Reversible male contraception by targeted inhibition of serine/threonine kinase 33.

Men or mice with homozygous serine/threonine kinase 33 (STK33) mutations are sterile owing to defective sperm morphology and motility. To chemically evaluate STK33 for male contraception with STK33-specific inhibitors, we screened our multibillion-compound collection of DNA-encoded chemical libraries, uncovered potent STK33-specific inhibitors, determined the STK33 kinase domain structure bound with a truncated hit CDD-2211, and generated an optimized hit CDD-2807 that demonstrates nanomolar cellular potency (half-maximal inhibitory concentration = 9.2 nanomolar) and favorable metabolic stability. In mice, CDD-2807 exhibited no toxicity, efficiently crossed the blood-testis barrier, did not accumulate in brain, and induced a reversible contraceptive effect that phenocopied genetic STK33 perturbations without altering testis size. Thus, STK33 is a chemically validated, nonhormonal contraceptive target, and CDD-2807 is an effective tool compound.

Stereospecific NANOG PEST Stabilization by Pin1.

NANOG protein levels correlate with stem cell pluripotency. NANOG concentrations fluctuate constantly with low NANOG levels leading to spontaneous cell differentiation. Previous literature implicated Pin1, a phosphorylation-dependent prolyl isomerase, as a key player in NANOG stabilization. Here, using NMR spectroscopy, we investigate the molecular interactions of Pin1 with the NANOG unstructured N-terminal domain that contains a PEST sequence with two phosphorylation sites. Phosphorylation of NANOG PEST peptides increases affinity to Pin1. By systematically increasing the amount of cis PEST conformers, we show that the peptides bind tighter to the prolyl isomerase domain (PPIase) of Pin1. Phosphorylation and cis Pro enhancement at both PEST sites lead to a 5-10-fold increase in NANOG binding to the Pin1 WW domain and PPIase domain, respectively. The cis-populated NANOG PEST peptides can be potential inhibitors for disrupting Pin1-dependent NANOG stabilization in cancer stem cells.

An auto-inhibited state of protein kinase G and implications for selective activation.

Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cyclic guanosine monophosphate (cGMP) signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs, complementing drugs that target nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory (AI) pseudo-substrate sequences to PKG Iα and Iß that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is not well understood. Here, we present a crystal structure of PKG Iß in which the AI sequence and the cyclic nucleotide-binding (CNB) domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG Iß AI sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I CNB domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wildtype cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG Iß auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and cooperative activation of PKGs.

Discovery of potent BET bromodomain 1 stereoselective inhibitors using DNA-encoded chemical library selections.

BRDT, BRD2, BRD3, and BRD4 comprise the bromodomain and extraterminal (BET) subfamily which contain two similar tandem bromodomains (BD1 and BD2). Selective BD1 inhibition phenocopies effects of tandem BET BD inhibition both in cancer models and, as we and others have reported of BRDT, in the testes. To find novel BET BD1 binders, we screened >4.5 billion molecules from our DNA-encoded chemical libraries with BRDT-BD1 or BRDT-BD2 proteins in parallel. A compound series enriched only by BRDT-BD1 was resynthesized off-DNA, uncovering a potent chiral compound, CDD-724, with >2,000-fold selectivity for inhibiting BRDT-BD1 over BRDT-BD2. CDD-724 stereoisomers exhibited remarkable differences in inhibiting BRDT-BD1, with the R-enantiomer (CDD-787) being 50-fold more potent than the S-enantiomer (CDD-786). From structure­activity relationship studies, we produced CDD-956, which maintained picomolar BET BD1 binding potency and high selectivity over BET BD2 proteins and had improved stability in human liver microsomes over CDD-787. BROMOscan profiling confirmed the excellent pan-BET BD1 affinity and selectivity of CDD-787 and CDD-956 on BD1 versus BD2 and all other BD-containing proteins. A cocrystal structure of BRDT-BD1 bound with CDD-956 was determined at 1.82 Å and revealed BRDT-BD1­specific contacts with the αZ and αC helices that explain the high affinity and selectivity for BET BD1 versus BD2. CDD-787 and CDD-956 maintain cellular BD1-selectivity in NanoBRET assays and show potent antileukemic activity in acute myeloid leukemia cell lines. These BET BD1-specific and highly potent compounds are structurally unique and provide insight into the importance of chirality to achieve BET specificity.

Liquid condensation of reprogramming factor KLF4 with DNA provides a mechanism for chromatin organization.

Expression of a few master transcription factors can reprogram the epigenetic landscape and three-dimensional chromatin topology of differentiated cells and achieve pluripotency. During reprogramming, thousands of long-range chromatin contacts are altered, and changes in promoter association with enhancers dramatically influence transcription. Molecular participants at these sites have been identified, but how this re-organization might be orchestrated is not known. Biomolecular condensation is implicated in subcellular organization, including the recruitment of RNA polymerase in transcriptional activation. Here, we show that reprogramming factor KLF4 undergoes biomolecular condensation even in the absence of its intrinsically disordered region. Liquid-liquid condensation of the isolated KLF4 DNA binding domain with a DNA fragment from the NANOG proximal promoter is enhanced by CpG methylation of a KLF4 cognate binding site. We propose KLF4-mediated condensation as one mechanism for selectively organizing and re-organizing the genome based on the local sequence and epigenetic state.

Dynamical Basis of Allosteric Activation for the Plasmodium falciparum Protein Kinase G.

The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is required for the progression of the Plasmodium's life cycle and is therefore a promising malaria drug target. PfPKG includes four cGMP-binding domains (CBD-A to CBD-D). CBD-D plays a crucial role in PfPKG regulation as it is the primary determinant for the inhibition and cGMP-dependent activation of the catalytic domain. Hence, it is critical to understand how CBD-D is allosterically regulated by cGMP. Although the apo versus holo conformational changes of CBD-D have been reported, information on the intermediates of the activation pathway is currently lacking. Here, we employed molecular dynamics simulations to model four key states along the thermodynamic cycle for the cGMP-dependent activation of the PfPKG CBD-D domain. The simulations were compared to NMR data, and they revealed that the PfPKG CBD-D activation pathway samples a compact intermediate in which the N- and C-terminal helices approach the central ß-barrel. In addition, by comparing the cGMP-bound active and inactive states, the essential binding interactions that differentiate these states were identified. The identification of structural and dynamical features unique to the cGMP-bound inactive state provides a promising basis to design PfPKG-selective allosteric inhibitors as a viable treatment for malaria.

Discovery and characterization of bromodomain 2-specific inhibitors of BRDT.

Bromodomain testis (BRDT), a member of the bromodomain and extraterminal (BET) subfamily that includes the cancer targets BRD2, BRD3, and BRD4, is a validated contraceptive target. All BET subfamily members have two tandem bromodomains (BD1 and BD2). Knockout mice lacking BRDT-BD1 or both bromodomains are infertile. Treatment of mice with JQ1, a BET BD1/BD2 nonselective inhibitor with the highest affinity for BRD4, disrupts spermatogenesis and reduces sperm number and motility. To assess the contribution of each BRDT bromodomain, we screened our collection of DNA-encoded chemical libraries for BRDT-BD1 and BRDT-BD2 binders. High-enrichment hits were identified and resynthesized off-DNA and examined for their ability to compete with JQ1 in BRDT and BRD4 bromodomain AlphaScreen assays. These studies identified CDD-1102 as a selective BRDT-BD2 inhibitor with low nanomolar potency and >1,000-fold selectivity over BRDT-BD1. Structure-activity relationship studies of CDD-1102 produced a series of additional BRDT-BD2/BRD4-BD2 selective inhibitors, including CDD-1302, a truncated analog of CDD-1102 with similar activity, and CDD-1349, an analog with sixfold selectivity for BRDT-BD2 versus BRD4-BD2. BROMOscan bromodomain profiling confirmed the great affinity and selectivity of CDD-1102 and CDD-1302 on all BET BD2 versus BD1 with the highest affinity for BRDT-BD2. Cocrystals of BRDT-BD2 with CDD-1102 and CDD-1302 were determined at 2.27 and 1.90 Å resolution, respectively, and revealed BRDT-BD2 specific contacts that explain the high affinity and selectivity of these compounds. These BD2-specific compounds and their binding to BRDT-BD2 are unique compared with recent reports and enable further evaluation of their nonhormonal contraceptive potential in vitro and in vivo.

Cyclic nucleotide selectivity of protein kinase G isozymes.

The intrinsic activity of the C-terminal catalytic (C) domain of cyclic guanosine monophosphate (cGMP)-dependent protein kinases (PKG) is inhibited by interactions with the N-terminal regulatory (R) domain. Selective binding of cGMP to cyclic nucleotide binding (CNB) domains within the R-domain disrupts the inhibitory R-C interaction, leading to the release and activation of the C-domain. Affinity measurements of mammalian and plasmodium PKG CNB domains reveal different degrees of cyclic nucleotide affinity and selectivity; the CNB domains adjacent to the C-domain are more cGMP selective and therefore critical for cGMP-dependent activation. Crystal structures of isolated CNB domains in the presence and absence of cyclic nucleotides reveal isozyme-specific contacts that explain cyclic nucleotide selectivity and conformational changes that accompany CNB. Crystal structures of tandem CNB domains identify two types of CNB-mediated dimeric contacts that indicate cGMP-driven reorganization of domain-domain interfaces that include large conformational changes. Here, we review the available structural and functional information of PKG CNB domains that further advance our understanding of cGMP mediated regulation and activation of PKG isozymes.

A substitution in cGMP-dependent protein kinase 1 associated with aortic disease induces an active conformation in the absence of cGMP.

Type 1 cGMP-dependent protein kinases (PKGs) play important roles in human cardiovascular physiology, regulating vascular tone and smooth-muscle cell phenotype. A mutation in the human PRKG1 gene encoding cGMP-dependent protein kinase 1 (PKG1) leads to thoracic aortic aneurysms and dissections. The mutation causes an arginine-to-glutamine (RQ) substitution within the first cGMP-binding pocket in PKG1. This substitution disrupts cGMP binding to the pocket, but it also unexpectedly causes PKG1 to have high activity in the absence of cGMP via an unknown mechanism. Here, we identified the molecular mechanism whereby the RQ mutation increases basal kinase activity in the human PKG1α and PKG1ß isoforms. Although we found that the RQ substitution (R177Q in PKG1α and R192Q in PKG1ß) increases PKG1α and PKG1ß autophosphorylation in vitro, we did not detect increased autophosphorylation of the PKG1α or PKG1ß RQ variant isolated from transiently transfected 293T cells, indicating that increased basal activity of the RQ variants in cells was not driven by PKG1 autophosphorylation. Replacement of Arg-177 in PKG1α with alanine or methionine also increased basal activity. PKG1 exists as a parallel homodimer linked by an N-terminal leucine zipper, and we show that the WT chain in WT-RQ heterodimers partly reduces basal activity of the RQ chain. Using hydrogen/deuterium-exchange MS, we found that the RQ substitution causes PKG1ß to adopt an active conformation in the absence of cGMP, similar to that of cGMP-bound WT enzyme. We conclude that the RQ substitution in PKG1 increases its basal activity by disrupting the formation of an inactive conformation.

Mechanism of allosteric inhibition in the Plasmodium falciparum cGMP-dependent protein kinase.

Most malaria deaths are caused by the protozoan parasite Plasmodium falciparum Its life cycle is regulated by a cGMP-dependent protein kinase (PfPKG), whose inhibition is a promising antimalaria strategy. Allosteric kinase inhibitors, such as cGMP analogs, offer enhanced selectivity relative to competitive kinase inhibitors. However, the mechanisms underlying allosteric PfPKG inhibition are incompletely understood. Here, we show that 8-NBD-cGMP is an effective PfPKG antagonist. Using comparative NMR analyses of a key regulatory domain, PfD, in its apo, cGMP-bound, and cGMP analog-bound states, we elucidated its inhibition mechanism of action. Using NMR chemical shift analyses, molecular dynamics simulations, and site-directed mutagenesis, we show that 8-NBD-cGMP inhibits PfPKG not simply by reverting a two-state active versus inactive equilibrium, but by sampling also a distinct inactive "mixed" intermediate. Surface plasmon resonance indicates that the ability to stabilize a mixed intermediate provides a means to effectively inhibit PfPKG, without losing affinity for the cGMP analog. Our proposed model may facilitate the rational design of PfPKG-selective inhibitors for improved management of malaria.

Correction to: Crystal structure of PKG Iß holoenzyme reveals a trans­inhibiting dimer assembly.

After publication of abstract S 2­06 in supplement [1], it was brought to our attention that the second author's name is spelled incorrectly. Originally the author name has been published as Rajesh Sarma. The correct author name is Rajesh Sharma.

FRET-based cyclic GMP biosensors measure low cGMP concentrations in cardiomyocytes and neurons.

Several FRET (fluorescence resonance energy transfer)-based biosensors for intracellular detection of cyclic nucleotides have been designed in the past decade. However, few such biosensors are available for cGMP, and even fewer that detect low nanomolar cGMP concentrations. Our aim was to develop a FRET-based cGMP biosensor with high affinity for cGMP as a tool for intracellular signaling studies. We used the carboxyl-terminal cyclic nucleotide binding domain of Plasmodium falciparum cGMP-dependent protein kinase (PKG) flanked by different FRET pairs to generate two cGMP biosensors (Yellow PfPKG and Red PfPKG). Here, we report that these cGMP biosensors display high affinity for cGMP (EC50 of 23 ± 3 nM) and detect cGMP produced through soluble guanylyl cyclase and guanylyl cyclase A in stellate ganglion neurons and guanylyl cyclase B in cardiomyocytes. These biosensors are therefore optimal tools for real-time measurements of low concentrations of cGMP in living cells.

Structures of the cGMP-dependent protein kinase in malaria parasites reveal a unique structural relay mechanism for activation.

The cyclic guanosine-3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) was identified >25 y ago; however, efforts to obtain a structure of the entire PKG enzyme or catalytic domain from any species have failed. In malaria parasites, cooperative activation of PKG triggers crucial developmental transitions throughout the complex life cycle. We have determined the cGMP-free crystallographic structures of PKG from Plasmodium falciparum and Plasmodium vivax, revealing how key structural components, including an N-terminal autoinhibitory segment (AIS), four predicted cyclic nucleotide-binding domains (CNBs), and a kinase domain (KD), are arranged when the enzyme is inactive. The four CNBs and the KD are in a pentagonal configuration, with the AIS docked in the substrate site of the KD in a swapped-domain dimeric arrangement. We show that although the protein is predominantly a monomer (the dimer is unlikely to be representative of the physiological form), the binding of the AIS is necessary to keep Plasmodium PKG inactive. A major feature is a helix serving the dual role of the N-terminal helix of the KD as well as the capping helix of the neighboring CNB. A network of connecting helices between neighboring CNBs contributes to maintaining the kinase in its inactive conformation. We propose a scheme in which cooperative binding of cGMP, beginning at the CNB closest to the KD, transmits conformational changes around the pentagonal molecule in a structural relay mechanism, enabling PKG to orchestrate rapid, highly regulated developmental switches in response to dynamic modulation of cGMP levels in the parasite.

DIRAS3-Derived Peptide Inhibits Autophagy in Ovarian Cancer Cells by Binding to Beclin1.

Autophagy can protect cancer cells from acute starvation and enhance resistance to chemotherapy. Previously, we reported that autophagy plays a critical role in the survival of dormant, drug resistant ovarian cancer cells using human xenograft models and correlated the up-regulation of autophagy and DIRAS3 expression in clinical samples obtained during "second look" operations. DIRAS3 is an imprinted tumor suppressor gene that encodes a 26 kD GTPase with homology to RAS that inhibits cancer cell proliferation and motility. Re-expression of DIRAS3 in ovarian cancer xenografts also induces dormancy and autophagy. DIRAS3 can bind to Beclin1 forming the Autophagy Initiation Complex that triggers autophagosome formation. Both the N-terminus of DIRAS3 (residues 15-33) and the switch II region of DIRAS3 (residues 93-107) interact directly with BECN1. We have identified an autophagy-inhibiting peptide based on the switch II region of DIRAS3 linked to Tat peptide that is taken up by ovarian cancer cells, binds Beclin1 and inhibits starvation-induced DIRAS3-mediated autophagy.

Structural basis for selective inhibition of human PKG Iα by the balanol-like compound N46.

Activation of protein kinase G (PKG) Iα in nociceptive neurons induces long-term hyperexcitability that causes chronic pain. Recently, a derivative of the fungal metabolite balanol, N46, has been reported to inhibit PKG Iα with high potency and selectivity and attenuate thermal hyperalgesia and osteoarthritic pain. Here we determined co-crystal structures of the PKG Iα C-domain and cAMP-dependent protein kinase (PKA) Cα, each bound with N46, at 1.98 Å and 2.65 Å, respectively. N46 binds the active site with its external phenyl ring, specifically interacting with the glycine-rich loop and the αC helix. Phe-371 at the PKG Iα glycine-rich loop is oriented parallel to the phenyl ring of N46, forming a strong π-stacking interaction, whereas the analogous Phe-54 in PKA Cα rotates 30° and forms a weaker interaction. Structural comparison revealed that steric hindrance between the preceding Ser-53 and the propoxy group of the phenyl ring may explain the weaker interaction with PKA Cα. The analogous Gly-370 in PKG Iα, however, causes little steric hindrance with Phe-371. Moreover, Ile-406 on the αC helix forms a hydrophobic interaction with N46 whereas its counterpart in PKA, Thr-88, does not. Substituting these residues in PKG Iα with those in PKA Cα increases the IC50 values for N46, whereas replacing these residues in PKA Cα with those in PKG Iα reduces the IC50, consistent with our structural findings. In conclusion, our results explain the structural basis for N46-mediated selective inhibition of human PKG Iα and provide a starting point for structure-guided design of selective PKG Iα inhibitors.

Neutron Crystallography Detects Differences in Protein Dynamics: Structure of the PKG II Cyclic Nucleotide Binding Domain in Complex with an Activator.

As one of the main receptors of a second messenger, cGMP, cGMP-dependent protein kinase (PKG) isoforms I and II regulate distinct physiological processes. The design of isoform-specific activators is thus of great biomedical importance and requires detailed structural information about PKG isoforms bound with activators, including accurate positions of hydrogen atoms and a description of the hydrogen bonding and water architecture. Here, we determined a 2.2 Å room-temperature joint X-ray/neutron (XN) structure of the human PKG II carboxyl cyclic nucleotide binding (CNB-B) domain bound with a potent PKG II activator, 8-pCPT-cGMP. The XN structure directly visualizes intermolecular interactions and reveals changes in hydrogen bonding patterns upon comparison to the X-ray structure determined at cryo-temperatures. Comparative analysis of the backbone hydrogen/deuterium exchange patterns in PKG II:8-pCPT-cGMP and previously reported PKG Iß:cGMP XN structures suggests that the ability of these agonists to activate PKG is related to how effectively they quench dynamics of the cyclic nucleotide binding pocket and the surrounding regions.

Mutations in Fibronectin Cause a Subtype of Spondylometaphyseal Dysplasia with "Corner Fractures".

Fibronectin is a master organizer of extracellular matrices (ECMs) and promotes the assembly of collagens, fibrillin-1, and other proteins. It is also known to play roles in skeletal tissues through its secretion by osteoblasts, chondrocytes, and mesenchymal cells. Spondylometaphyseal dysplasias (SMDs) comprise a diverse group of skeletal dysplasias and often manifest as short stature, growth-plate irregularities, and vertebral anomalies, such as scoliosis. By comparing the exomes of individuals with SMD with the radiographic appearance of "corner fractures" at metaphyses, we identified three individuals with fibronectin (FN1) variants affecting highly conserved residues. Furthermore, using matching tools and the SkelDys emailing list, we identified other individuals with de novo FN1 variants and a similar phenotype. The severe scoliosis in most individuals and rare developmental coxa vara distinguish individuals with FN1 mutations from those with classical Sutcliffe-type SMD. To study functional consequences of these FN1 mutations on the protein level, we introduced three disease-associated missense variants (p.Cys87Phe [c.260G>T], p.Tyr240Asp [c.718T>G], and p.Cys260Gly [c.778T>G]) into a recombinant secreted N-terminal 70 kDa fragment (rF70K) and the full-length fibronectin (rFN). The wild-type rF70K and rFN were secreted into the culture medium, whereas all mutant proteins were either not secreted or secreted at significantly lower amounts. Immunofluorescence analysis demonstrated increased intracellular retention of the mutant proteins. In summary, FN1 mutations that cause defective fibronectin secretion are found in SMD, and we thus provide additional evidence for a critical function of fibronectin in cartilage and bone.

Switching Cyclic Nucleotide-Selective Activation of Cyclic Adenosine Monophosphate-Dependent Protein Kinase Holoenzyme Reveals Distinct Roles of Tandem Cyclic Nucleotide-Binding Domains.

The cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-dependent protein kinases (PKA and PKG) are key effectors of cyclic nucleotide signaling. Both share structural features that include tandem cyclic nucleotide-binding (CNB) domains, CNB-A and CNB-B, yet their functions are separated through preferential activation by either cAMP or cGMP. Based on structural studies and modeling, key CNB contact residues have been identified for both kinases. In this study, we explored the requirements for conversion of PKA activation from cAMP-dependent to cGMP-dependent. The consequences of the residue substitutions T192R/A212T within CNB-A or G316R/A336T within CNB-B of PKA-RIα on cyclic nucleotide binding and holoenzyme activation were assessed in vitro using purified recombinant proteins, and ex vivo using RIα-deficient mouse embryonic fibroblasts genetically reconstituted with wild-type or mutant PKA-RIα. In vitro, a loss of binding and activation selectivity was observed when residues in either one of the CNB domains were mutated, while mutations in both CNB domains resulted in a complete switch of selectivity from cAMP to cGMP. The switch in selectivity was also recapitulated ex vivo, confirming their functional roles in cells. Our results highlight the importance of key cyclic nucleotide contacts within each CNB domain and suggest that these domains may have evolved from an ancestral gene product to yield two distinct cyclic nucleotide-dependent protein kinases.

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 ß4 with the PET moiety, whereas the A site of PKG II has a larger ß5/ß6 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.

Mutations of PKA cyclic nucleotide-binding domains reveal novel aspects of cyclic nucleotide selectivity.

Cyclic AMP and cyclic GMP are ubiquitous second messengers that regulate the activity of effector proteins in all forms of life. The main effector proteins, the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), are preferentially activated by cAMP and cGMP, respectively. However, the molecular basis of this cyclic nucleotide selectivity is still not fully understood. Analysis of isolated cyclic nucleotide-binding (CNB) domains of PKA regulatory subunit type Iα (RIα) reveals that the C-terminal CNB-B has a higher cAMP affinity and selectivity than the N-terminal CNB-A. Here, we show that introducing cGMP-specific residues using site-directed mutagenesis reduces the selectivity of CNB-B, while the combination of two mutations (G316R/A336T) results in a cGMP-selective binding domain. Furthermore, introducing the corresponding mutations (T192R/A212T) into the PKA RIα CNB-A turns this domain into a highly cGMP-selective domain, underlining the importance of these contacts for achieving cGMP specificity. Binding data with the generic purine nucleotide 3',5'-cyclic inosine monophosphate (cIMP) reveal that introduced arginine residues interact with the position 6 oxygen of the nucleobase. Co-crystal structures of an isolated CNB-B G316R/A336T double mutant with either cAMP or cGMP reveal that the introduced threonine and arginine residues maintain their conserved contacts as seen in PKG I CNB-B. These results improve our understanding of cyclic nucleotide binding and the molecular basis of cyclic nucleotide specificity.