7 research outputs found
Site-directed spin label mutants of GRP1 PH domain and their measured parameters.
1<p>For spin-labeled mutants (R1), the indicated residue in the Cysless PH domain is changed to Cys and labeled with MTSSL. Also indicated is the secondary structure element in which each spin label is located.</p>2<p>The qualitative ranking of spectral changes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033640#pone-0033640-g004" target="_blank">Figure 4</a> utilizes three categories: large change (++), detectable change (+) and no detectable change (−).</p
Protein-membrane interactions in the optimized, self-consistent EPR docking model.
<p>Shown is the optimized, self-consistent EPR docking model for GRP1 PH domain co-complexed with IP<sub>4</sub> (1FGY <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033640#pone.0033640-Lietzke1" target="_blank">[22]</a>) and docked to a target bilayer. The schematic target bilayer highlights transient positions of backbone phosphates (red-brown spheres) and headgroups (PC or PS, black spheres) from a snapshot of a simulated bilayer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033640#pone.0033640-Hoff1" target="_blank">[50]</a>. (A) Views of the PIP<sub>3</sub> headgroup relative to the mean backbone phosphate plane in both its lowest energy conformation (left) and its PH domain-bound conformation (right), illustrating the effect of PH domain binding on the target headgroup depth and orientation. (B) The PH domain docked to the schematic target bilayer in the optimized geometry. (C) Basic residues of the PH domain (dark blue spheres for R277, K279, K282, R283, R322, K323, R349) that can contact the negatively charged target bilayer in the optimized docking geometry. In some cases, the indicated side chain rotomer was adjusted to enhance membrane contact. (D) Hydrophobic and polar residues (light blue spheres for V278, T280, W281, P321, A346) that can contact the bilayer. Y298 obstructs the view and is not shown; it also contacts the bilayer and, perhaps more importantly, contacts multiple side chains responsible for specific PIP<sub>3</sub> binding. (E) Acidic residues (red spheres for D320, E345, D347) that contact the anionic bilayer surface and are thus proposed to limit protein penetration into the target bilayer.</p
Hyperbolic relationship between spin label depth parameters and membrane penetration depths in the optimized, self-consistent EPR docking model.
<p>As described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033640#s4" target="_blank">Methods</a>, the crystal structure of the GRP1 PH domain co-complex with IP<sub>4</sub> (1FGY <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033640#pone.0033640-Lietzke1" target="_blank">[22]</a>) was modeled with MTSSL spin labels at the 18 chosen positions, then docked to the target bilayer using an interactive procedure that optimizes the known hyperbolic relationship between the measured spin label EPR depth parameters and the calculated spin label membrane penetration depths. Shown are the measured depth parameters for the protein spin labels (filled symbols) and the calibration lipid spin labels (open symbols), as well as the calculated membrane depth for each spin label in the final optimized, self-consistent EPR membrane docking model (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033640#pone-0033640-g006" target="_blank">Figure 6</a>). The excellent agreement with the best-fit hyperbola (solid curve) emphasizes the high quality of the docking model. Depth parameters were measured by EPR power saturation (Methods) at 23°C and samples contained 10–200 µM protein, 40 mM total lipid as SUVs, 25 mM HEPES, 140 mM KCl, 15 mM NaCl, 0.5 mM MgCl<sub>2</sub>, pH 7.4. Except where otherwise indicated, errors are propagated from the errors of the accessibility parameters (Π(NiEDDA) and Π(O<sub>2</sub>)) used to calculate the depth parameter (Eq. 1), n≥15 power settings were used for each accessibility parameter measurement.</p
Effect of Target Membrane Docking on EPR Spectra.
<p>Each spectral overlay shows the effects of target membrane docking on the EPR spectrum of a given MTSSL spin-labeled GRP1 PH domain. The free PH domain was saturated with 200 µM IP<sub>6</sub> and spectra were acquired in the absence and presence of target PC∶ PS∶ PIP<sub>3</sub> (74∶ 24∶ 2) membranes. A spectral change is observed when the free IP<sub>6</sub>-PH domain complex docks to a target PIP<sub>3</sub> headgroup on the membrane surface, releasing IP<sub>6</sub>. Since the ligand binding cleft is occupied in both states the spectral changes are triggered primarly by membrane docking rather than by cleft occupancy (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033640#pone-0033640-g003" target="_blank">Figure 3</a>). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033640#pone-0033640-t001" target="_blank">Table 1</a> qualitatively ranks the magnitudes of the target membrane-induced spectral changes (++, +, −). Each pair of overlayed spectra were collected as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033640#pone-0033640-g003" target="_blank">Figure 3</a> legend thus their relative intensities can be directly compared. Spectra were acquired at 23°C and samples contained 10–200 µM protein, 0 or 40 mM total lipid as SUVs, and 200 µM IP<sub>6</sub> in 25 mM HEPES, 140 mM KCl, 15 mM NaCl, 0.5 mM MgCl<sub>2</sub>, pH 7.4.</p
Control EPR spectra for a representative mutant.
<p>Shown are reproducible EPR spectral overlays for the MTSSL spin-labeled GRP1 PH domain V278R1, illustrating the strategy employed to analyze the spectral effects of membrane docking. (A) V278R1 PH domain in the absence and presence of control PC∶ PS (3∶1) membranes lacking PIP<sub>3</sub>, illustrating spectral broadening due to nonspecific membrane association. (B) V278R1 PH domain saturated with 200 µM IP<sub>6</sub>, both in the absence and presence of control PC∶ PS (3∶1) membranes, showing that unlike the apo PH domain the IP<sub>6</sub>-PH domain complex does not bind nonspecifically to membranes when PIP<sub>3</sub> is absent. (C) V278R1 PH domain saturated with 200 µM IP<sub>6</sub>, both in the absence and presence of target PC∶ PS∶ PIP<sub>3</sub> (74∶ 24∶ 2) membranes, showing the spectral change upon docking of the IP<sub>6</sub>-PH domain complex to membrane-bound PIP<sub>3</sub> (with release of IP<sub>6</sub>). This is the standard comparison carried out for all spin-labeled PH domains (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033640#pone-0033640-g004" target="_blank">Fig. 4</a>), since the free IP<sub>6</sub>-PH domain complex does not dock to background lipids and use of this complex as a reference point ensures that spectral changes are due to the environmental effects of membrane docking, rather than to the conformational effects of ligand binding cleft occupancy. (D) V278R1 PH domain binding to target PC∶ PS∶ PIP<sub>3</sub> (74∶ 24∶ 2) membranes in the absence and presence of saturating 200 µM IP<sub>6</sub>, showing that the competitive inhibitor IP<sub>6</sub> does not perturb PH domain binding to target membrane PIP<sub>3</sub> under these conditions. Each pair of overlayed spectra were obtained for two samples made from the same protein stock to ensure nearly identical spin concentrations, for which the same number of scans were collected and plotted in absolute intensity mode. Double integrations confirmed that each pair of spectra represented virtually identical numbers of spins. Thus, the relative intensities of each spectral pair can be directly compared. Spectra were acquired at 23°C and samples contained 10–200 µM protein, 0 or 40 mM total lipid as SUVs, and 0 or 200 µM IP<sub>6</sub>, in 25 mM HEPES, 140 mM KCl, 15 mM NaCl, 0.5 mM MgCl<sub>2</sub>, pH 7.4.</p
Effect of spin labeling on target membrane binding.
<p>Shown are representative competitive displacement curves for three GRP1 PH domains: Wild Type, Cysless and V278R1. Each PH domain was added to PC∶ PS∶ PIP<sub>3</sub>∶ dansylPE (mole ratios 70∶ 23∶ 2∶ 5) target membrane and allowed to form the PIP<sub>3</sub>-protein complex on the membrane surface. Subsequently, using a standard competition assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033640#pone.0033640-Corbin1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033640#pone.0033640-Landgraf1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033640#pone.0033640-Pilling1" target="_blank">[25]</a>, the competitive inhibitor IP<sub>6</sub> was titrated into the sample, thereby displacing PH domain from the membrane as revealed by decreasing protein-to-membrane FRET. The resulting competition curve was best fit for a homogeneous population of PIP<sub>3</sub>/IP<sub>6</sub> binding sites (solid curves) to determine the K<sub>i</sub> for IP<sub>6</sub>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033640#pone-0033640-t001" target="_blank">Table 1</a> summarizes the measured K<sub>i</sub>(IP<sub>6</sub>) values, which are directly proportional to the affinity of each PH domain for membrane-embedded PIP<sub>3</sub>. Experimental conditions: 0.2 µM PH domain and 200 µM total lipid in 25 mM HEPES, 140 mM KCl, 15 mM NaCl, 0.5 mM MgCl<sub>2</sub>, pH 7.4, 25°C.</p
Single-Molecule Studies Reveal a Hidden Key Step in the Activation Mechanism of Membrane-Bound Protein Kinase C‑α
Protein
kinase C-α (PKCα) is a member of the conventional
family of protein kinase C isoforms (cPKCs) that regulate diverse
cellular signaling pathways, share a common activation mechanism,
and are linked to multiple pathologies. The cPKC domain structure
is modular, consisting of an N-terminal pseudosubstrate peptide, two
inhibitory domains (C1A and C1B), a targeting domain (C2), and a kinase
domain. Mature, cytoplasmic cPKCs are inactive until they are switched
on by a multistep activation reaction that occurs largely on the plasma
membrane surface. Often, this activation begins with a cytoplasmic
Ca<sup>2+</sup> signal that triggers C2 domain targeting to the plasma
membrane where it binds phosphatidylserine (PS) and phosphatidylinositol
4,5-bisphosphate (PIP<sub>2</sub>). Subsequently, the appearance of
the signaling lipid diacylglycerol (DAG) activates the membrane-bound
enzyme by recruiting the inhibitory pseudosubstrate and one or both
C1 domains away from the kinase domain. To further investigate this
mechanism, this study has utilized single-molecule total internal
reflection fluorescence microscopy (TIRFM) to quantitate the binding
and lateral diffusion of full-length PKCα and fragments missing
specific domain(s) on supported lipid bilayers. Lipid binding events,
and events during which additional protein is inserted into the bilayer,
were detected by their effects on the equilibrium bound particle density
and the two-dimensional diffusion rate. In addition to the previously
proposed activation steps, the findings reveal a major, undescribed,
kinase-inactive intermediate. On bilayers containing PS or PS and
PIP<sub>2</sub>, full-length PKCα first docks to the membrane
via its C2 domain, and then its C1A domain embeds itself in the bilayer
even before DAG appears. The resulting pre-DAG intermediate with membrane-bound
C1A and C2 domains is the predominant state of PKCα while it
awaits the DAG signal. The newly detected, membrane-embedded C1A domain
of this pre-DAG intermediate confers multiple useful features, including
enhanced membrane affinity and longer bound state lifetime. The findings
also identify the key molecular step in kinase activation: because
C1A is already membrane-embedded in the kinase off state, recruitment
of C1B to the bilayer by DAG or phorbol ester is the key regulatory
event that stabilizes the kinase on state. More broadly, this study
illustrates the power of single-molecule methods in elucidating the
activation mechanisms and hidden regulatory states of membrane-bound
signaling proteins