The αC-ß4 loop controls the allosteric cooperativity between nucleotide and substrate in the catalytic subunit of protein kinase A.

Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic subunit of protein kinase A (PKA-C). This long-range synergistic action is involved in substrate recognition and fidelity, and it may also regulate PKA's association with regulatory subunits and other binding partners. To date, a complete understanding of this intramolecular mechanism is still lacking. Here, we integrated NMR(Nuclear Magnetic Resonance)-restrained molecular dynamics simulations and a Markov State Model to characterize the free energy landscape and conformational transitions of PKA-C. We found that the apoenzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the αC-ß4 loop. We validated the second excited state by analyzing the F100A mutant of PKA-C, assessing its structural response to ATP and substrate binding. While PKA-CF100A preserves its catalytic efficiency with Kemptide, this mutation rearranges the αC-ß4 loop conformation, interrupting the coupling of the two lobes and abolishing the allosteric binding cooperativity. The highly conserved αC-ß4 loop emerges as a pivotal element to control the synergistic binding of nucleotide and substrate, explaining how mutations or insertions near or within this motif affect the function and drug sensitivity in homologous kinases.

Protein Kinase Structure and Dynamics: Role of the αC-ß4 Loop.

Although the αC-ß4 loop is a stable feature of all protein kinases, the importance of this motif as a conserved element of secondary structure, as well as its links to the hydrophobic architecture of the kinase core, has been underappreciated. We first review the motif and then describe how it is linked to the hydrophobic spine architecture of the kinase core, which we first discovered using a computational tool, Local Spatial Pattern (LSP) alignment. Based on NMR predictions that a mutation in this motif abolishes the synergistic high-affinity binding of ATP and a pseudo substrate inhibitor, we used LSP to interrogate the F100A mutant. This comparison highlights the importance of the αC-ß4 loop and key residues at the interface between the N- and C-lobes. In addition, we delved more deeply into the structure of the apo C-subunit, which lacks ATP. While apo C-subunit showed no significant changes in backbone dynamics of the αC-ß4 loop, we found significant differences in the side chain dynamics of K105. The LSP analysis suggests disruption of communication between the N- and C-lobes in the F100A mutant, which would be consistent with the structural changes predicted by the NMR spectroscopy.

The αC-ß4 loop controls the allosteric cooperativity between nucleotide and substrate in the catalytic subunit of protein kinase A.

Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic (C) subunit of protein kinase A (PKA). Not only this long-range synergistic action is involved in substrate recognition and fidelity, but it is likely to regulate PKA association with regulatory subunits and other binding partners. To date, a complete understanding of the molecular determinants for this intramolecular mechanism is still lacking. Here, we used an integrated NMR-restrained molecular dynamics simulations and a Markov Model to characterize the free energy landscape and conformational transitions of the catalytic subunit of protein kinase A (PKA-C). We found that the apo-enzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the tip of the αC-ß4 loop. To experimentally validate the second excited state, we mutated F100 into alanine and used NMR spectroscopy to characterize the binding thermodynamics and structural response of ATP and a prototypical peptide substrate. While the activity of PKA-CF100A toward a prototypical peptide substrate is unaltered and the enzyme retains its affinity for ATP and substrate, this mutation rearranges the αC-ß4 loop conformation interrupting the allosteric coupling between nucleotide and substrate. The highly conserved αC-ß4 loop emerges as a pivotal element able to modulate the synergistic binding between nucleotide and substrate and may affect PKA signalosome. These results may explain how insertion mutations within this motif affect drug sensitivity in other homologous kinases.

AI-designed NMR spectroscopy RF pulses for fast acquisition at high and ultra-high magnetic fields.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful high-resolution tool for characterizing biomacromolecular structure, dynamics, and interactions. However, the lengthy longitudinal relaxation of the nuclear spins significantly extends the total experimental time, especially at high and ultra-high magnetic field strengths. Although longitudinal relaxation-enhanced techniques have sped up data acquisition, their application has been limited by the chemical shift dispersion. Here we combined an evolutionary algorithm and artificial intelligence to design 1H and 15N radio frequency (RF) pulses with variable phase and amplitude that cover significantly broader bandwidths and allow for rapid data acquisition. We re-engineered the basic transverse relaxation optimized spectroscopy experiment and showed that the RF shapes enhance the spectral sensitivity of well-folded proteins up to 180 kDa molecular weight. These RF shapes can be tailored to re-design triple-resonance experiments for accelerating NMR spectroscopy of biomacromolecules at high fields.

An NMR portrait of functional and dysfunctional allosteric cooperativity in cAMP-dependent protein kinase A.

The cAMP-dependent protein kinase A (PKA) is the archetypical eukaryotic kinase. The catalytic subunit (PKA-C) structure is highly conserved among the AGC-kinase family. PKA-C is a bilobal enzyme with a dynamic N-lobe, harbouring the Adenosine-5'-triphosphate (ATP) binding site and a more rigid helical C-lobe. The substrate-binding groove resides at the interface of the two lobes. A distinct feature of PKA-C is the positive binding cooperativity between nucleotide and substrate. Several PKA-C mutations lead to the development of adenocarcinomas, myxomas, and other rare forms of liver tumours. Nuclear magnetic resonance (NMR) spectroscopy shows that these mutations disrupt the allosteric communication between the two lobes, causing a drastic decrease in binding cooperativity. The loss of cooperativity correlates with changes in substrate fidelity and reduced kinase affinity for the endogenous protein kinase inhibitor (PKI). The similarity between PKI and the inhibitory sequence of the kinase regulatory subunits suggests that the overall mechanism of regulation of the kinase may be disrupted. We surmise that a reduced or obliterated cooperativity may constitute a common trait for both orthosteric and allosteric mutations of PKA-C that may lead to dysregulation and disease.

Water irradiation devoid pulses enhance the sensitivity of 1H,1H nuclear Overhauser effects.

The nuclear Overhauser effect (NOE) is one of NMR spectroscopy's most important and versatile parameters. NOE is routinely utilized to determine the structures of medium-to-large size biomolecules and characterize protein-protein, protein-RNA, protein-DNA, and protein-ligand interactions in aqueous solutions. Typical [1H,1H] NOESY pulse sequences incorporate water suppression schemes to reduce the water signal that dominates 1H-detected spectra and minimize NOE intensity losses due to unwanted polarization exchange between water and labile protons. However, at high- and ultra-high magnetic fields, the excitation of the water signal during the execution of the NOESY pulse sequences may cause significant attenuation of NOE cross-peak intensities. Using an evolutionary algorithm coupled with artificial intelligence, we recently designed high-fidelity pulses [Water irrAdiation DEvoid (WADE) pulses] that elude water excitation and irradiate broader bandwidths relative to commonly used pulses. Here, we demonstrate that WADE pulses, implemented into the 2D [1H,1H] NOESY experiments, increase the intensity of the NOE cross-peaks for labile and, to a lesser extent, non-exchangeable protons. We applied the new 2D [1H,1H] WADE-NOESY pulse sequence to two well-folded, medium-size proteins, i.e., the K48C mutant of ubiquitin and the Raf kinase inhibitor protein. We observed a net increase of the NOE intensities varying from 30 to 170% compared to the commonly used NOESY experiments. The new WADE pulses can be easily engineered into 2D and 3D homo- and hetero-nuclear NOESY pulse sequences to boost their sensitivity.

A Case of Multidisciplinary Approach to Post-Radiotherapy Dilative Cardiomyopathy Undergoing Elective Cesarean Delivery: Anesthetic and Intensive Care Management.

Background: Cardiovascular diseases are the most common non-obstetric cause of maternal death. These cases became more common thanks to the improvement in cardiovascular therapies. A multidisciplinary team is necessary to manage these pregnancies. Case Report: A 32 years old women at the 25th week of gestation for acute heart failure in pre-existing left ventricular dysfunction induced by radio-chemotherapy admitted to the Coronary Unit of IRCCS Policlinico Universitario Agostino Gemelli for worsening of dyspneic symptoms and anuria not responding to diuretic therapy. At the echocardiogram: ejection fraction 30%, enlarged left atrium, systolic pulmonary arterial pressure 38 mmHg, bilateral pleural effusion, bilateral diffused pulmonary B lines. A multidisciplinary team composed by cardiologists, gynecologists, anesthesiologists, cardiac surgeons, neonatologists and bioethicists decided for an elective cesarean delivery at the 27th week of gestation in the hybrid cardio-thoracic operating theater. Anesthesia was provided by combined spinal-epidural technique under invasive continuous hemodynamic monitoring with the Edwards Lifesciences HemoSphere with Hypotension Prediction Index (HPI) and ForeSight technology (Edwards Lifesciences, Irvine, USA) through catheterization of the left radial artery. The femoral arteries were left available for extracorporeal circulation. Continuous norepinephrine infusion was started once liquor was collected in the spinal needle at a 0.1 mcg/kg/minute through a central line and was continued until the end of surgery. Fluid management consisted of a total of 200 ml of crystalloids. HPI values never reached alarm values (maximum value =10). The patient was discharged home on the 5th day after delivery with good hemodynamic compensation. The baby was intubated at birth and then gradually weaned from mechanical ventilation, then discharged.

A PKA inhibitor motif within SMOOTHENED controls Hedgehog signal transduction.

The Hedgehog (Hh) cascade is central to development, tissue homeostasis and cancer. A pivotal step in Hh signal transduction is the activation of glioma-associated (GLI) transcription factors by the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO). How SMO activates GLI remains unclear. Here we show that SMO uses a decoy substrate sequence to physically block the active site of the cAMP-dependent protein kinase (PKA) catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular and organismal models, we demonstrate that interfering with SMO-PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism uncovered echoes one used by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, our findings define a mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases.

High-fidelity control of spin ensemble dynamics via artificial intelligence: from quantum computing to NMR spectroscopy and imaging.

High-fidelity control of spin ensemble dynamics is essential for many research areas, spanning from quantum computing and radio-frequency (RF) engineering to NMR spectroscopy and imaging. However, attaining robust and high-fidelity spin operations remains an unmet challenge. Using an evolutionary algorithm and artificial intelligence (AI), we designed new RF pulses with customizable spatial or temporal field inhomogeneity compensation. Compared with the standard RF shapes, the new AI-generated pulses show superior performance for bandwidth, robustness, and tolerance to field imperfections. As a benchmark, we constructed a spin entanglement operator for the weakly coupled two-spin-1/2 system of 13CHCl3, achieving high-fidelity transformations under multiple inhomogeneity sources. We then generated band-selective and ultra-broadband RF pulses typical of biomolecular NMR spectroscopy. When implemented in multipulse NMR experiments, the AI-generated pulses significantly increased the sensitivity of medium-size and large protein spectra relative to standard pulse sequences. Finally, we applied the new pulses to typical imaging experiments, showing a remarkable tolerance to changes in the RF field. These AI-generated RF pulses can be directly implemented in quantum information, NMR spectroscopy of biomolecules, magnetic resonance imaging techniques for in vivo and materials sciences.

Design and applications of water irradiation devoid RF pulses for ultra-high field biomolecular NMR spectroscopy.

Water suppression is of paramount importance for many biological and analytical NMR spectroscopy applications. Here, we report the design of a new set of binomial-like radio frequency (RF) pulses that elude water irradiation while exciting or refocusing the remainder of the 1H spectrum. These pulses were generated using a combination of an evolutionary algorithm and artificial intelligence. They display higher sensitivity relative to classical water suppression schemes and tunable water selectivity to avoid suppressing 1H resonances near the water signal. The broad bandwidth excitation obtained with these RF pulses makes them suitable for several NMR applications at high and ultra-high-field magnetic fields.

ATP-competitive inhibitors modulate the substrate binding cooperativity of a kinase by altering its conformational entropy.

ATP-competitive inhibitors are currently the largest class of clinically approved drugs for protein kinases. By targeting the ATP-binding pocket, these compounds block the catalytic activity, preventing substrate phosphorylation. A problem with these drugs, however, is that inhibited kinases may still recognize and bind downstream substrates, acting as scaffolds or binding hubs for signaling partners. Here, using protein kinase A as a model system, we show that chemically different ATP-competitive inhibitors modulate the substrate binding cooperativity by tuning the conformational entropy of the kinase and shifting the populations of its conformationally excited states. Since we found that binding cooperativity and conformational entropy of the enzyme are correlated, we propose a new paradigm for the discovery of ATP-competitive inhibitors, which is based on their ability to modulate the allosteric coupling between nucleotide and substrate-binding sites.

Raf Kinase Inhibitory Protein regulates the cAMP-dependent protein kinase signaling pathway through a positive feedback loop.

Raf Kinase Inhibitory Protein (RKIP) maintains cellular robustness and prevents the progression of diseases such as cancer and heart disease by regulating key kinase cascades including MAP kinase and protein kinase A (PKA). Phosphorylation of RKIP at S153 by Protein Kinase C (PKC) triggers a switch from inhibition of Raf to inhibition of the G protein coupled receptor kinase 2 (GRK2), enhancing signaling by the ß-adrenergic receptor (ß-AR) that activates PKA. Here we report that PKA-phosphorylated RKIP promotes ß-AR-activated PKA signaling. Using biochemical, genetic, and biophysical approaches, we show that PKA phosphorylates RKIP at S51, increasing S153 phosphorylation by PKC and thereby triggering feedback activation of PKA. The S51V mutation blocks the ability of RKIP to activate PKA in prostate cancer cells and to induce contraction in primary cardiac myocytes in response to the ß-AR activator isoproterenol, illustrating the functional importance of this positive feedback circuit. As previously shown for other kinases, phosphorylation of RKIP at S51 by PKA is enhanced upon RKIP destabilization by the P74L mutation. These results suggest that PKA phosphorylation at S51 may lead to allosteric changes associated with a higher-energy RKIP state that potentiates phosphorylation of RKIP at other key sites. This allosteric regulatory mechanism may have therapeutic potential for regulating PKA signaling in disease states.

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.

Comparison of ropivacaine plasma concentration after posterior Quadratus Lumborum Block in Cesarean Section with ropivacaine with epinephrine vs. plane.

BACKGROUND: The posterior quadratus lumborum block (pQLB) has been used in postoperative pain management after cesarean section (CS). However, clinicians have no data about pQLB safety in pregnants, at increased risk of local anesthetic systemic toxicity (LAST). The purpose of the present study was to explore the efficacy and the safety of adding epinephrine to ropivacaine for bilateral pQLB vs. bilateral pQLB performed with ropivacaine alone in CS. METHODS: In this prospective trial 52 pregnants, ASA 2 physiological status, were consecutively allocated to one of two groups, e-pQLB and pQLB; e-pQLB group received 0.375% ropivacaine+100 mcg epinephrine, 20 mL each side; pQLB received 0.375% ropivacaine alone, 20 mL each side. The primary and secondary outcomes were to evaluate if the adjunct of epinephrine to ropivacaine increases efficacy and safety of pQLB, respectively. RESULTS: Authors found in e-pQLB group vs. p-QLB group: a total mean morphine consumption statistically lower during the first 24 postoperative hours (5.08±3.12, vs. 9.11±4.67 SD mg, P=0.0002); NRS values statistically lower at six hours from block, both at rest (1.73±1.88 SD vs. 2.88±2.53, P=0.03) and with movement (3.03±1.98 SD vs. 4.23±2.87, P=0.04); a longer time between block and the first opioid request (5.92±2.48 vs. 3.78±2.68 SD hrs, P<0.003); venous ropivacaine concentrations significantly lower at any time of samples but at 120 minutes. CONCLUSIONS: Adding epinephrine to ropivacaine increases efficacy and duration of pQLB. Moreover it increases block safety, reducing peak and mean venous ropivacaine concentration.

Anesthesia management of pediatric dentistry patients with cardiofaciocutaneous syndrome: a case report.

Cardiofaciocutaneous syndrome is a rare syndrome characterized by particular craniofacial features, cardiac abnormalities, and multiple organ diseases. Patients present with pulmonary stenosis, hypertrophic cardiomyopathy, short neck, micrognathia, laryngomalacia, and tracheomalacia. These conditions may strongly influence patient perioperative outcomes. We describe a 15-year-old child with cardiofaciocutaneous syndrome presenting for a dentistry procedure. She had an uneventful perioperative and postoperative course except for difficult airway management.

Defective internal allosteric network imparts dysfunctional ATP/substrate-binding cooperativity in oncogenic chimera of protein kinase A.

An aberrant fusion of the DNAJB1 and PRKACA genes generates a chimeric protein kinase (PKA-CDNAJB1) in which the J-domain of the heat shock protein 40 is fused to the catalytic α subunit of cAMP-dependent protein kinase A (PKA-C). Deceivingly, this chimeric construct appears to be fully functional, as it phosphorylates canonical substrates, forms holoenzymes, responds to cAMP activation, and recognizes the endogenous inhibitor PKI. Nonetheless, PKA-CDNAJB1 has been recognized as the primary driver of fibrolamellar hepatocellular carcinoma and is implicated in other neoplasms for which the molecular mechanisms remain elusive. Here we determined the chimera's allosteric response to nucleotide and pseudo-substrate binding. We found that the fusion of the dynamic J-domain to PKA-C disrupts the internal allosteric network, causing dramatic attenuation of the nucleotide/PKI binding cooperativity. Our findings suggest that the reduced allosteric cooperativity exhibited by PKA-CDNAJB1 alters specific recognitions and interactions between substrates and regulatory partners contributing to dysregulation.

CHESPA/CHESCA-SPARKY: automated NMR data analysis plugins for SPARKY to map protein allostery.

MOTIVATION: Correlated Nuclear Magnetic Resonance (NMR) chemical shift changes identified through the CHEmical Shift Projection Analysis (CHESPA) and CHEmical Shift Covariance Analysis (CHESCA) reveal pathways of allosteric transitions in biological macromolecules. To address the need for an automated platform that implements CHESPA and CHESCA and integrates them with other NMR analysis software packages, we introduce here integrated plugins for NMRFAM-SPARKY that implement the seamless detection and visualization of allosteric networks. AVAILABILITY AND IMPLEMENTATION: CHESCA-SPARKY and CHESPA-SPARKY are available in the latest version of NMRFAM-SPARKY from the National Magnetic Resonance Facility at Madison (http://pine.nmrfam.wisc.edu/download_packages.html), the NMRbox Project (https://nmrbox.org) and to subscribers to the SBGrid (https://sbgrid.org). The assigned spectra involved in this study and tutorial videos using this dataset are available at https://sites.google.com/view/chescachespa-sparky. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics Online.

Multi-state recognition pathway of the intrinsically disordered protein kinase inhibitor by protein kinase A.

In the nucleus, the spatiotemporal regulation of the catalytic subunit of cAMP-dependent protein kinase A (PKA-C) is orchestrated by an intrinsically disordered protein kinase inhibitor, PKI, which recruits the CRM1/RanGTP nuclear exporting complex. How the PKA-C/PKI complex assembles and recognizes CRM1/RanGTP is not well understood. Using NMR, SAXS, fluorescence, metadynamics, and Markov model analysis, we determined the multi-state recognition pathway for PKI. After a fast binding step in which PKA-C selects PKI's most competent conformations, PKI folds upon binding through a slow conformational rearrangement within the enzyme's binding pocket. The high-affinity and pseudo-substrate regions of PKI become more structured and the transient interactions with the kinase augment the helical content of the nuclear export sequence, which is then poised to recruit the CRM1/RanGTP complex for nuclear translocation. The multistate binding mechanism featured by PKA-C/PKI complex represents a paradigm on how disordered, ancillary proteins (or protein domains) are able to operate multiple functions such as inhibiting the kinase while recruiting other regulatory proteins for nuclear export.