Jeong Joo Kim

Recurrent Gain-of-Function Mutation in PRKG1 Causes Thoracic Aortic Aneurysms and Acute Aortic Dissections

Gene mutations that lead to decreased contraction of vascular smooth-muscle cells (SMCs) can cause inherited thoracic aortic aneurysms and dissections. Exome sequencing of distant relatives affected by thoracic aortic disease and subsequent Sanger sequencing of additional probands with familial thoracic aortic disease identified the same rare variant, PRKG1 c.530G>A (p.Arg177Gln), in four families. This mutation segregated with aortic disease in these families with a combined two-point LOD score of 7.88. The majority of affected individuals presented with acute aortic dissections (63%) at relatively young ages (mean 31 years, range 17-51 years). PRKG1 encodes type I cGMP-dependent protein kinase (PKG-1), which is activated upon binding of cGMP and controls SMC relaxation. Although the p.Arg177Gln alteration disrupts binding to the high-affinity cGMP binding site within the regulatory domain, the altered PKG-1 is constitutively active even in the absence of cGMP. The increased PKG-1 activity leads to decreased phosphorylation of the myosin regulatory light chain in fibroblasts and is predicted to cause decreased contraction of vascular SMCs. Thus, identification of a gain-of-function mutation in PRKG1 as a cause of thoracic aortic disease provides further evidence that proper SMC contractile function is critical for maintaining the integrity of the thoracic aorta throughout a lifetime.

Co-Crystal Structures of PKG Iβ (92–227) with cGMP and cAMP Reveal the Molecular Details of Cyclic-Nucleotide Binding

Background Cyclic GMP-dependent protein kinases (PKGs) are central mediators of the NO-cGMP signaling pathway and phosphorylate downstream substrates that are crucial for regulating smooth muscle tone, platelet activation, nociception and memory formation. As one of the main receptors for cGMP, PKGs mediate most of the effects of cGMP elevating drugs, such as nitric oxide-releasing agents and phosphodiesterase inhibitors which are used for the treatment of angina pectoris and erectile dysfunction, respectively. Methodology/Principal Findings We have investigated the mechanism of cyclic nucleotide binding to PKG by determining crystal structures of the amino-terminal cyclic nucleotide-binding domain (CNBD-A) of human PKG I bound to either cGMP or cAMP. We also determined the structure of CNBD-A in the absence of bound nucleotide. The crystal structures of CNBD-A with bound cAMP or cGMP reveal that cAMP binds in either syn or anti configurations whereas cGMP binds only in a syn configuration, with a conserved threonine residue anchoring both cyclic phosphate and guanine moieties. The structure of CNBD-A in the absence of bound cyclic nucleotide was similar to that of the cyclic nucleotide bound structures. Surprisingly, isothermal titration calorimetry experiments demonstrated that CNBD-A binds both cGMP and cAMP with a relatively high affinity, showing an approximately two-fold preference for cGMP. Conclusions/Significance Our findings suggest that CNBD-A binds cGMP in the syn conformation through its interaction with Thr193 and an unusual cis-peptide forming residues Leu172 and Cys173. Although these studies provide the first structural insights into cyclic nucleotide binding to PKG, our ITC results show only a two-fold preference for cGMP, indicating that other domains are required for the previously reported cyclic nucleotide selectivity.

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 cyclic nucleotide selectivity in cGMP dependent protein kinase II.

Membrane-bound cGMP-dependent protein kinase (PKG) II is a key regulator of bone growth, renin secretion, and memory formation. Despite its crucial physiological roles, little is known about its cyclic nucleotide selectivity mechanism due to a lack of structural information. Here, we find that the C-terminal cyclic nucleotide binding (CNB-B) domain of PKG II binds cGMP with higher affinity and selectivity when compared with its N-terminal CNB (CNB-A) domain. To understand the structural basis of cGMP selectivity, we solved co-crystal structures of the CNB domains with cyclic nucleotides. Our structures combined with mutagenesis demonstrate that the guanine-specific contacts at Asp-412 and Arg-415 of the αC-helix of CNB-B are crucial for cGMP selectivity and activation of PKG II. Structural comparison with the cGMP selective CNB domains of human PKG I and Plasmodium falciparum PKG (PfPKG) shows different contacts with the guanine moiety, revealing a unique cGMP selectivity mechanism for PKG II.

Long-Acting Beta Agonists Enhance Allergic Airway Disease.

Asthma is one of the most common of medical illnesses and is treated in part by drugs that activate the beta-2-adrenoceptor (β2-AR) to dilate obstructed airways. Such drugs include long acting beta agonists (LABAs) that are paradoxically linked to excess asthma-related mortality. Here we show that LABAs such as salmeterol and structurally related β2-AR drugs such as formoterol and carvedilol, but not short-acting agonists (SABAs) such as albuterol, promote exaggerated asthma-like allergic airway disease and enhanced airway constriction in mice. We demonstrate that salmeterol aberrantly promotes activation of the allergic disease-related transcription factor signal transducer and activator of transcription 6 (STAT6) in multiple mouse and human cells. A novel inhibitor of STAT6, PM-242H, inhibited initiation of allergic disease induced by airway fungal challenge, reversed established allergic airway disease in mice, and blocked salmeterol-dependent enhanced allergic airway disease. Thus, structurally related β2-AR ligands aberrantly activate STAT6 and promote allergic airway disease. This untoward pharmacological property likely explains adverse outcomes observed with LABAs, which may be overcome by agents that antagonize STAT6.

Construction of novel cGMP FRET-sensors based on PKG from Plasmodium falciparum

Background Several sensors for cyclic nucleotides have been developed the past decade. However, there are few sensors for cGMP available and even fewer that detect low concentrations of cGMP. Currently, only the CFP/YFP sensors Cygnet 2.1, cGES-DE5 and cGi-500 (EC50 ~1.7 μM, 1.5 μM and 500 nM, respectively) and the T-sapphire/Dimer2 sensor Red cGES-DE5 (EC50 ~40 nM) are available [1-3]. We aim to measure localized pools of cGMP in single adult cardiac myocytes, and have previously found that such sensors should have an EC50 less than 50 nM, thus excluding the CFP/YFP sensors above. The Red cGES-DE5 has an acceptable EC50, but tagging this sensor to locate in distinct compartments within cardiac myocytes yielded sensors with a smaller dynamic range. We therefore decided to construct new FRET-based cGMP-sensors with high affinity for cGMP that could be better candidates for subcellular localization.

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.

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.