Effect of Phosphorylation on Interactions between Transmembrane Domains of SERCA and Phospholamban.

We have used site-directed spin labeling and electron paramagnetic resonance (EPR) to map interactions between the transmembrane (TM) domains of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) and phospholamban (PLB) as affected by PLB phosphorylation. In the cardiac sarcoplasmic reticulum, PLB binding to SERCA results in Ca-dependent enzyme inhibition, which is reversed by PLB phosphorylation at Ser16. Previous spectroscopic studies on SERCA-PLB have largely focused on the cytoplasmic domain of PLB, showing that phosphorylation induces a structural shift in this domain relative to SERCA. However, SERCA inhibition is due entirely to TM domain interactions. Therefore, we focus here on PLB's TM domain, attaching Cys-reactive spin labels at five different positions. In each case, continuous-wave EPR indicated moderate spin-label mobility, with the addition of SERCA revealing two populations, one indistinguishable from PLB alone and another with more restricted rotational mobility, presumably due to SERCA-binding. Phosphorylation had no effect on the rotational mobility of either component but significantly decreased the mole fraction of the restricted component. Solvent-accessibility experiments using power-saturation EPR and saturation-recovery EPR confirmed that these two spectral components were SERCA-bound and unbound PLB and showed that phosphorylation increased the overall lipid accessibility of the TM domain by increasing the fraction of unbound PLB. However-based on these results-at physiological levels of SERCA and PLB, most SERCA would have bound PLB even after phosphorylation. Additionally, no structural shift in the TM domain of SERCA-bound PLB was detected, as there were no significant changes in membrane insertion depth or its accessibility. Therefore, we conclude that under physiological conditions, the phosphorylation of PLB induces little or no change in the interaction of the TM domain with SERCA, so relief of inhibition is predominantly due to the previously observed structural shift in the cytoplasmic domain.

Dynamic rearrangement of the intrinsic ligand regulates KCNH potassium channels.

KCNH voltage-gated potassium channels (EAG, ERG, and ELK) play significant roles in neuronal and cardiac excitability. They contain cyclic nucleotide-binding homology domains (CNBHDs) but are not directly regulated by cyclic nucleotides. Instead, the CNBHD ligand-binding cavity is occupied by an intrinsic ligand, which resides at the intersubunit interface between the N-terminal eag domain and the C-terminal CNBHD. We show that, in Danio rerio ELK channels, this intrinsic ligand is critical for voltage-dependent potentiation (VDP), a process in which channel opening is stabilized by prior depolarization. We demonstrate that an exogenous peptide corresponding to the intrinsic ligand can bind to and regulate zebrafish ELK channels. This exogenous intrinsic ligand inhibits the channels before VDP and potentiates the channels after VDP. Furthermore, using transition metal ion fluorescence resonance energy transfer and a fluorescent noncanonical amino acid L-Anap, we show that there is a rearrangement of the intrinsic ligand relative to the CNBHD during VDP. We propose that the intrinsic ligand switches from antagonist to agonist as a result of a rearrangement of the eag domain-CNBHD interaction during VDP.

Structural insights into the mechanisms of CNBD channel function.

Cyclic nucleotide-binding domain (CNBD) channels are a family of ion channels in the voltage-gated K+ channel superfamily that play crucial roles in many physiological processes. CNBD channels are structurally similar but functionally very diverse. This family includes three subfamilies: (1) the cyclic nucleotide-gated (CNG) channels, which are cation-nonselective, voltage-independent, and cyclic nucleotide-gated; (2) the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are weakly K+ selective, hyperpolarization-activated, and cyclic nucleotide-gated; and (3) the ether-à-go-go-type (KCNH) channels, which are strongly K+ selective, depolarization-activated, and cyclic nucleotide-independent. Recently, several high-resolution structures have been reported for intact CNBD channels, providing a structural framework to better understand their diverse function. In this review, we compare and contrast the recent structures and discuss how they inform our understanding of ion selectivity, voltage-dependent gating, and cyclic nucleotide-dependent gating within this channel family.

The Therapeutic Antibody LM609 Selectively Inhibits Ligand Binding to Human αVß3 Integrin via Steric Hindrance.

The LM609 antibody specifically recognizes αVß3 integrin and inhibits angiogenesis, bone resorption, and viral infections in an arginine-glycine-aspartate-independent manner. LM609 entered phase II clinical trials for the treatment of several cancers and was also used for αVß3-targeted radioimmunotherapy. To elucidate the mechanisms of recognition and inhibition of αVß3 integrin, we solved the structure of the LM609 antigen-binding fragment by X-ray crystallography and determined its binding affinity for αVß3. Using single-particle electron microscopy, we show that LM609 binds at the interface between the ß-propeller domain of the αV chain and the ßI domain of the ß3 chain, near the RGD-binding site, of all observed integrin conformational states. Integrating these data with fluorescence size-exclusion chromatography, we demonstrate that LM609 sterically hinders access of large ligands to the RGD-binding pocket, without obstructing it. This work provides a structural framework to expedite future efforts utilizing LM609 as a diagnostic or therapeutic tool.

CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel.

Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) ion channels play crucial physiological roles in phototransduction, olfaction, and cardiac pace making. These channels are characterized by the presence of a carboxyl-terminal cyclic nucleotide-binding domain (CNBD) that connects to the channel pore via a C-linker domain. Although cyclic nucleotide binding has been shown to promote CNG and HCN channel opening, the precise mechanism underlying gating remains poorly understood. Here we used cryoEM to determine the structure of the intact LliK CNG channel isolated from Leptospira licerasiae-which shares sequence similarity to eukaryotic CNG and HCN channels-in the presence of a saturating concentration of cAMP. A short S4-S5 linker connects nearby voltage-sensing and pore domains to produce a non-domain-swapped transmembrane architecture, which appears to be a hallmark of this channel family. We also observe major conformational changes of the LliK C-linkers and CNBDs relative to the crystal structures of isolated C-linker/CNBD fragments and the cryoEM structures of related CNG, HCN, and KCNH channels. The conformation of our LliK structure may represent a functional state of this channel family not captured in previous studies.

A bifunctional spin label reports the structural topology of phospholamban in magnetically-aligned bicelles.

We have applied a bifunctional spin label and EPR spectroscopy to determine membrane protein structural topology in magnetically-aligned bicelles, using monomeric phospholamban (PLB) as a model system. Bicelles are a powerful tool for studying membrane proteins by NMR and EPR spectroscopies, where magnetic alignment yields topological constraints by resolving the anisotropic spectral properties of nuclear and electron spins. However, EPR bicelle studies are often hindered by the rotational mobility of monofunctional Cys-linked spin labels, which obscures their orientation relative to the protein backbone. The rigid and stereospecific TOAC label provides high orientational sensitivity but must be introduced via solid-phase peptide synthesis, precluding its use in large proteins. Here we show that a bifunctional methanethiosulfonate spin label attaches rigidly and stereospecifically to Cys residues at i and i+4 positions along PLB's transmembrane helix, thus providing orientational resolution similar to that of TOAC, while being applicable to larger membrane proteins for which synthesis is impractical. Computational modeling and comparison with NMR data shows that these EPR experiments provide accurate information about helix tilt relative to the membrane normal, thus establishing a robust method for determining structural topology in large membrane proteins with a substantial advantage in sensitivity over NMR.

Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation.

We showed previously that phosphorylation of Noxa, a 54-residue Bcl-2 protein, at serine 13 (Ser13) inhibited its ability to promote apoptosis through interactions with canonical binding partner, Mcl-1. Using EPR spectroscopy, molecular dynamics (MD) simulations and binding assays, we offer evidence that a structural alteration caused by phosphorylation partially masks Noxa's BH3 domain, inhibiting the Noxa-Mcl-1 interaction. EPR of unphosphorylated Noxa, with spin-labeled amino acid TOAC incorporated within the BH3 domain, revealed equilibrium between ordered and dynamically disordered states. Mcl-1 further restricted the ordered component for non-phosphorylated Noxa, but left the pSer13 Noxa profile unchanged. Microsecond MD simulations indicated that the BH3 domain of unphosphorylated Noxa is housed within a flexible loop connecting two antiparallel ß-sheets, flanked by disordered N- and C-termini and Ser13 phosphorylation creates a network of salt-bridges that facilitate the interaction between the N-terminus and the BH3 domain. EPR showed that a spin label inserted near the N-terminus was weakly immobilized in unphosphorylated Noxa, consistent with a solvent-exposed helix/loop, but strongly constrained in pSer13 Noxa, indicating a more ordered peptide backbone, as predicted by MD simulations. Together these studies reveal a novel mechanism by which phosphorylation of a distal serine inhibits a pro-apoptotic BH3 domain and promotes cell survival.

Optimization of bicelle lipid composition and temperature for EPR spectroscopy of aligned membranes.

We have optimized the magnetic alignment of phospholipid bilayered micelles (bicelles) for EPR spectroscopy, by varying lipid composition and temperature. Bicelles have been extensively used in NMR spectroscopy for several decades, in order to obtain aligned samples in a near-native membrane environment and take advantage of the intrinsic sensitivity of magnetic resonance to molecular orientation. Recently, bicelles have also seen increasing use in EPR, which offers superior sensitivity and orientational resolution. However, the low magnetic field strength (less than 1 T) of most conventional EPR spectrometers results in homogeneously oriented bicelles only at a temperature well above physiological. To optimize bicelle composition for magnetic alignment at reduced temperature, we prepared bicelles containing varying ratios of saturated (DMPC) and unsaturated (POPC) phospholipids, using EPR spectra of a spin-labeled fatty acid to assess alignment as a function of lipid composition and temperature. Spectral analysis showed that bicelles containing an equimolar mixture of DMPC and POPC homogeneously align at 298 K, 20 K lower than conventional DMPC-only bicelles. It is now possible to perform EPR studies of membrane protein structure and dynamics in well-aligned bicelles at physiological temperatures and below.

Synthetic phosphopeptides enable quantitation of the content and function of the four phosphorylation states of phospholamban in cardiac muscle.

We have studied the differential effects of phospholamban (PLB) phosphorylation states on the activity of the sarcoplasmic reticulum Ca-ATPase (SERCA). It has been shown that unphosphorylated PLB (U-PLB) inhibits SERCA and that phosphorylation of PLB at Ser-16 or Thr-17 relieves this inhibition in cardiac sarcoplasmic reticulum. However, the levels of the four phosphorylation states of PLB (U-PLB, P16-PLB, P17-PLB, and doubly phosphorylated 2P-PLB) have not been measured quantitatively in cardiac tissue, and their functional effects on SERCA have not been determined directly. We have solved both problems through the chemical synthesis of all four PLB species. We first used the synthetic PLB as standards for a quantitative immunoblot assay, to determine the concentrations of all four PLB phosphorylation states in pig cardiac tissue, with and without left ventricular hypertrophy (LVH) induced by aortic banding. In both LVH and sham hearts, all phosphorylation states were significantly populated, but LVH hearts showed a significant decrease in U-PLB, with a corresponding increase in the ratio of total phosphorylated PLB to U-PLB. To determine directly the functional effects of each PLB species, we co-reconstituted each of the synthetic peptides in phospholipid membranes with SERCA and measured calcium-dependent ATPase activity. SERCA inhibition was maximally relieved by P16-PLB (the most highly populated PLB state in cardiac tissue homogenates), followed by 2P-PLB, then P17-PLB. These results show that each PLB phosphorylation state uniquely alters Ca(2+) homeostasis, with important implications for cardiac health, disease, and therapy.

Open and closed conformations of the isolated transmembrane domain of death receptor 5 support a new model of activation.

It has long been presumed that activation of the apoptosis-initiating Death Receptor 5, as well as other structurally homologous members of the TNF-receptor superfamily, relies on ligand-stabilized trimerization of noninteracting receptor monomers. We and others have proposed an alternate model in which the TNF-receptor dimer-sitting at the vertices of a large supramolecular receptor network of ligand-bound receptor trimers-undergoes a closed-to-open transition, propagated through a scissorslike conformational change in a tightly bundled transmembrane (TM) domain dimer. Here we have combined electron paramagnetic resonance spectroscopy and potential-of-mean force calculations on the isolated TM domain of the long isoform of DR5. The experiments and calculations both independently validate that the opening transition is intrinsic to the physical character of the TM domain dimer, with a significant energy barrier separating the open and closed states.

Protein-protein interactions in calcium transport regulation probed by saturation transfer electron paramagnetic resonance.

We have used electron paramagnetic resonance (EPR) to probe the homo- and heterooligomeric interactions of reconstituted sarcoplasmic reticulum Ca-ATPase (SERCA) and its regulator phospholamban (PLB). SERCA is responsible for restoring calcium to the sarcoplasmic reticulum to allow muscle relaxation, whereas PLB inhibits cardiac SERCA unless phosphorylated at Ser(16). To determine whether changes in protein association play essential roles in regulation, we detected the microsecond rotational diffusion of both proteins using saturation transfer EPR. Peptide synthesis was used to create a fully functional and monomeric PLB mutant with a spin label rigidly coupled to the backbone of the transmembrane helix, while SERCA was reacted with a Cys-specific spin label. Saturation transfer EPR revealed that sufficiently high lipid/protein ratios minimized self-association for both proteins. Under these dilute conditions, labeled PLB was substantially immobilized after co-reconstitution with unlabeled SERCA, reflecting their association to form the regulatory complex. Ser(16) phosphorylation slightly increased this immobilization. Complementary measurements with labeled SERCA showed no change in mobility after co-reconstitution with unlabeled PLB, regardless of its phosphorylation state. We conclude that phosphorylating monomeric PLB can relieve SERCA inhibition without changes in the oligomeric states of these proteins, indicating a structural rearrangement within the heterodimeric regulatory complex.

Structural and functional dynamics of an integral membrane protein complex modulated by lipid headgroup charge.

We have used membrane surface charge to modulate the structural dynamics of an integral membrane protein, phospholamban (PLB), and thereby its functional inhibition of the sarcoplasmic reticulum Ca-ATPase (SERCA). It was previously shown by electron paramagnetic resonance, in vesicles of neutral lipids, that the PLB cytoplasmic domain is in equilibrium between an ordered T state and a dynamically disordered R state and that phosphorylation of PLB increases the R state and relieves SERCA inhibition, suggesting that R is less inhibitory. Here, we sought to control the T/R equilibrium by an alternative means-varying the lipid headgroup charge, thus perturbing the electrostatic interaction of PLB's cationic cytoplasmic domain with the membrane surface. We resolved the T and R states not only by electron paramagnetic resonance in the absence of SERCA but also by time-resolved fluorescence resonance energy transfer from SERCA to PLB, thus probing directly the SERCA-PLB complex. Compared to neutral lipids, anionic lipids increased both the T population and SERCA inhibition, while cationic lipids had the opposite effects. In contrast to conventional models, decreased inhibition was not accompanied by decreased binding. We conclude that PLB binds to SERCA in two distinct structural states of the cytoplasmic domain: an inhibitory T state that interacts strongly with the membrane surface and a less inhibitory R state that interacts more strongly with the anionic SERCA cytoplasmic domain. Modulating membrane surface charge provides an effective way of investigating the correlation between structural dynamics and function of integral membrane proteins.

Large-scale opening of utrophin's tandem calponin homology (CH) domains upon actin binding by an induced-fit mechanism.

We have used site-directed spin labeling and pulsed electron paramagnetic resonance to resolve a controversy concerning the structure of the utrophin-actin complex, with implications for the pathophysiology of muscular dystrophy. Utrophin is a homolog of dystrophin, the defective protein in Duchenne and Becker muscular dystrophies, and therapeutic utrophin derivatives are currently being developed. Both proteins have a pair of N-terminal calponin homology (CH) domains that are important for actin binding. Although there is a crystal structure of the utrophin actin-binding domain, electron microscopy of the actin-bound complexes has produced two very different structural models, in which the CH domains are in open or closed conformations. We engineered a pair of labeling sites in the CH domains of utrophin and used dipolar electron-electron resonance to determine the distribution of interdomain distances with high resolution. We found that the two domains are flexibly connected in solution, indicating a dynamic equilibrium between two distinct open structures. Upon actin binding, the two domains become dramatically separated and ordered, indicating a transition to a single open and extended conformation. There is no trace of this open conformation of utrophin in the absence of actin, providing strong support for an induced-fit model of actin binding.