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 indicate that a population shift/conformational selection rather than an induced-fit mechanism governs substrate recognition.

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.allostery | cooperativity | phospholamban | substrate recognition | intrinsically disordered proteins

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.allostery ͉ NMR ͉ signaling ͉ enzymes ͉ chemical shift mapping T he protein kinase superfamily is one of the largest gene families in eukaryotes, representing 2-4% of most genomes (1, 2). The human genome, for example, has Ͼ500 predicted protein kinases. Protein kinases relay an extracellular signal into a biological response by phosphoryl transfer on specific substrates resulting in regulation of cell division, memory, differentiation, cell growth, and most other cell processes with exquisite precision. Since the first structure of the catalytic subunit of protein kinase A (PKA-C) was determined in 1991 (3, 4), there has been significant progress in filling the structural space of this family. Over 60 unique kinase structures are now available in the Protein Data Bank (PDB; www.rcsb.org). All of these structures share a highly conserved kidney-shaped core that in PKA-C comprises residues 40-300. The core consists of a small N-terminal lobe formed by ␤-strands that binds and positions ATP during catalysis and a large lobe that provides a docking surface for the substrate. The N terminus ends with a single helix of Ϸ40 residues (A helix). The 50 residues comprising the C terminus wrap around the two lobes, docking into a hydrophobic pocket located in the small lobe (3, 4).Despite sharing a highly conserved core, protein kinases are remarkably diverse in terms of their regulation, activation, and substrate recognition. Crystal structures suggest that kinases are highly dynamic, with allosteric networks that radiate throughout the molecule. Unfortunately, these structures are static and often only include the kinase core. Although there have been extensive studies of kinases in solution, including hydrogen/ deuterium exchange coupled with mass spectrometry (H/D-MS), fluorescence anisotropy (5-7), and small-angle x-ray scattering (8, 9), these results lack atomic-level resolution and offer an incomplete picture of protein dynamics and recognition mechanisms, presenting a substantial limitation in understanding the enzymatic cycle of kinases, and leaving many questions unanswered. How do these enzymes discriminate cognate and noncognate substrates? What is the role of the nucleotide in substrate recognition? Does allostery play a fundamental role in these recognition processes?Here, we anal...

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 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 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 methanobactin’s 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.

Phospholamban (PLN) and sarcolipin (SLN) are two single-pass membrane proteins that regulate Ca2+-ATPase (SERCA), an ATP-driven pump that translocates calcium ions into the lumen of the sarcoplasmic reticulum, initiating muscle relaxation. Both proteins bind SERCA through intramembrane interactions, impeding calcium translocation. While phosphorylation of PLN at Ser-16 and/or Thr-17 reestablishes calcium flux, the regulatory mechanism of SLN remains elusive. SERCA has been crystallized in several different states along the enzymatic reaction coordinates, providing remarkable mechanistic information; however, the lack of high-resolution crystals in the presence of PLN and SLN limits the current understanding of the regulatory mechanism. This brief review offers a survey of our hybrid structural approach using solution and solid-state NMR methodologies to understand SERCA regulation from the point of view of PLN and SLN. These results have improved our understanding of the calcium translocation process and are the basis for designing new therapeutic approaches to ameliorate muscle malfunctions.