Sandeep Pallikkuth

Phosphomimetic mutations enhance oligomerization of phospholemman and modulate its interaction with the Na/K-ATPase.

Na/K-ATPase (NKA) activity is dynamically regulated by an inhibitory interaction with a small transmembrane protein, phospholemman (PLM). Inhibition is relieved upon PLM phosphorylation. Phosphorylation may alter how PLM interacts with NKA and/or itself, but details of these interactions are unknown. To address this, we quantified FRET between PLM and its regulatory target NKA in live cells. Phosphorylation of PLM was mimicked by mutation S63E (PKC site), S68E (PKA/PKC site), or S63E/S68E. The dependence of FRET on protein expression in live cells yielded information about the structure and binding affinity of the PLM-NKA regulatory complex. PLM phosphomimetic mutations altered the quaternary structure of the regulatory complex and reduced the apparent affinity of the PLM-NKA interaction. The latter effect was likely due to increased oligomerization of PLM phosphomimetic mutants, as suggested by PLM-PLM FRET measurements. Distance constraints obtained by FRET suggest that phosphomimetic mutations slightly alter the oligomer quaternary conformation. Photon-counting histogram measurements revealed that the major PLM oligomeric species is a tetramer. We conclude that phosphorylation of PLM increases its oligomerization into tetramers, decreases its binding to NKA, and alters the structures of both the tetramer and NKA regulatory complex.

Phosphorylated Phospholamban Stabilizes a Compact Conformation of the Cardiac Calcium-ATPase

The sarcoendoplasmic reticulum calcium ATPase (SERCA) plays a key role in cardiac calcium handling and is considered a high-value target for the treatment of heart failure. SERCA undergoes conformational changes as it harnesses the chemical energy of ATP for active transport. X-ray crystallography has provided insight into SERCA structural substates, but it is not known how well these static snapshots describe in vivo conformational dynamics. The goals of this work were to quantify the direction and magnitude of SERCA motions as the pump performs work in live cardiac myocytes, and to identify structural determinants of SERCA regulation by phospholamban. We measured intramolecular fluorescence resonance energy transfer (FRET) between fluorescent proteins fused to SERCA cytoplasmic domains. We detected four discrete structural substates for SERCA expressed in cardiac muscle cells. The relative populations of these discrete states oscillated with electrical pacing. Low FRET states were most populated in low Ca (diastole), and were indicative of an open, disordered structure for SERCA in the E2 (Ca-free) enzymatic substate. High FRET states increased with Ca (systole), suggesting rigidly closed conformations for the E1 (Ca-bound) enzymatic substates. Notably, a special compact E1 state was observed after treatment with β-adrenergic agonist or with coexpression of phosphomimetic mutants of phospholamban. The data suggest that SERCA calcium binding induces the pump to undergo a transition from an open, dynamic conformation to a closed, ordered structure. Phosphorylated phospholamban stabilizes a unique conformation of SERCA that is characterized by a compact architecture.

Dysferlin Forms a Dimer Mediated by the C2 Domains and the Transmembrane Domain In Vitro and in Living Cells

Dysferlin was previously identified as a key player in muscle membrane repair and its deficiency leads to the development of muscular dystrophy and cardiomyopathy. However, little is known about the oligomerization of this protein in the plasma membrane. Here we report for the first time that dysferlin forms a dimer in vitro and in living adult skeletal muscle fibers isolated from mice. Endogenous dysferlin from rabbit skeletal muscle exists primarily as a ∼460 kDa species in detergent-solubilized muscle homogenate, as shown by sucrose gradient fractionation, gel filtration and cross-linking assays. Fluorescent protein (YFP) labeled human dysferlin forms a dimer in vitro, as demonstrated by fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analyses. Dysferlin also dimerizes in living cells, as probed by fluorescence resonance energy transfer (FRET). Domain mapping FRET experiments showed that dysferlin dimerization is mediated by its transmembrane domain and by multiple C2 domains. However, C2A did not significantly contribute to dimerization; notably, this is the only C2 domain in dysferlin known to engage in a Ca-dependent interaction with cell membranes. Taken together, the data suggest that Ca-insensitive C2 domains mediate high affinity self-association of dysferlin in a parallel homodimer, leaving the Ca-sensitive C2A domain free to interact with membranes.

Phosphorylated Phospholamban Stabilizes a Distinct Compact Conformation of Sarco(endo)Plasmic Reticulum Ca-ATPase

Sarco(endo)plasmic reticulum Ca-ATPase (SERCA) is inhibited by the 52 amino acid protein phospholamban (PLB). Inhibition of SERCA is relieved when PLB is phosphorylated by protein kinase A (PKA). To better understand the mechanism of this regulatory mechanism on SERCA structure, we designed a “2-color” SERCA labeled with donor and acceptor fluorescent proteins in the A- and N-domain. Intramolecular fluorescence resonance energy transfer (FRET) between these two domains was quantified as an index of SERCA conformation. Fluorescence lifetime distribution analysis (FLDA) showed that SERCA sampled three discrete conformations in the absence of PLB or in the presence of non-phosphorylatable PLB (S16A). Co-transfection of SERCA with phosphomimetic PLB (S16E) revealed an additional state characterized by a highly compact conformation. This unique state was only observed under conditions of high intracellular calcium. The data support the hypothesis that PLB remains bound to SERCA after phosphorylation by PKA, inducing the pump to assume a very compact E1 conformation.

Dynamic Conformational Transitions of Sarcoendoplasmic Reticulum Ca-ATPase (SERCA) Quantified by Single Molecule FRET

We present here the measurement of dynamic transition of SERCA among its various conformational states during its Ca pumping cycle. This is achieved by monitoring single-molecule FRET (smFRET) between fluorescent proteins fused to the N and A domains of SERCA. We observe fluctuations in the FRET efficiency by measuring the changes in the fluorescence lifetime of the donor fluorescent protein as a function of time as detergent solubilized SERCA single molecules transit through the laser excitation spot. Making histograms of these FRET efficiency fluctuations, we detected multiple discrete FRET levels indicating different conformational states. The mean FRET efficiency values determined from the histograms are used to assign conformational states of SERCA to the FRET efficiency time trajectories. The fluctuations in these time trajectories then provide us the timescales of the transition of SERCA between its different conformational states. We detect fast transitions (μs) between the long lived (ms) conformational states of SERCA. We also measure decrease in these transition timescales in the presence of Ca. This information provides new insight to the structural dynamics of SERCA during the Ca transport cycle.

Conformational Dynamics of Sarcoendoplasmic Reticulum Ca-ATPase (SERCA) Quantified by Intramolecular FRET Fluctuations

To quantify the dynamic transitions of SERCA between various structural substates, we measured intramolecular FRET between fluorescent proteins fused to the N and A domains with fluorescence lifetime fluctuation analysis. This ‘2-color’ SERCA was subjected to pulsed laser excitation and fluorescence was detected with time correlated single photon counting. The arrival times of photons from short fluorescence bursts were analyzed using maximum likelihood estimation for determination of the donor fluorescence lifetimes. By monitoring lifetimes of a few molecules at a time, we detected multiple discrete FRET levels indicating different conformational states. A broad range of conformations was observed, including structures not detected by steady state experiments. We also recorded lifetime trajectories indicating changes in FRET efficiency consistent with rapid transitions between long-lived conformational states. The data provide insight into SERCA structural dynamics during the Ca transport cycle.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Conformational Changes of SERCA Revealed by Intramolecular FRET

The first structures of the sarcoendoplasmic reticulum ATPase (SERCA) determined by X-ray crystallography suggested that pump undergoes a large conformational change during catalytic cycling. The transition from the E1 (Ca-bound) state to the E2 (Ca-free) state was predicted to decrease inter-domain separation distance with closure of the SERCA cytoplasmic headpiece. To test this model, we fused Cerulean to the A-domain and YFP to the N or P or TM-domain of SERCA2a. These “2-color” SERCA constructs were expressed in AAV cells, and SERCA structure transitions were detected by changes in intramolecular fluorescence resonance energy transfer (FRET). FRET decreased with thapsigargin for the two N-domain fusion sites (residues 510, 577), while the P- (residue 610) and TM- domain (C-terminus) fusions showed increased FRET with thapsigargin. Unexpectedly, FRET in permeabilized cells was higher in Ca [10 μM] than in EGTA for all constructs, suggesting an increase in domain separation distance with the E1 to E2 transition. These observations were supported by parallel experiments in fluorescence lifetime distribution (FLD) analysis. FLD resolved two broad distributions of fluorescence lifetimes for 2-color SERCA expressed in live cardiac myocytes, consistent with two major FRET states. The relative populations of these states oscillated with electrical pacing, favoring the high FRET (short distance) state during systole (contraction) and the low FRET (long distance) state during diastole (relaxation). We expect 2-color SERCA constructs to be useful for exploring the magnitude, direction, and kinetics of calcium pump conformational changes. They may also be useful for screening candidate compounds for modulation of SERCA pump structure and function.