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.

Isothermal titration calorimetry and vesicle leakage assays highlight the differential behaviors of tau repeat segments upon interaction with anionic lipid membranes.

Tau misfolding has been implicated in a variety of tauopathies, including Alzheimer's disease. The microtubule binding domain of tau consists of four repeat segments (R1-R4), and aggregation of these segments leads to the formation of neurofibrillary tangles. Previous studies indicate that misfolded tau associates with anionic phospholipid membranes, invoking structural transformations that could play a role in aggregation. Here, we investigated the role of membrane surface charge on the binding affinity of individual tau repeat segments, and whether these segments exhibit lytic activity. We quantified the thermodynamics of this process in terms of the affinity (Kd), enthalpy (ΔH), entropy (ΔS), and change in specific heat capacity (ΔCp). While neutral membranes exhibited weak interactions with each tau repeat segment, segments R2 and R3 exhibited relatively strong binding with anionic membranes with favorable ΔS and a negative value of ΔCp. Calcein leakage assays show that each repeat segment displays lytic activity, but only upon the interaction with anionic membranes. Taken together, these results distinguish the relative selectivity for anionic membranes by each repeat segment and the degree of membrane disruption that results.

Dysfunctional conformational dynamics of protein kinase A induced by a lethal mutant of phospholamban hinder phosphorylation.

The dynamic interplay between kinases and substrates is crucial for the formation of catalytically committed complexes that enable phosphoryl transfer. However, a clear understanding on how substrates modulate kinase structural dynamics to control catalytic efficiency is still missing. Here, we used solution NMR spectroscopy to study the conformational dynamics of two complexes of the catalytic subunit of the cAMP-dependent protein kinase A with WT and R14 deletion phospholamban, a lethal human mutant linked to familial dilated cardiomyopathy. Phospholamban is a central regulator of heart muscle contractility, and its phosphorylation by protein kinase A constitutes a primary response to ß-adrenergic stimulation. We found that the single deletion of arginine in phospholamban's recognition sequence for the kinase reduces its binding affinity and dramatically reduces phosphorylation kinetics. Structurally, the mutant prevents the enzyme from adopting conformations and motions committed for catalysis, with concomitant reduction in catalytic efficiency. Overall, these results underscore the importance of a well-tuned structural and dynamic interplay between the kinase and its substrates to achieve physiological phosphorylation levels for proper Ca(2+) signaling and normal cardiac function.

Synchronous opening and closing motions are essential for cAMP-dependent protein kinase A signaling.

Conformational fluctuations play a central role in enzymatic catalysis. However, it is not clear how the rates and the coordination of the motions affect the different catalytic steps. Here, we used NMR spectroscopy to analyze the conformational fluctuations of the catalytic subunit of the cAMP-dependent protein kinase (PKA-C), a ubiquitous enzyme involved in a myriad of cell signaling events. We found that the wild-type enzyme undergoes synchronous motions involving several structural elements located in the small lobe of the kinase, which is responsible for nucleotide binding and release. In contrast, a mutation (Y204A) located far from the active site desynchronizes the opening and closing of the active cleft without changing the enzyme's structure, rendering it catalytically inefficient. Since the opening and closing motions govern the rate-determining product release, we conclude that optimal and coherent conformational fluctuations are necessary for efficient turnover of protein kinases.

Structure and dynamics of a primordial catalytic fold generated by in vitro evolution.

Engineering functional protein scaffolds capable of carrying out chemical catalysis is a major challenge in enzyme design. Starting from a noncatalytic protein scaffold, we recently generated a new RNA ligase by in vitro directed evolution. This artificial enzyme lost its original fold and adopted an entirely new structure with substantially enhanced conformational dynamics, demonstrating that a primordial fold with suitable flexibility is sufficient to carry out enzymatic function.

Conformational equilibrium of N-myristoylated cAMP-dependent protein kinase A by molecular dynamics simulations.

The catalytic subunit of protein kinase A (PKA-C) is subject to several post- or cotranslational modifications that regulate its activity both spatially and temporally. Among those, N-myristoylation increases the kinase affinity for membranes and might also be implicated in substrate recognition and allosteric regulation. Here, we investigated the effects of N-myristoylation on the structure, dynamics, and conformational equilibrium of PKA-C using atomistic molecular dynamics simulations. We found that the myristoyl group inserts into the hydrophobic pocket and leads to a tighter packing of the A-helix against the core of the enzyme. As a result, the conformational dynamics of the A-helix are reduced and its motions are more coupled with the active site. Our simulations suggest that cation-π interactions among W30, R190, and R93 are responsible for coupling these motions. Two major conformations of the myristoylated N-terminus are the most populated: a long loop (LL conformation), similar to Protein Data Bank (PDB) entry 1CMK , and a helix-turn-helix structure (HTH conformation), similar to PDB entry 4DFX , which shows stronger coupling between the conformational dynamics observed at the A-helix and active site. The HTH conformation is stabilized by S10 phosphorylation of the kinase via ionic interactions between the protonated amine of K7 and the phosphate group on S10, further enhancing the dynamic coupling to the active site. These results support a role of N-myristoylation in the allosteric regulation of PKA-C.

Isotope labeling for solution and solid-state NMR spectroscopy of membrane proteins.

In this chapter, we summarize the isotopic labeling strategies used to obtain high-quality solution and solid-state NMR spectra of biological samples, with emphasis on integral membrane proteins (IMPs). While solution NMR is used to study IMPs under fast tumbling conditions, such as in the presence of detergent micelles or isotropic bicelles, solid-state NMR is used to study the structure and orientation of IMPs in lipid vesicles and bilayers. In spite of the tremendous progress in biomolecular NMR spectroscopy, the homogeneity and overall quality of the sample is still a substantial obstacle to overcome. Isotopic labeling is a major avenue to simplify overlapped spectra by either diluting the NMR active nuclei or allowing the resonances to be separated in multiple dimensions. In the following we will discuss isotopic labeling approaches that have been successfully used in the study of IMPs by solution and solid-state NMR spectroscopy.

Allostery and binding cooperativity of the catalytic subunit of protein kinase A by NMR spectroscopy and molecular dynamics simulations.

The catalytic subunit of cAMP-dependent protein kinase A (PKA-C) is an exquisite example of a single molecule allosteric enzyme, where classical and modern views of allosteric signaling merge. In this chapter, we describe the mapping of PKA-C conformational dynamics and allosteric signaling in the free and bound states using a combination of NMR spectroscopy and molecular dynamics simulations. We show that ligand binding affects the enzyme's conformational dynamics, shaping the free-energy landscape toward the next stage of the catalytic cycle. While nucleotide and substrate binding enhance the enzyme's conformational entropy and define dynamically committed states, inhibitor binding attenuates the internal dynamics in favor of enthalpic interactions and delineates dynamically quenched states. These studies support a central role of conformational dynamics in many aspects of enzymatic turnover and suggest future avenues for controlling enzymatic function.

Heteronuclear Adiabatic Relaxation Dispersion (HARD) for quantitative analysis of conformational dynamics in proteins.

NMR relaxation methods probe biomolecular motions over a wide range of timescales. In particular, the rotating frame spin-lock R(1ρ) and Carr-Purcell-Meiboom-Gill (CPMG) R(2) experiments are commonly used to characterize µs to ms dynamics, which play a critical role in enzyme folding and catalysis. In an effort to complement these approaches, we introduced the Heteronuclear Adiabatic Relaxation Dispersion (HARD) method, where dispersion in rotating frame relaxation rate constants (longitudinal R(1ρ) and transverse R(2ρ)) is created by modulating the shape and duration of adiabatic full passage (AFP) pulses. Previously, we showed the ability of the HARD method to detect chemical exchange dynamics in the fast exchange regime (k(ex)∼10(4)-10(5) s(-1)). In this article, we show the sensitivity of the HARD method to slower exchange processes by measuring R(1ρ) and R(2ρ) relaxation rates for two soluble proteins (ubiquitin and 10C RNA ligase). One advantage of the HARD method is its nominal dependence on the applied radio frequency field, which can be leveraged to modulate the dispersion in the relaxation rate constants. In addition, we also include product operator simulations to define the dynamic range of adiabatic R(1ρ) and R(2ρ) that is valid under all exchange regimes. We conclude from both experimental observations and simulations that this method is complementary to CPMG-based and rotating frame spin-lock R(1ρ) experiments to probe conformational exchange dynamics for biomolecules. Finally, this approach is germane to several NMR-active nuclei, where relaxation rates are frequency-offset independent.

FLAMEnGO: a fuzzy logic approach for methyl group assignment using NOESY and paramagnetic relaxation enhancement data.

Building on a recent method by Matthews and co-workers [1], we developed a new and efficient algorithm to assign methyl resonances from sparse and ambiguous NMR data. The new algorithm (FLAMEnGO: Fuzzy Logic Assignment of MEthyl GrOups) uses Monte Carlo sampling in conjunction with fuzzy logic to obtain the assignment of methyl resonances at high fidelity. Furthermore, we demonstrate that the inclusion of paramagnetic relaxation enhancement (PRE) data in the assignment strategy increases the percentage of correct assignments with sparse NOE data. Using synthetic tests and experimental data we show that this new approach provides up to ∼80% correct assignments with only 30% of methyl-methyl NOE data. In the experimental case of ubiquitin, PRE data from two spin labeled sites improve the percentage of assigned methyl groups up to ∼91%. This new strategy promises to further expand methyl group NMR spectroscopy to very large macromolecular systems.

Templating α-amylase peptide inhibitors with organotin compounds.

Metal centers have been widely used to nucleate secondary structures in linear peptides. However, very few examples have been reported for peptide/organometal complexes. Here, we illustrate the use of organotin compounds as nucleation centers for secondary structures of linear peptide inhibitors of α-amylase. Specifically, we utilized methyl-substituted tin compounds to template short type I ß-turns similar to the binding loop of tendamistat, the natural inhibitor of the enzyme, which are able to bind and inhibit α-amylase. We show that enzyme activity is inhibited by neither the unstructured peptide nor the organotin compounds, but rather the peptide/organotin complex, which inhibits the enzyme with K (i) ~ 0.5 µM. The results delineate a strategy to use organometallic compounds to drive the active conformation in small linear peptides.

A myristoyl/phosphoserine switch controls cAMP-dependent protein kinase association to membranes.

The cAMP-dependent protein kinase [protein kinase A (PKA)] mediates a myriad of cellular signaling events, and its activity is tightly regulated in both space and time. Among these regulatory mechanisms is N-myristoylation, whose biological role has been elusive. Using a combination of thermodynamics, kinetics, and spectroscopic methods, we analyzed the effects of N-myristoylation and phosphorylation at Ser10 on the interactions of PKA with model membranes. We found that, in the absence of lipids, the myristoyl group is tucked into the hydrophobic binding pocket of the enzyme (myr-in state). Upon association with lipid bilayers, the myristoyl group is extruded and inserts into the hydrocarbon region of the lipid bilayer (myr-out state). NMR data indicate that the enzyme undergoes conformational equilibrium between myr-in and myr-out states, which can be shifted byeither interaction with membranes and/or phosphorylation at Ser10. Our results provide evidence that the membrane binding motif of the myristoylated C-subunit of PKA (PKA-C) steers the enzyme toward lipids independent of its regulatory subunit or an A-kinase anchoring protein, providing an additional mechanism to localize the enzyme near membrane-bound substrates.

cAMP-dependent protein kinase A selects the excited state of the membrane substrate phospholamban.

Phosphorylation of membrane proteins is a central regulatory and signaling mechanism across cell compartments. However, the recognition process and phosphorylation mechanism of membrane-bound substrates by kinases are virtually unknown. cAMP-dependent protein kinase A (PKA) is a ubiquitous enzyme that phosphorylates several soluble and membrane-bound substrates. In cardiomyocytes, PKA targets phospholamban (PLN), a membrane protein that inhibits the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA). In the unphosphorylated state, PLN binds SERCA, reducing the calcium uptake and generating muscle contraction. PKA phosphorylation of PLN at S16 in the cytoplasmic helix relieves SERCA inhibition, initiating muscle relaxation. Using steady-state kinetic assays, NMR spectroscopy, and molecular modeling, we show that PKA recognizes and phosphorylates the excited, membrane-detached R-state of PLN. By promoting PLN from a ground state to an excited state, we obtained a linear relationship between rate of phosphorylation and population of the excited state of PLN. The conformational equilibrium of PLN is crucial to regulate the extent of PLN phosphorylation and SERCA inhibition.

Dynamically committed, uncommitted, and quenched states encoded in protein kinase A revealed by NMR spectroscopy.

Protein kinase A (PKA) is a ubiquitous phosphoryl transferase that mediates hundreds of cell signaling events. During turnover, its catalytic subunit (PKA-C) interconverts between three major conformational states (open, intermediate, and closed) that are dynamically and allosterically activated by nucleotide binding. We show that the structural transitions between these conformational states are minimal and allosteric dynamics encode the motions from one state to the next. NMR and molecular dynamics simulations define the energy landscape of PKA-C, with the substrate allowing the enzyme to adopt a broad distribution of conformations (dynamically committed state) and the inhibitors (high magnesium and pseudosubstrate) locking it into discrete minima (dynamically quenched state), thereby reducing the motions that allow turnover. These results unveil the role of internal dynamics in both kinase function and regulation.

Lethal Arg9Cys phospholamban mutation hinders Ca2+-ATPase regulation and phosphorylation by protein kinase A.

The regulatory interaction of phospholamban (PLN) with Ca(2+)-ATPase controls the uptake of calcium into the sarcoplasmic reticulum, modulating heart muscle contractility. A missense mutation in PLN cytoplasmic domain (R9C) triggers dilated cardiomyopathy in humans, leading to premature death. Using a combination of biochemical and biophysical techniques both in vitro and in live cells, we show that the R9C mutation increases the stability of the PLN pentameric assembly via disulfide bridge formation, preventing its binding to Ca(2+)-ATPase as well as phosphorylation by protein kinase A. These effects are enhanced under oxidizing conditions, suggesting that oxidative stress may exacerbate the cardiotoxic effects of the PLN(R9C) mutant. These results reveal a regulatory role of the PLN pentamer in calcium homeostasis, going beyond the previously hypothesized role of passive storage for active monomers.

Dynamics connect substrate recognition to catalysis in protein kinase A.

Atomic resolution studies of protein kinases have traditionally been carried out in the inhibitory state, limiting our current knowledge on the mechanisms of substrate recognition and catalysis. Using NMR, X-ray crystallography and thermodynamic measurements, we analyzed the substrate recognition process of cAMP-dependent protein kinase (PKA), finding that entropy and protein dynamics play a prominent role. The nucleotide acts as a dynamic and allosteric activator by coupling the two lobes of apo PKA, enhancing the enzyme dynamics synchronously and priming it for catalysis. The formation of the ternary complex is entropically driven, and NMR spin relaxation data reveal that both substrate and PKA are dynamic in the closed state. Our results show that the enzyme toggles between open and closed states, which indicates that a conformational selection rather than an induced-fit mechanism governs substrate recognition.

One-sample approach to determine the relative orientations of proteins in ternary and binary complexes from residual dipolar coupling measurements.

We present a procedure that supports the acquisition of (1)H-(15)N residual dipolar coupling (RDC) values for individual subunits in binary or ternary protein assemblies from a single experimental sample. Our method relies on asymmetric labeling of each subunit with the following scheme: species A uniformly with (15)N, species B uniformly with (15)N and (13)C, and species C uniformly with (15)N but selectively with (13)C' or (13)C(alpha). Because only a single sample is required, the approach obviates the need for preparing multiple samples and eliminates potential errors introduced from differences in sample conditions. Because numerous biological processes rely on protein assemblies or transient interactions, this method should be well suited for a wide range of future applications.

Backbone NMR resonance assignment of the catalytic subunit of cAMP-dependent protein kinase A in complex with AMP-PNP.

The catalytic subunit of protein kinase A is involved with a number of signal transduction pathways and has been used as a benchmark to study the structural biology and biochemistry for the entire kinase family of enzymes. Here, we report the backbone assignment of the intact 41 kDa catalytic subunit bound to AMP-PNP.

Pseudoenzymatic dealkylation of alkyltins by biological dithiols.

We investigated the time dependence of the degradation of three alkyltin derivatives by a nine amino acid linear peptide (I(1)LGCWCYLR(9)) containing a CXC motif derived from the primary sequence of stannin, a membrane protein involved in alkyltin toxicity. We monitored the reaction kinetics using the intrinsic fluorescence of the tryptophan residue in position 5 of the peptide and found that all of the alkyltins analyzed are progressively degraded to dialkyl derivatives, following a pseudoenzymatic reaction mechanism. The end point of the reactions is the formation of a covalent complex between the disubstituted alkyltin and the peptide cysteines. These data agree with the speciation profiles proposed for polysubstituted alkyltins in the environment and reveal a possible biotic degradation pathway for these toxic compounds.

Expression and purification of isotopically labeled peptide inhibitors and substrates of cAMP-dependant protein kinase A for NMR analysis.

Extensive X-ray crystallographic studies carried out on the catalytic-subunit of protein kinase A (PKA-C) enabled the atomic characterization of inhibitor and/or substrate peptide analogues trapped at its active site. Yet, the structural and dynamic transitions of these peptides from the free to the bound state are missing. These conformational transitions are central to understanding molecular recognition and the enzymatic cycle. NMR spectroscopy allows one to study these phenomena under functionally relevant conditions. However, the amounts of isotopically labeled peptides required for this technique present prohibitive costs for solid-phase peptide synthesis. To enable NMR studies, we have optimized both expression and purification of isotopically enriched substrate/inhibitor peptides using a recombinant fusion protein system. Three of these peptides correspond to the cytoplasmic regions of the wild-type and lethal mutants of the membrane protein phospholamban, while the fourth peptide correspond to the binding epitope of the heat-stable protein kinase inhibitor (PKI(5-24)). The target peptides were fused to the maltose binding protein (MBP), which is further purified using a His(6) tag approach. This convenient protocol allows for the purification of milligram amounts of peptides necessary for NMR analysis.