Table_1_Binding of the Human 14-3-3 Isoforms to Distinct Sites in the Leucine-Rich Repeat Kinase 2.pdf

Proteins of the 14-3-3 family are well known modulators of the leucine-rich repeat kinase 2 (LRRK2) regulating kinase activity, cellular localization, and ubiquitylation. Although binding between those proteins has been investigated, a comparative study of all human 14-3-3 isoforms interacting with LRRK2 is lacking so far. In a comprehensive approach, we quantitatively analyzed the interaction between the seven human 14-3-3 isoforms and LRRK2-derived peptides covering both, reported and putative 14-3-3 binding sites. We observed that phosphorylation is an absolute prerequisite for 14-3-3 binding and generated binding patterns of 14-3-3 isoforms to interact with peptides derived from the N-terminal phosphorylation cluster (S910 and S935), the Roc domain (S1444) and the C-terminus. The tested 14-3-3 binding sites in LRRK2 preferentially were recognized by the isoforms γ and η, whereas the isoforms ϵ and especially σ showed the weakest or no binding. Interestingly, the possible pathogenic mutation Q930R in LRRK2 drastically increases binding affinity to a peptide encompassing pS935. We then identified the autophosphorylation site T2524 as a so far not described 14-3-3 binding site at the very C-terminus of LRRK2. Binding affinities of all seven 14-3-3 isoforms were quantified for all three binding regions with pS1444 displaying the highest affinity of all measured singly phosphorylated peptides. The strongest binding was detected for the combined phosphosites S910 and S935, suggesting that avidity effects are important for high affinity interaction between 14-3-3 proteins and LRRK2.</p

Cyclic Nucleotide Mapping of Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a central role in the regulation of cardiac and neuronal firing rate, and these channels can be dually activated by membrane hyperpolarization and by binding of cyclic nucleotides. cAMP has been shown to directly bind HCN channels and modulate their activity. Despite this, while there are selective inhibitors that block the activation potential of the HCN channels, regulation by cAMP analogs has not been well investigated. A comprehensive screen of 47 cyclic nucleotides with modifications in the nucleobase, ribose moiety, and cyclic phosphate was tested on the three isoforms HCN1, HCN2, and HCN4. 7-CH-cAMP was identified to be a high affinity binder for HCN channels and crosschecked for its ability to act on other cAMP receptor proteins. While 7-CH-cAMP is a general activator for cAMP- and cGMP-dependent protein kinases as well as for the guanine nucleotide exchange factors Epac1 and Epac2, it displays the highest affinity to HCN channels. The molecular basis of the high affinity was investigated by determining the crystal structure of 7-CH-cAMP in complex with the cyclic nucleotide binding domain of HCN4. Electrophysiological studies demonstrate a strong activation potential of 7-CH-cAMP for the HCN4 channel in vivo. So, this makes 7-CH-cAMP a promising activator of the HCN channels in vitro whose functionality can be translated in living cells

Data_Sheet_1.pdf

<p>S-adenosyl-L-homocysteine (SAH) hydrolases (SAHases) are involved in the regulation of methylation reactions in many organisms and are thus crucial for numerous cellular functions. Consequently, their dysregulation is associated with severe health problems. The SAHase-catalyzed reaction is reversible and both directions depend on the redox activity of nicotinamide adenine dinucleotide (NAD⁺) as a cofactor. Therefore, nicotinamide cofactor biomimetics (NCB) are a promising tool to modulate SAHase activity. In the present in vitro study, we investigated 10 synthetic truncated NAD⁺ analogs against a SAHase from the root-nodulating bacterium Bradyrhizobium elkanii. Among this set of analogs, one was identified to inhibit the SAHase in both directions. Isothermal titration calorimetry (ITC) and crystallography experiments suggest that the inhibitory effect is not mediated by a direct interaction with the protein. Neither the apo-enzyme (i.e., deprived of the natural cofactor), nor the holo-enzyme (i.e., in the NAD⁺-bound state) were found to bind the inhibitor. Yet, enzyme kinetics point to a non-competitive inhibition mechanism, where the inhibitor acts on both, the enzyme and enzyme-SAH complex. Based on our experimental results, we hypothesize that the NCB inhibits the enzyme via oxidation of the enzyme-bound NADH, which may be accessible through an open molecular gate, leaving the enzyme stalled in a configuration with oxidized cofactor, where the reaction intermediate can be neither converted nor released. Since the reaction mechanism of SAHase is quite uncommon, this kind of inhibition could be a viable pharmacological route, with a low risk of off-target effects. The NCB presented in this work could be used as a template for the development of more potent SAHase inhibitors.</p

Divalent Metal Ions Mg²⁺ and Ca²⁺ Have Distinct Effects on Protein Kinase A Activity and Regulation

cAMP-dependent protein kinase (PKA) is regulated primarily in response to physiological signals while nucleotides and metals may provide fine-tuning. PKA can use different metal ions for phosphoryl transfer, yet some, like Ca²⁺, do not support steady-state catalysis. Fluorescence Polarization (FP) and Surface Plasmon Resonance (SPR) were used to study inhibitor and substrate interactions with PKA. The data illustrate how metals can act differentially as a result of their inherent coordination properties. We found that Ca²⁺, in contrast to Mg²⁺, does not induce high-affinity binding of PKA to pseudosubstrate inhibitors. However, Ca²⁺ works in a single turnover mode to allow for phosphoryl-transfer. Using a novel SPR approach, we were able to directly monitor the interaction of PKA with a substrate in the presence of Mg²⁺ATP. This allows us to depict the entire kinase reaction including complex formation as well as release of the phosphorylated substrate. In contrast to Mg²⁺, Ca²⁺ apparently slows down the enzymatic reaction. A focus on individual reaction steps revealed that Ca²⁺ is not as efficient as Mg²⁺ in stabilizing the enzyme:substrate complex. The opposite holds true for product dissociation where Mg²⁺ easily releases the phospho-substrate while Ca²⁺ traps both reaction products at the active site. This explains the low steady-state activity in the presence of Ca²⁺. Furthermore, Ca²⁺ is able to modulate kinase activity as well as inhibitor binding even in the presence of Mg²⁺. We therefore hypothesize that the physiological metal ions Mg²⁺ and Ca²⁺ both play a role in kinase activity and regulation. Since PKA is localized close to calcium channels and may render PKA activity susceptible to Ca²⁺, our data provide a possible mechanism for novel crosstalk between cAMP and calcium signaling

Role of the unique capping triad at <i>Pf</i>CNB-D in <i>Pf</i>PKG activation and <i>Plasmodium</i> parasite life cycle.

<p>(A) Role of the capping motif forming residues in kinase activation. Individual curves with error bars denoting standard error of mean are shown on the left and corresponding <i>Ka</i> values for WT and capping triad mutants are shown on the right. Each data curve was normalized by designating the lowest value of the data set as 0% and the highest value as 100%. (B) The specific activities of the WT and mutants at 10 μM cGMP are shown as bar graphs with error bars denoting standard error of mean. (C) Immunoblot showing co-expression of endogenous <i>Pf</i>PKG and ectopic <i>Pf</i>PKG-HA in transgenic schizonts. 3D7/attB is the parental line and 3D7/PKG-HA is a previously established line where the endogenous <i>Pf</i>PKG gene has been HA-tagged. Blots were incubated with anti-HA, anti-human PKG, and anti-<i>Pf</i>GAPDH as a loading control. Note that the anti-human PKG antibody does not react with HA-tagged PKG (lane 2) because the free carboxyl terminus is crucial for antibody binding. This allows the differentiation between endogenous and HA-tagged PKG. (D) Growth of the transgenic lines in the presence of 1 μM compound 2 over 8 days. The established compound 2-resistant line 3D7/T618Q was included as a positive control. (E) Late trophozoites/early schizonts of the three transgenics were cultured for 12 hours in the presence of 1 μM compound 2 (lower panels) or DMSO (upper panels) and parasite development examined on Giemsa-stained blood smears. (F) Quantification of <i>(E)</i>. >1000 cells were counted for each culture and condition and ring stage parasitaemia determined. Data represent the mean of three experiments (error bars = SD). Dark grey bars are DMSO controls, light grey bars 1 μM compound 2 treated samples. For each sample, parasitaemia was adjusted to make the DMSO control 10% to eliminate variability from differences in parasitaemia between experiments. (G) Sequence alignment of apicomplexan PKGs. Conserved residues are shaded in yellow (identical), in green (functionally similar), and in gray (identical in >66%). The capping triad residues are typed in red and marked with arrows. <i>Toxoplasma gondii</i> PKG, TgPKG; <i>Theileria orientalis</i> PKG, ToPKG; <i>Eimeria tenella</i> PKG, EtPKG; <i>Babesia bovis</i> PKG, BbPKG, and <i>Cryptosporidium hominis</i>, ChPKG.</p

Conformational changes upon cGMP binding.

<p>Conformational changes are depicted in a step-wise fashion. The hydrophobic core, hinge, and cap forming residues are shown with transparent surface. Their side chain carbons are colored in black, oxygen in red, and nitrogen in blue.. (A) Step 1: PBC assuming a closed conformation upon cGMP binding. (B) Step 2: A cogwheel-like motion between the αP- and αB-helices allowing the αB-helix to move toward the PBC. Zoomed in views highlight changes in hydrophobic interactions. (C) Step 3: The N3A motif moving away from the PBC. (D) Step 4: The αC-helix shielding the bottom of the cGMP pocket and enabling the capping triad formation.</p

Domain organization and overall structures of <i>Pf</i>CNB-D.

<p>(A) Domain organization of <i>Pf</i>PKG and sequence alignment between <i>Pf</i>CNB-D and <i>Hs</i>CNB-B (Human PKG I). Identical residues are highlighted in yellow and the capping residues in both proteins are highlighted in red. The capping triad residues are also marked with arrows. (B) cGMP and cAMP affinities of <i>Pf</i>CNB domains. Competition FP curves for cGMP are shown on the left and EC₅₀ values on the right. (C) Overall structure of <i>Pf</i>CNB-D without cGMP. The secondary structure elements are labeled. The phosphate binding cassette (PBC) is colored in yellow, the αB and αC helices in light cyan and blue, the N-terminal helices in light green and the β-barrel in gray. The N- and C-termini are labeled with their corresponding residue number seen in the final model. The sulfate ion co-crystallized with the protein is colored with its sulfur in yellow and oxygen in red. (D) Overall structure of the <i>Pf</i>CNB-D:cGMP complex. The structure is shown with the same color scheme as above except for cGMP. The cGMP is colored by atom type (carbon, white; nitrogen, blue; oxygen, red; and phosphorus, orange). All structure images were generated using <i>PyMOL</i> (Delano Scientific).</p

cAMP-dependent signaling pathways as potential targets for inhibition of Plasmodium falciparum blood stages.

We review the role of signaling pathways in regulation of the key processes of merozoite egress and red blood cell invasion by Plasmodium falciparum and, in particular, the importance of the second messengers, cAMP and Ca2+, and cyclic nucleotide dependent kinases. cAMP-dependent protein kinase (PKA) is comprised of cAMP-binding regulatory, and catalytic subunits. The less well conserved cAMP-binding pockets should make cAMP analogs attractive drug leads, but this approach is compromised by the poor membrane permeability of cyclic nucleotides. We discuss how the conserved nature of ATP-binding pockets makes ATP analogs inherently prone to off-target effects and how ATP analogs and genetic manipulation can be useful research tools to examine this. We suggest that targeting PKA interaction partners as well as substrates, or developing inhibitors based on PKA interaction sites or phosphorylation sites in PKA substrates, may provide viable alternative approaches for the development of anti-malarial drugs. Proximity of PKA to a substrate is necessary for substrate phosphorylation, but the P. falciparum genome encodes few recognizable A-kinase anchor proteins (AKAPs), suggesting the importance of PKA-regulatory subunit myristylation and membrane association in determining substrate preference. We also discuss how Pf14-3-3 assembles a phosphorylation-dependent signaling complex that includes PKA and calcium dependent protein kinase 1 (CDPK1) and how this complex may be critical for merozoite invasion, and a target to block parasite growth. We compare altered phosphorylation levels in intracellular and egressed merozoites to identify potential PKA substrates. Finally, as host PKA may have a critical role in supporting intracellular parasite development, we discuss its role at other stages of the life cycle, as well as in other apicomplexan infections. Throughout our review we propose possible new directions for the therapeutic exploitation of cAMP-PKA-signaling in malaria and other diseases caused by apicomplexan parasites

Structural comparison between the apo- and cGMP bound <i>Pf</i>CNB-D.

<p>The apo and <i>Pf</i>CNB-D:cGMP complex structures are aligned at the β-barrel region (not colored). The helical subdomain of the apo structure is colored in light cyan and that of the cGMP complex structure in yellow.</p

Structural comparison between <i>Pf</i>CNB-D and CNB-B and cGMP binding pocket of <i>Pf</i>CNB-D.

<p>(A) The cGMP pockets <i>Pf</i>CNB-D and CNB-B from human PKG Iβ (PDB code: 4KU7) are shown. The cGMP pocket of <i>Pf</i>CNB-D is colored in yellow (left) and the pocket of PKG Iβ CNB-B in gray (right). Key residues that stabilize the C-helix including the capping residues are shown with transparent surface in the following color theme: side chain carbon, black; oxygen, red; nitrogen. A water molecule captured between E483, R484, and Q532 is shown as a blue sphere. The C atoms of glycine residues located between at the αB and αC helices are shown as black spheres. Hydrogen bonds are shown as dotted lines. (B) Detailed interactions between <i>Pf</i>CNB-D and cGMP. Zoomed in views for each cGMP binding site are shown on either side. The backbone amide of A485 is marked with a blue dot. The individual cGMP interacting residues are shown with the following color theme: side chain carbon, black; oxygen, red; nitrogen, blue. The residues binds cGMP with VDW contacts including the capping residues are shown with transparent surface. Hydrogen bonds are shown as dotted lines with their distances in Å units.</p