PKA and PKG emerge as "pan-sensors" going well beyond canonical cyclic nucleotide activation, revealing their versatile roles as central signaling hubs.

A target fishing study to spot possible biological targets of fusaric acid: Inhibition of protein kinase-A and insights on the underpinning mechanisms.

Fusaric acid is a secondary metabolite produced by various Fusarium fungi, present with relatively high incidence in Fusarium-contaminated foods. It was already described as phytotoxic and cytotoxic. However, the understanding of its molecular mechanisms is still fragmentary and further data are needed to ensure an informed assessment of the risk related to its presence in food. This work applied an integrated in silico/in vitro approach to reveal novel potential biological activities of fusaric acid and to investigate the underpinning mechanisms. An in silico reverse screening was used to identify novel biological targets for fusaric acid. Computational results indicated as target protein kinase-A, which was confirmed with biochemical cell-free assays providing evidence of its actual inhibitory potential. Cell-based experiments on intestinal cells (HCEC-1CT cells) identified the mitochondrial network and cell membranes as potentially affected organelles, possibly resulting from PKA inhibition. The integration of 3D molecular modeling supported the plausibility of fusaric acid-dependent inhibition. From the hazard identification perspective, considering the Low Observed Adverse Effect Level described here (0.1 mM) and the possible level of contamination in food, fusaric acid might raise concern from a food safety standpoint and the gastrointestinal tract was described as a meaningful system to investigate with priority.

Cloning, expression, solubilization, and purification of a functionally active recombinant cAMP-dependent protein kinase catalytic subunit-like protein PKAC1 from Trypanosoma equiperdum.

The gene encoding the cAMP-dependent protein kinase (PKA) catalytic subunit-like protein PKAC1 from the Venezuelan TeAp-N/D1 strain of Trypanosoma equiperdum was cloned, and the recombinant TeqPKAC1 protein was overexpressed in bacteria. A major polypeptide with an apparent molecular mass of ∼38 kDa was detected by SDS-polyacrylamide gel electrophoresis, and immunoblotting using antibodies against the human PKA catalytic subunit α. Unfortunately, most of the expressed TeqPKAC1 was highly insoluble. Polypeptides of 36-38 kDa and 45-50 kDa were predominantly seen by immunoblotting in the bacterial particulate and cytosolic fractions, respectively. Since the incorporation of either 4% Triton X-100 or 3% sarkosyl or a mixture of 10 mM MgCl2 and 1 mM ATP (MgATP) improved the solubilization of TeqPKAC1, we used a combination of Triton X-100, sarkosyl and MgATP to solubilize the recombinant protein. TeqPKAC1 was purified by first reconstituting a hybrid holoenzyme between the recombinant protein and a mammalian poly-His-tagged PKA regulatory subunit that was immobilized on a Ni2+-chelating affinity resin, and then by eluting TeqPKAC1 using cAMP. TeqPKAC1 was functional given that it was capable of phosphorylating PKA catalytic subunit substrates, such as kemptide (LRRASLG), histone type II-AS, and the peptide SP20 (TTYADFIASGRTGRRNSIHD), and was inhibited by the peptide IP20 (TTYADFIASGRTGRRNAIHD), which contains the inhibitory motif of the PKA-specific heat-stable inhibitor PKI-α. Optimal enzymatic activity was obtained at 37 °C and pH 8.0-9.0; and the order of effectiveness of nucleotide triphosphates and divalent cations was ATP ¼ GTP â‰… ITP and Mg2+ â‰… Mn2+ â‰… Fe2+ ¼ Ca2+ â‰… Zn2, respectively.

Phosphorylation of human CEACAM1-LF by PKA and GSK3ß promotes its interaction with ß-catenin.

CEACAM1-LF, a homotypic cell adhesion adhesion molecule, transduces intracellular signals via a 72 amino acid cytoplasmic domain that contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a binding site for ß-catenin. Phosphorylation of Ser503 by PKC in rodent CEACAM1 was shown to affect bile acid transport or hepatosteatosis via the level of ITIM phosphorylation, but the phosphorylation of the equivalent residue in human CEACAM1 (Ser508) was unclear. Here we studied this analogous phosphorylation by NMR analysis of the 15N labeled cytoplasmic domain peptide. Incubation with a variety of Ser/Thr kinases revealed phosphorylation of Ser508 by GSK3bß but not by PKC. The lack of phosphorylation by PKC is likely due to evolutionary sequence changes between the rodent and human genes. Phosphorylation site assignment by mass spectrometry and NMR revealed phosphorylation of Ser472, Ser461 and Ser512 by PKA, of which Ser512 is part of a conserved consensus site for GSK3ß binding. We showed here that only after phosphorylation of Ser512 by PKA was GSK3ß able to phosphorylate Ser508. Phosphorylation of Ser512 by PKA promoted a tight association with the armadillo repeat domain of ß-catenin at an extended region spanning the ITIMs of CEACAM1. The kinetics of phosphorylation of the ITIMs by Src, as well dephosphorylation by SHP2, were affected by the presence of Ser508/512 phosphorylation, suggesting that PKA and GSK3ß may regulate the signal transduction activity of human CEACAM1-LF. The interaction of CEACAM1-LF with ß-catenin promoted by PKA is suggestive of a tight association between the two ITIMs of CEACAM1-LF.

KARATE: PKA-induced KRAS4B-RHOA-mTORC2 supercomplex phosphorylates AKT in insulin signaling and glucose homeostasis.

AKT is a serine/threonine kinase that plays an important role in metabolism, cell growth, and cytoskeletal dynamics. AKT is activated by two kinases, PDK1 and mTORC2. Although the regulation of PDK1 is well understood, the mechanism that controls mTORC2 is unknown. Here, by investigating insulin receptor signaling in human cells and biochemical reconstitution, we found that insulin induces the activation of mTORC2 toward AKT by assembling a supercomplex with KRAS4B and RHOA GTPases, termed KARATE (KRAS4B-RHOA-mTORC2 Ensemble). Insulin-induced KARATE assembly is controlled via phosphorylation of GTP-bound KRAS4B at S181 and GDP-bound RHOA at S188 by protein kinase A. By developing a KARATE inhibitor, we demonstrate that KRAS4B-RHOA interaction drives KARATE formation. In adipocytes, KARATE controls insulin-dependent translocation of the glucose transporter GLUT4 to the plasma membrane for glucose uptake. Thus, our work reveals a fundamental mechanism that activates mTORC2 toward AKT in insulin-regulated glucose homeostasis.

Neuroprotective effects of NDEELNK from sea cucumber ovum against scopolamine-induced PC12 cell damage through enhancing energy metabolism and upregulation of the PKA/BDNF/NGF signaling pathway.

The aim of the study was to evaluate the neuroprotective function of sea cucumber ovum peptide-derived NDEELNK and explore the underlying molecular mechanisms. NDEELNK exerted the neuroprotective effect by improving the acetylcholine (ACh) level and reducing the acetylcholinesterase (AChE) activity in PC12 cells. By molecular docking, we confirmed that the NDEELNK backbone and AChE interacted through hydrophobic and hydrogen bonds in contact with the amino acid residues of the cavity wall. NDEELNK increased superoxide dismutase (SOD) activity and decreased reactive oxygen species (ROS) production, thereby reducing mitochondrial dysfunction and enhancing energy metabolism. Our results demonstrated that NDEELNK supplementation alleviated scopolamine-induced PC12 cell damage by improving the cholinergic system, increasing energy metabolism and upregulating the expression of phosphorylated protein kinase A (p-PKA), brain-derived neurotrophic factor (BNDF) and nerve growth factor (NGF) signaling proteins in in vitro experiments. These results demonstrated that the sea cucumber ovum peptide-derived NDEELNK might play a protective role in PC12 cells.

Is Disrupted Nucleotide-Substrate Cooperativity a Common Trait for Cushing's Syndrome Driving Mutations of Protein Kinase A?

Somatic mutations in the PRKACA gene encoding the catalytic α subunit of protein kinase A (PKA-C) are responsible for cortisol-producing adrenocortical adenomas. These benign neoplasms contribute to the development of Cushing's syndrome. The majority of these mutations occur at the interface between the two lobes of PKA-C and interfere with the enzyme's ability to recognize substrates and regulatory (R) subunits, leading to aberrant phosphorylation patterns and activation. Rarely, patients with similar phenotypes carry an allosteric mutation, E31V, located at the C-terminal end of the αA-helix and adjacent to the αC-helix, but structurally distinct from the PKA-C/R subunit interface mutations. Using a combination of solution NMR, thermodynamics, kinetic assays, and molecular dynamics simulations, we show that the E31V allosteric mutation disrupts central communication nodes between the N- and C- lobes of the enzyme as well as nucleotide-substrate binding cooperativity, a hallmark for kinases' substrate fidelity and regulation. For both orthosteric (L205R and W196R) and allosteric (E31V) Cushing's syndrome mutants, the loss of binding cooperativity is proportional to the density of the intramolecular allosteric network. This structure-activity relationship suggests a possible common mechanism for Cushing's syndrome driving mutations in which decreased nucleotide/substrate binding cooperativity is linked to loss in substrate fidelity and dysfunctional regulation.

Beyond PKA: Evolutionary and structural insights that define a docking and dimerization domain superfamily.

Protein-interaction domains can create unique macromolecular complexes that drive evolutionary innovation. By combining bioinformatic and phylogenetic analyses with structural approaches, we have discovered that the docking and dimerization (D/D) domain of the PKA regulatory subunit is an ancient and conserved protein fold. An archetypal function of this module is to interact with A-kinase-anchoring proteins (AKAPs) that facilitate compartmentalization of this key cell-signaling enzyme. Homology searching reveals that D/D domain proteins comprise a superfamily with 18 members that function in a variety of molecular and cellular contexts. Further in silico analyses indicate that D/D domains segregate into subgroups on the basis of their similarity to type I or type II PKA regulatory subunits. The sperm autoantigenic protein 17 (SPA17) is a prototype of the type II or R2D2 subgroup that is conserved across metazoan phyla. We determined the crystal structure of an extended D/D domain from SPA17 (amino acids 1-75) at 1.72 Å resolution. This revealed a four-helix bundle-like configuration featuring terminal ß-strands that can mediate higher order oligomerization. In solution, SPA17 forms both homodimers and tetramers and displays a weak affinity for AKAP18. Quantitative approaches reveal that AKAP18 binding occurs at nanomolar affinity when SPA17 heterodimerizes with the ropporin-1-like D/D protein. These findings expand the role of the D/D fold as a versatile protein-interaction element that maintains the integrity of macromolecular architectures within organelles such as motile cilia.

A most versatile kinase: The catalytic subunit of PKA in T-cell biology.

The cAMP-dependent protein kinase, more commonly referred to as protein kinase A (PKA), is one of the most-studied enzymes in biology. PKA is ubiquitously expressed in mammalian cells, can be activated in response to a plethora of biological stimuli, and phosphorylates more than 250 known substrates. Indeed, PKA is of central importance to a wide range of organismal processes, including energy homeostasis, memory formation and immunity. It serves as the primary effector of the second-messenger molecule 3',5'-cyclic adenosine monophosphate (cAMP), which is believed to have mostly inhibitory effects on the adaptive immune response. In particular, elevated levels of intracellular cAMP inhibit the activation of conventional T cells by limiting signal transduction through the T-cell receptor and altering gene expression, primarily in a PKA-dependent manner. Regulatory T cells have been shown to increase the cAMP levels in adjacent T cells by direct and indirect means, but the role of cAMP within regulatory T cells themselves remains incompletely understood. Paradoxically, cAMP has been implicated in promoting T-cell activation as well, adding another functional dimension beyond its established immunosuppressive effects. Furthermore, PKA can phosphorylate the NF-κB subunit p65, a transcription factor that is essential for T-cell activation, independently of cAMP. This phosphorylation of p65 drastically enhances NF-κB-dependent transcription and thus is likely to facilitate immune activation. How these immunosuppressive and immune-activating properties of PKA balance in vivo remains to be elucidated. This review provides a brief overview of PKA regulation, its ability to affect NF-κB activation, and its diverse functions in T-cell biology.

From structure to the dynamic regulation of a molecular switch: A journey over 3 decades.

It is difficult to imagine where the signaling community would be today without the Protein Data Bank. This visionary resource, established in the 1970s, has been an essential partner for sharing information between academics and industry for over 3 decades. We describe here the history of our journey with the protein kinases using cAMP-dependent protein kinase as a prototype. We summarize what we have learned since the first structure, published in 1991, why our journey is still ongoing, and why it has been essential to share our structural information. For regulation of kinase activity, we focus on the cAMP-binding protein kinase regulatory subunits. By exploring full-length macromolecular complexes, we discovered not only allostery but also an essential motif originally attributed to crystal packing. Massive genomic data on disease mutations allows us to now revisit crystal packing as a treasure chest of possible protein:protein interfaces where the biological significance and disease relevance can be validated. It provides a new window into exploring dynamic intrinsically disordered regions that previously were deleted, ignored, or attributed to crystal packing. Merging of crystallography with cryo-electron microscopy, cryo-electron tomography, NMR, and millisecond molecular dynamics simulations is opening a new world for the signaling community where those structure coordinates, deposited in the Protein Data Bank, are just a starting point!

The crystal structure of yeast regulatory subunit reveals key evolutionary insights into Protein Kinase A oligomerization.

Protein Kinase A (PKA) is a widespread enzyme that plays a key role in many signaling pathways from lower eukaryotes to metazoans. In mammals, the regulatory (R) subunits sequester and target the catalytic (C) subunits to proper subcellular locations. This targeting is accomplished by the dimerization and docking (D/D) domain of the R subunits. The activation of the holoenzyme depends on the binding of the second messenger cAMP. The only available structures of the D/D domain proceed from mammalian sources. Unlike dimeric mammalian counterparts, the R subunit from Saccharomyces cerevisiae (Bcy1) forms tetramers in solution. Here we describe the first high-resolution structure of a non-mammalian D/D domain. The tetramer in the crystals of the Bcy1 D/D domain is a dimer of dimers that retain the classical D/D domain fold. By using phylogenetic and structural analyses combined with site-directed mutagenesis, we found that fungal R subunits present an insertion of a single amino acid at the D/D domain that shifts the position of a downstream, conserved arginine. This residue participates in intra-dimer interactions in mammalian D/D domains, while due to this insertion it is involved in inter-dimer contacts in Bcy1, which are crucial for the stability of the tetramer. This surprising finding challenges well-established concepts regarding the oligomeric state within the PKAR protein family and provides important insights into the yet unexplored structural diversity of the D/D domains and the molecular determinants of R subunit oligomerization.

Myristoylation alone is sufficient for PKA catalytic subunits to associate with the plasma membrane to regulate neuronal functions.

Myristoylation is a posttranslational modification that plays diverse functional roles in many protein species. The myristate moiety is considered insufficient for protein-membrane associations unless additional membrane-affinity motifs, such as a stretch of positively charged residues, are present. Here, we report that the electrically neutral N-terminal fragment of the protein kinase A catalytic subunit (PKA-C), in which myristoylation is the only functional motif, is sufficient for membrane association. This myristoylation can associate a fraction of PKA-C molecules or fluorescent proteins (FPs) to the plasma membrane in neuronal dendrites. The net neutral charge of the PKA-C N terminus is evolutionally conserved, even though its membrane affinity can be readily tuned by changing charges near the myristoylation site. The observed membrane association, while moderate, is sufficient to concentrate PKA activity at the membrane by nearly 20-fold and is required for PKA regulation of AMPA receptors at neuronal synapses. Our results indicate that myristoylation may be sufficient to drive functionally significant membrane association in the absence of canonical assisting motifs. This provides a revised conceptual base for the understanding of how myristoylation regulates protein functions.

Reversible phosphorylation of a protein from Trypanosoma equiperdum that exhibits homology with the regulatory subunits of mammalian cAMP-dependent protein kinases.

Homologous proteins of the cAMP-dependent protein kinase (PKA) regulatory and catalytic subunits have been identified in Trypanosoma equiperdum (TeqR-like and TeqC-like, respectively). Partially purified TeqR-like from parasites isolated in the presence of glucose migrated as an apparent 55 kDa/57 kDa polypeptide doublet when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, a single polypeptide of 57 kDa was obtained when parasites were deprived of glucose, a condition that has been shown to activate a TeqC-like enzyme. As revealed by immunoblots using anti-phospothreonine antibodies, the 57 kDa band corresponded to a form of TeqR-like that was phosphorylated in threonine residues. TeqR-like phosphorylation was reversible since the level of phospho-TeqR-like decreased once glucose was readded to glucose starved-parasites. Dephospho- and phospho-TeqR-like proteins are monomers with native molecular masses of 54.93-57.41 kDa, Stokes radii of 3.42-3.37 nm, and slightly asymmetric shapes (frictional ratio f/fo = 1.36-1.32). A protein kinase of ∼40 kDa was also partially purified from glucose deprived-trypanosomes, which corresponded to the TeqC-like enzyme by its ability to phosphorylate kemptide, its inhibition by PKA-specific inhibitors, and its immunorecognition by anti-PKA catalytic subunit antibodies. TeqR-like and TeqC-like did not coelute following anion-exchange chromatography, revealing that these proteins are not associated forming a complex in T. equiperdum. Yet, when TeqR-like was incubated in vitro with TeqC-like in the presence of Mg2+ and ATP, the 55 kDa dephospho form of the 55kDa/57 kDa polypeptide doublet of TeqR-like was converted into the 57 kDa phospho form, demonstrating that TeqR-like is a substrate for TeqC-like.

Inhibitor binding to mutants of protein kinase A with GGGxxG and GxGxxA glycine-rich loop motifs.

The conserved GxGxxG motif of protein kinases forms a beta turn at the tip of the flexible glycine-rich loop and creates much of the ATP pocket binding surface. Notable exceptions to this sequence include GGGxxG in ABL kinase and GxGxxA in protein kinase C isoforms. We constructed the corresponding mutants of PKA, T51G, and G55A, and tested quinazoline inhibitors that were designed to bind via glycine-rich loop interactions, testing also staurosporine for comparison. The quinazoline inhibitors have significantly reduced binding strengths in both mutants. In striking contrast to these results, the binding of the "pan-kinome" inhibitor staurosporine is strengthened in the mutants. Surface plasmon resonance (SPR) shows that the tightened binding of staurosporine arises from increased kon rates, changes not offset by more moderately increased koff rates. The SPR results fit best to a two step binding process for staurosporine in wild type PKA, but not the mutants.

A prion-like domain of Tpk2 catalytic subunit of protein kinase A modulates P-body formation in response to stress in budding yeast.

Low complexity regions are involved in the assembly and disassembly of P-bodies (PBs). Saccharomyces cerevisiae contains three genes encoding the protein kinase A (PKA) catalytic subunit: TPK1, TPK2 and TPK3. Tpk2 and Tpk3 isoforms localize to PBs upon glucose starvation showing different mechanisms and kinetics of accumulation. In contrast to the other two isoforms, Tpk2 harbors a glutamine-rich prion-like domain (PrLD) at the N-terminus. Here we show that the appearance of Tpk2 foci in response to glucose starvation, heat stress or stationary phase was dependent on its PrLD. Moreover, the PrLD of Tpk2 was necessary for efficient PB and stress granule aggregation during stress conditions and in quiescent cells. Deletion of PrLD does not affect the in vitro and in vivo kinase activity of Tpk2 or its interaction with the regulatory subunit Bcy1. We present evidence that the PrLD of Tpk2 serves as a scaffold domain for PB assembly in a manner that is independent of Pat1 phosphorylation by PKA. In addition, a mutant strain where Tpk2 lacks PrLD showed a decrease of turnover of mRNA during glucose starvation. This work therefore provides new insight into the mechanism of stress-induced cytoplasmic mRNP assembly, and the role of isoform specific domains in the regulation of PKA catalytic subunit specificity and dynamic localization to cytoplasmic RNPs granules.

Two Methods, One Goal: Structural Differences between Cocrystallization and Crystal Soaking to Discover Ligand Binding Poses.

In lead optimization, protein crystallography is an indispensable tool to analyze drug binding. Binding modes and non-covalent interaction inventories are essential to design follow-up synthesis candidates. Two protocols are commonly applied to produce protein-ligand complexes: cocrystallization and soaking. Because of its time and cost effectiveness, soaking is the more popular method. Taking eight ligand hinge binders of protein kinase A, we demonstrate that cocrystallization is superior. Particularly for flexible proteins, such as kinases, and larger ligands cocrystallization captures more reliable the correct binding pose and induced protein adaptations. The geometrical discrepancies between soaking and cocrystallization appear smaller for fragment-sized ligands. For larger flexible ligands that trigger conformational changes of the protein, soaking can be misleading and underestimates the number of possible polar interactions due to inadequate, highly impaired positions of protein amino-acid side and main chain atoms. Thus, if applicable cocrystallization should be the gold standard to study protein-ligand complexes.