Larry R. Masterson

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.

Allosteric cooperativity in protein kinase A

Allosteric signaling in proteins requires long-range communication mediated by highly conserved residues, often triggered by ligand binding. In this article, we map the allosteric network in the catalytic subunit of protein kinase A using NMR spectroscopy. We show that positive allosteric cooperativity is generated by nucleotide and substrate binding during the transitions through the major conformational states: apo, intermediate, and closed. The allosteric network is disrupted by a single site mutation (Y204A), which also decouples the cooperativity of ligand binding. Because protein kinase A is the prototype for the entire kinome, these findings may serve as a paradigm for describing long-range coupling in other protein kinases.

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.

NMR, Mass Spectrometry and Chemical Evidence Reveal a Different Chemical Structure for Methanobactin That Contains Oxazolone Rings

Methanobactin (mb) is a small copper-binding peptide produced by methanotrophic bacteria and is intimately involved in both their copper metabolism and their role in the global carbon cycle. The structure for methanobactin comprises seven amino acids plus two chromophoric residues that appear unique to methanobactin. In a previously published structure, both chromophoric residues contain a thiocarbonyl attached to a hydroxyimidazolate ring. In addition, one is attached to a pyrrolidine ring, while the other is attached to an isopropyl ester. A published X-ray determined structure for methanobactin shows these two chromophoric groups forming an N2S2 binding site for a single Cu(I) ion with a distorted tetrahedral geometry. In this report we show that NMR, mass spectrometry, and chemical data reveal a chemical structure that is significantly different than the previously published one. Specifically, the 1H and 13C NMR assignments are inconsistent with an N-terminal isopropyl ester and point instead to a 3-methylbutanoyl group. Our data also indicate that oxazolone rings instead of hydroxyimidazolate rings form the core of the two chromophoric residues. Because these rings are directly involved in the binding of Cu(I) and other metals by methanobactin and are likely involved in the many chemical activities displayed by methanobactin, their correct identity is central to developing an accurate and detailed understanding of methanobactins many chemical and biological roles. For example, the oxazolone rings make methanobactin structurally more similar to other bacterially produced bactins and siderophores and suggest pathways for its biosynthesis.

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 non-catalytic protein scaffold, we recently generated a novel RNA ligase by in vitro directed evolution. This artificial enzyme lost its original fold and adopted an entirely novel structure with dramatically enhanced conformational dynamics, demonstrating that a primordial fold with suitable flexibility is sufficient to carry out enzymatic function.

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

The regulatory interaction of phospholamban (PLN) with Ca2+-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 Ca2+-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 PLNR9C 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.

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.

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 enzymes 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.

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.

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.