Eugen Franz

Isoform-selective disruption of AKAP-localized PKA using hydrocarbon stapled peptides.

A-kinase anchoring proteins (AKAPs) play an important role in the spatial and temporal regulation of protein kinase A (PKA) by scaffolding critical intracellular signaling complexes. Here we report the design of conformationally constrained peptides that disrupt interactions between PKA and AKAPs in an isoform-selective manner. Peptides derived from the A Kinase Binding (AKB) domain of several AKAPs were chemically modified to contain an all-hydrocarbon staple and target the docking/dimerization domain of PKA-R, thereby occluding AKAP interactions. The peptides are cell-permeable against diverse human cell lines, are highly isoform-selective for PKA-RII, and can effectively inhibit interactions between AKAPs and PKA-RII in intact cells. These peptides can be applied as useful reagents in cell-based studies to selectively disrupt AKAP-localized PKA-RII activity and block AKAP signaling complexes. In summary, the novel hydrocarbon-stapled peptides developed in this study represent a new class of AKAP disruptors to study compartmentalized RII-regulated PKA signaling in cells.

PKA-Type I Selective Constrained Peptide Disruptors of AKAP Complexes

A-Kinase Anchoring Proteins (AKAPs) coordinate complex signaling events by serving as spatiotemporal modulators of cAMP-dependent protein kinase activity in cells. Although AKAPs organize a plethora of diverse pathways, their cellular roles are often elusive due to the dynamic nature of these signaling complexes. AKAPs can interact with the type I or type II PKA holoenzymes by virtue of high-affinity interactions with the R-subunits. As a means to delineate AKAP-mediated PKA signaling in cells, we sought to develop isoform-selective disruptors of AKAP signaling. Here, we report the development of conformationally constrained peptides named RI-STapled Anchoring Disruptors (RI-STADs) that target the docking/dimerization domain of the type 1 regulatory subunit of PKA. These high-affinity peptides are isoform-selective for the RI isoforms, can outcompete binding by the classical AKAP disruptor Ht31, and can selectively displace RIα, but not RIIα, from binding the dual-specific AKAP149 complex. Importantly, these peptides are cell-permeable and disrupt Type I PKA-mediated phosphorylation events in the context of live cells. Hence, RI-STAD peptides are versatile cellular tools to selectively probe anchored type I PKA signaling events.

Crystal Structures of the Carboxyl cGMP Binding Domain of the Plasmodium falciparum cGMP-dependent Protein Kinase Reveal a Novel Capping Triad Crucial for Merozoite Egress.

The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is a key regulator across the malaria parasite life cycle. Little is known about PfPKG’s activation mechanism. Here we report that the carboxyl cyclic nucleotide binding domain functions as a “gatekeeper” for activation by providing the highest cGMP affinity and selectivity. To understand the mechanism, we have solved its crystal structures with and without cGMP at 2.0 and 1.9 Å, respectively. These structures revealed a PfPKG-specific capping triad that forms upon cGMP binding, and disrupting the triad reduces kinase activity by 90%. Furthermore, mutating these residues in the parasite prevents blood stage merozoite egress, confirming the essential nature of the triad in the parasite. We propose a mechanism of activation where cGMP binding allosterically triggers the conformational change at the αC-helix, which bridges the regulatory and catalytic domains, causing the capping triad to form and stabilize the active conformation.

Structural Basis of Analog Specificity in PKG I and II.

Cyclic GMP analogs, 8-Br, 8-pCPT, and PET-cGMP, have been widely used for characterizing cellular functions of cGMP-dependent protein kinase (PKG) I and II isotypes. However, interpreting results obtained using these analogs has been difficult due to their low isotype specificity. Additionally, each isotype has two binding sites with different cGMP affinities and analog selectivities, making understanding the molecular basis for isotype specificity of these compounds even more challenging. To determine isotype specificity of cGMP analogs and their structural basis, we generated the full-length regulatory domains of PKG I and II isotypes with each binding site disabled, determined their affinities for these analogs, and obtained cocrystal structures of both isotypes bound with cGMP analogs. Our affinity and activation measurements show that PET-cGMP is most selective for PKG I, whereas 8-pCPT-cGMP is most selective for PKG II. Our structures of cyclic nucleotide binding (CNB) domains reveal that the B site of PKG I is more open and forms a unique π/π interaction through Arg285 at β4 with the PET moiety, whereas the A site of PKG II has a larger β5/β6 pocket that can better accommodate the bulky 8-pCPT moiety. Our structural and functional results explain the selectivity of these analogs for each PKG isotype and provide a starting point for the rational design of isotype selective activators.

Transforming PKA into PKG – a structure-function approach to understand cyclic nucleotide selectivity

BackgroundcAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) are the main effectors ofdistinct cyclic nucleotide pathways and are preferentiallyactivated by cAMP or cGMP, respectively.We recently characterized the isolated C-terminal cyc-lic nucleotide binding domain (CNB-B) of the humanPKG Ib as highly cGMP-selective (manuscript in pre-paration). In a crystal structure of the CNB-B two novelcGMP-specific interaction sites were identified in addi-tion to the previously described threonine residue (T317)in the phosphate binding cassette [1]. Mutation of eachindividual site resulted in reduced cGMP-selectivity andinterfered with cGMP-dependent activation of PKG Ib.To gain further insight into the molecular basis of cyc-lic nucleotide selectivity, we inserted two cGMP-specificinteraction sites into the CNB-B of human PKA RIa bymutating corresponding residues. We hypothesize thatthis way cGMP-specific interaction contacts can be cre-ated in PKA and thereby modulate cAMP-selectivity[1,2].ResultsWe characterized a deletion construct of the PKA hRIaCNB-B as cAMP-selective using fluorescence polarization(FP) and surface plasmon resonance (SPR).In comparison to the wildtype PKA hRIa CNB-B, singlemutant constructs showed similar affinities for cAMP-and cGMP-analogs, revealing a loss of selectivity. Thecombination of two mutations led to a construct withhigher affinity for cGMP compared to cAMP.Co-crystal structures of this double mutant with cAMPor cGMP, respectively, showed that the cGMP-specificinteraction contacts retained their function in the contextof the PKA hRIa CNB-B.ConclusionThegeneralstructureofcyclicnucleotidebindingdomains is conserved. However, varying amino acids inthe binding pocket enable the distinction between cAMPand cGMP. Here we show that cGMP interaction sitesfound in PKG do restore their specific binding mechan-isms when introduced into PKA.The results underline the relevance of the describednovel binding sites in mediating cGMP-selectivity. Still,other features of CNB domains involved in the specificbinding mechanism as well as the detailed mechanism ofkinase activation need to be investigated.

Crystal structures of the carboxyl cGMP binding domain of plasmodium falciparum cGMP- dependent protein kinase reveals a novel salt bridge crucial for activation

Background Plasmodium falciparum cGMP-dependent protein kinase (pfPKG) is a validated therapeutic target of malaria. As a key regulator of its life cycle, pfPKG plays a crucial role in both the sexual and asexual blood-stages that cause malaria pathology. Inhibiting pfPKG blocks proliferation and transmission of the parasite [1,2]. However the development of pfPKG-specific inhibitor has been greatly hampered by the lack of high-resolution structure information to guide drug design. Targeting the ATP binding site of pfPKG is an approach commonly associated with low specificity and toxicity [3]. Therefore, we aim to target a domain that is unique to this kinase, the cyclic nucleotide binding (CNB) domain. Since previous studies demonstrated the fourth-cyclic nucleotide binding (CNB-D) domain of pfPKG to be the most important for the kinase activation [4] we focused on this domain to understand its role in cGMP dependent activation.

cGMP Binding Domain D Mediates a Unique Activation Mechanism in Plasmodium falciparum PKG

cGMP-dependent protein kinase from Plasmodium falciparum ( PfPKG) plays a crucial role in the sexual as well as the asexual proliferation of this human malaria causing parasite. However, function and regulation of PfPKG are largely unknown. Previous studies showed that the domain organization of PfPKG significantly differs from human PKG ( hPKG) and indicated a critical role of the cyclic nucleotide binding domain D (CNB-D). We identified a novel mechanism, where the CNB-D controls activation and regulation of the parasite specific protein kinase. Here, kinase activity is not dependent on a pseudosubstrate autoinhibitory sequence (IS), as reported for human PKG. A construct lacking the putative IS and containing only the CNB-D and the catalytic domain is inactive in the absence of cGMP and can efficiently be activated with cGMP. On the basis of structural evidence, we describe a regulatory mechanism, whereby cGMP binding to CNB-D induces a conformational change involving the αC-helix of the CNB-D. The inactive state is defined by a unique interaction between Asp597 of the catalytic domain and Arg528 of the αC-helix. The same arginine (R528), however, stabilizes cGMP binding by interacting with Tyr480 of the phosphate binding cassette (PBC). This represents the active state of PfPKG. Our results unveil fundamental differences in the activation mechanism between PfPKG and hPKG, building the basis for the development of strategies for targeted drug design in fighting malaria.

The role of a parasite-specific D-site in activation of Plasmodium falciparum cGMP-dependent protein kinase

Background Malaria is one of the most dangerous tropical diseases worldwide, resulting in approximately 1.5-2.7 million deaths per year [1]. Furthermore, malaria belongs to the four major infectious diseases also including HIV, tuberculosis and hepatitis. In humans, malaria is transmitted by four species of the genus Plasmodium. However, most malaria deaths are caused by Plasmodium falciparum [2]. The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is one of the key regulators of the malaria parasite life cycle in both sexual and asexual blood-stages. Inhibition of PfPKG stops differentiation and transmission of the parasites, indicating that this kinase is a promising drug target for malaria [3-5]. However, despite its physiological importance, the activation mechanism of PfPKG is not fully understood. Recently, our group discovered that disrupting cGMP binding at the C-terminal cyclic nucleotide-binding (CNB-D) domain almost completely abolishes the kinase activation [6]. Therefore, we investigate the functional role of the PfCNB-D in PfPKG activation.