Mapping the Hydrogen Bond Networks in the Catalytic Subunit of Protein Kinase A Using H/D Fractionation Factors

Protein kinase A is a prototypical phosphoryl transferase, sharing its catalytic core (PKA-C) with the entire kinase family. PKA-C substrate recognition, active site organization, and product release depend on the enzyme’s conformational transitions from the open to the closed state, which regulate its allosteric cooperativity. Here, we used equilibrium nuclear magnetic resonance hydrogen/deuterium (H/D) fractionation factors (φ) to probe the changes in the strength of hydrogen bonds within the kinase upon binding the nucleotide and a pseudosubstrate peptide (PKI_5–24). We found that the φ values decrease upon binding both ligands, suggesting that the overall hydrogen bond networks in both the small and large lobes of PKA-C become stronger. However, we observed several important exceptions, with residues displaying higher φ values upon ligand binding. Notably, the changes in φ values are not localized near the ligand binding pockets; rather, they are radiated throughout the entire enzyme. We conclude that, upon ligand and pseudosubstrate binding, the hydrogen bond networks undergo extensive reorganization, revealing that the open-to-closed transitions require global rearrangements of the internal forces that stabilize the enzyme’s fold

Structures of the Excited States of Phospholamban and Shifts in Their Populations upon Phosphorylation

Phospholamban is an integral membrane protein that controls the calcium balance in cardiac muscle cells. As the function and regulation of this protein require the active involvement of low populated states in equilibrium with the native state, it is of great interest to acquire structural information about them. In this work, we calculate the conformations and populations of the ground state and the three main excited states of phospholamban by incorporating nuclear magnetic resonance residual dipolar couplings as replica-averaged structural restraints in molecular dynamics simulations. We then provide a description of the manner in which phosphorylation at Ser16 modulates the activity of the protein by increasing the sizes of the populations of its excited states. These results demonstrate that the approach that we describe provides a detailed characterization of the different states of phospholamban that determine the function and regulation of this membrane protein. We anticipate that the knowledge of conformational ensembles enable the design of new dominant negative mutants of phospholamban by modulating the relative populations of its conformational substates

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

Formaldehyde impairs SERCA activity.

<p>A) SERCA activity is impaired by pre-exposure to formaldehyde for 30 min as measured by NADH-coupled enzyme assay (n = 3 for each point). Activity as a function of the calcium concentration (negative logarithm pCa) was fit using the Hill equation. B) V_max was decreased to 70% of the initial SERCA control value upon pretreatment by 1 mM formaldehyde and SERCA activity was abolished by ≥10 mM formaldehyde. *p < 0.05, and ***p < 0.001.</p

Formaldehyde does not release calcium in HEK293t via ryanodine or IP3 receptor activation.

<p>A) In HEK293t cells no responses were induced by ryanodine at the given concentrations. B) Dantrolene increased intracellular calcium at 2 μM, but reduced calcium levels at 20 μM. C) The magnitude of repetitive formaldehyde-induced calcium responses was not altered by co-administration of ryanodine 30 μM or D) dantrolene 20 μM.</p

Formaldehyde activates TRPA1-deficient DRG neurons by release of calcium from the endoplasmatic reticulum.

<p>A) Formaldehyde increases intracellular calcium levels in cultured TRPA1^-/- DRG neurons. B) Concentration-response of calcium transients, normalized to formaldehyde 400 mM (n = 71). A second protocol including formaldehyde 126 mM (not shown) provided an additional data point for a robust concentration-response fit. C) In the absence of extracellular calcium formaldehyde 40 mM induced intracellular calcium increases in DRG neurons from TRPA1^-/- mice (n = 212). D) Calcium transients in the absence of extracellular calcium were 81% of the subsequent response in the presence of extracellular calcium. E) In the absence of extracellular calcium, formaldehyde was applied twice (Control, no application during the period indicated by the hatched bar, n = 143). In experiments depleting mitochondrial calcium stores (hatched bar: incubation with CCCP 2 μM, n = 147), the responses induced by formaldehyde had a similar magnitude as the control. However, if the calcium stores of the endoplasmatic reticulum were depleted by SERCA inhibition (hatched bar: Thapsigargin 2 μM, n = 148), the second response was reduced to 19% of the first response. F) Second formaldehyde-induced calcium responses of TRPA1^-/- neurons normalized to the first response.</p

Formaldehyde-induced calcium release in cell lines.

<p>A) Cells of common cell lines were exposed to increasing concentrations of formaldehyde. Chinese hamster ovary (CHO-K1, n = 58) cells were more sensitive to formaldehyde than a mouse neuroblastoma rat neuron hybrid cell line (ND7/23, n = 370) and HEK293t cells (n = 276). Responses in CHO-K1 cells were similar compared to DRG neurons [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123762#pone.0123762.ref003" target="_blank">3</a>]. Formaldehyde concentrations up to 40 mM show slowly reversible responses. B) Responses are normalized to formaldehyde 400 mM, which causes a permanent submaximal calcium increase. Note that in the original formalin test, 616 mM are injected into the paw.</p

Ca²⁺ ATPase Conformational Transitions in Lipid Bilayers Mapped by Site-directed Ethylation and Solid-State NMR

To transmit signals across cellular compartments, many membrane-embedded enzymes undergo extensive conformational rearrangements. Monitoring these events in lipid bilayers by NMR at atomic resolution has been challenging due to the large size of these systems. It is further exacerbated for large mammalian proteins that are difficult to express and label with NMR-active isotopes. Here, we synthesized and engineered ¹³C ethyl groups on native cysteines to map the structural transitions of the sarcoplasmic reticulum Ca²⁺-ATPase, a 110 kDa transmembrane enzyme that transports Ca²⁺ into the sarcoplasmic reticulum. Using magic angle spinning NMR, we monitored the chemical shifts of the methylene and methyl groups of the derivatized cysteine residues along the major steps of the enzymatic cycle. The methylene chemical shifts are sensitive to the ATPase conformational changes induced upon nucleotide and Ca²⁺ ion binding and are ideal probes for active and inactive states of the enzyme. This new approach is extendable to large mammalian enzymes and signaling proteins with native or engineered cysteine residues in their amino acid sequence

Formaldehyde releases calcium from intracellular stores in untransfected HEK293t cells.

<p>A) HEK293t cells were patch-clamped at—60 mV, voltage ramps were applied at an interval of 5 seconds. The currents at ±60 mV of these voltage ramps are displayed; exposure to formaldehyde did not change transmembrane currents (n = 9). B) The steady-state current-voltage relationships 30 s before (open symbols) and during (closed symbols) exposure to formaldehyde are not significantly different (n = 12). C) Calcium transients evoked by formaldehyde 40 mM in HEK293t cells (n = 235) are similar in absence and presence of extracellular calcium, indicating an intracellular release and explaining the lack of a detectable transmembrane current. D) Calcium transients evoked by formaldehyde 40 mM are decreased after exposure to Thapsigargin 5 μM. Formaldehyde responses recovered after replenishing intracellular calcium stores (n = 111). Note the slight delay in calcium clearance due to the application of Thapsigargin, indicating a slow calcium efflux out of the stores when SERCA is inhibited.</p

Formaldehyde activates mouse keratinocytes by release of calcium from the endoplasmatic reticulum.

<p>A) Formaldehyde increases intracellular calcium levels in primary cultured C57BL/6 keratinocytes. B) Concentration-response of calcium transients, normalized to formaldehyde 400 mM (n = 142). C) In the absence of extracellular calcium formaldehyde 40 mM induced intracellular calcium increases in mouse keratinocytes (n = 170). D) Calcium transients in the absence of extracellular calcium were 82% of the subsequent response in the presence of extracellular calcium. E) In the absence of extracellular calcium, formaldehyde was applied twice (Control, no application during period indicated by the hatched bar, n = 165). In experiments depleting mitochondrial calcium stores (hatched bar: incubation with CCCP 2 μM, n = 119) the responses induced by formaldehyde had a similar magnitude as the control. However, when the calcium stores of the endoplasmatic reticulum were depleted by SERCA inhibition (hatched bar: thapsigargin 2 μM, n = 181), the second response was reduced to 58% of the first response. F) Amplitude of the second formaldehyde-induced calcium responses of keratinocytes, normalized to the first response.</p