The role of phosphorylation on the structure and dynamics of phospholamban: a model from molecular simulations

Phospholamban (PLB) is a small membrane protein that regulates the activity of the calcium ATP-ase in the cardiac, slow-twitch, and smooth muscle sarcoplasmic reticulum through the reversible phosphorylation of Ser16. We present here a comparative molecular dynamics study of unmodified and phosphorylated PLB immersed in a phospholipid membrane. The study has been performed under different ionic strength conditions, using the NMR structures of two PLB variants determined in mixed organic solvent and dodecylphosphocholine micelles. The simulations indicate that all PLB forms studied display a highly dynamic behavior of the N-terminal cytoplasmic moiety, with a decrease of its helical content in the phosphorylated forms. The cytoplasmic domain undergoes large collective motions sampling conformations parallel as well as perpendicular to the membrane surface in all the simulations. The transmembrane domain retains a tightly folded helical conformation with a small tilt with respect to the membrane plane probably induced by the presence of Asn30 and Asn34 within the hydrophobic environment. Furthermore, the phosphoric group on Ser16 establishes transient electrostatic interactions with the phospholipid heads. We propose a model in which phosphorylation diminishes the probability of interactions of PLB with residues near Lys400 in the SERCA pump, thus relieving its inhibition.     

Role of conformational sampling of Ser16 and Thr17-phosphorylated phospholamban in interactions with SERCA

Phosphorylation of phospholamban (PLB) at Ser16 and/ or Thr17 is believed to release its inhibitory effect on sarcoplasmic reticulum calcium ATPase. Ser16 phosphorylation of PLB has been suggested to cause a conformational change that alters the interaction between the enzyme and protein. Using computer simulations, the conformational sampling of Ser16 phosphorylated PLB in implicit membrane environment is compared here with the unphosphorylated PLB system to investigate these conformational changes. The results suggest that conformational changes in the cytoplasmic domain of PLB upon phosphorylation at Ser16 increase the likelihood of unfavorable interactions with SERCA in the E2 state prompting a conformational switch of SERCA from E2 to E1. Phosphorylation of PLB at Thr17 on the other hand does not appear to affect interactions with SERCA significantly suggesting that the mechanism of releasing the inhibitory effect is different between Thr17 phosphorylated and Ser16 phosphorylated PLB.     

Pores formed by the nicotinic receptor m2delta Peptide: a molecular dynamics simulation study

The M2delta peptide self-assembles to form a pentameric bundle of transmembrane alpha-helices that is a model of the pore-lining region of the nicotinic acetylcholine receptor. Long (>15 ns) molecular dynamics simulations of a model of the M2delta(5) bundle in a POPC bilayer have been used to explore the conformational dynamics of the channel assembly. On the timescale of the simulation, the bundle remains relatively stable, with the polar pore-lining side chains remaining exposed to the lumen of the channel. Fluctuations at the helix termini, and in the helix curvature, result in closing/opening transitions at both mouths of the channel, on a timescale of approximately 10 ns. On average, water within the pore lumen diffuses approximately 4x more slowly than water outside the channel. Examination of pore water trajectories reveals both single-file and path-crossing regimes to occur at different times within the simulation.     

Molecular dynamics simulations of the ErbB-2 transmembrane domain within an explicit membrane environment: comparison with vacuum simulations

Two 500-ps molecular dynamics simulations performed on the single transmembrane domain of the ErbB-2 tyrosine kinase receptor immersed in a fully solvated dilauroylphosphatidyl-ethanolamine bilayer (DLPE) are compared to vacuum simulations. One membrane simulation shows that the initial alpha helix undergoes a local pi helix conversion in the peptide part embedded in the membrane core similar to that found in simulation vacuum. Lipid/water/peptide interaction analysis shows that in the helix core, the intramolecular peptide interactions are largely dominant compared to the interactions with water and lipids whereas the helix extremities are much more sensitive to these interactions at the membrane interfaces. Our results suggest that simulations in a lipid environment are required to understand the dynamics of transmembrane helices, but can be reasonably supplemented by in vacuo simulations to explore rapidly its conformational space and to describe the internal deformation of the hydrophobic core.     

Molecular dynamics investigation of membrane-bound bundles of the channel-forming transmembrane domain of viral protein U from the human immunodeficiency virus HIV-1

Molecular dynamics (MD) simulations have been carried out on bundles of the channel-forming transmembrane (TM) domain of the viral protein U (VPU(1-27) and VPU(6-27)) from the human immunodeficiency virus (HIV-1). Simulations of hexameric and pentameric bundles of VPU(6-27) in an octane/water membrane mimetic system suggested that the pentamer is the preferred oligomer. Accordingly, an unconstrained pentameric helix bundle of VPU(1-27) was then placed in a hydrated palmitoyl-oleyl-3-n-glycero-phosphatidylethanolamine (POPE) lipid bilayer and its structural properties calculated from a 3-ns MD run. Some water molecules, initially inside the channel lumen, were expelled halfway through the simulation and the bundle adopted a conical structure reminiscent of previous MD results obtained for VPU(6-27) in an octane/water system. The pore constriction generated may correspond to a closed state of the channel and underlies the relocation of the W residue toward the pore lumen. The relative positions of the helices with respect to the bilayer and their interactions with the lipids are discussed. The observed structure is stabilized via specific interactions between the VPU helices and the carbonyl oxygen atoms of the lipid molecules, particularly at the Q and S residues.     

Molecular dynamics simulations of a bacterial autotransporter: NalP from Neisseria meningitidis

NalP is an autotransporter secretory protein found in the outer membrane of Neisseria meningitidis. The crystal structure of the NalP translocator domain revealed a transmembrane beta-barrel containing a central alpha-helix. The role of this alpha-helix, and of the conformational dynamics of the beta-barrel pore have been studied via atomistic molecular dynamics simulations. Three simulations, each of 10 ns duration, of NalP embedded within a solvated DMPC bilayer were performed. The helix was removed from the barrel interior in one simulation. The conformational stability of the protein is similar to that of other outer membrane proteins, e.g., OmpA, in comparable simulations. The transmembrane beta-barrel is stable even in the absence of the alpha-helix. Removal of the helix results in an influx of water into the pore region, suggesting the helix acts as a 'plug'. Water molecules entering the resultant pore form hydrogen bonds with the barrel lining that compensate for the loss of helix-barrel hydrogen bonds. The dimensions of the pore fluctuate over the course of the simulation revealing it to be flexible, but only wide enough to allow transport of the passenger domain in an unfolded or extended conformation. The simulations help us to understand the role of the central helix in plugging the pore and in maintaining the width of the barrel, and show that the NalP monomer is sufficient for the transport of the passenger domain in an unfolded or extended conformation.     

Electron paramagnetic resonance reveals a large-scale conformational change in the cytoplasmic domain of phospholamban upon binding to the sarcoplasmic reticulum Ca-ATPase

We used EPR spectroscopy to probe directly the interaction between phospholamban (PLB) and its regulatory target, the sarcoplasmic reticulum Ca-ATPase (SERCA). Synthetic monomeric PLB was prepared with a single cytoplasmic cysteine at residue 11, which was then spin labeled. PLB was reconstituted into membranes in the presence or absence of SERCA, and spin label mobility and accessibility were measured. The spin label was quite rotationally mobile in the absence of SERCA, but became more restricted in the presence of SERCA. SERCA also decreased the dependence of spin label mobility on PLB concentration in the membrane, indicating that SERCA reduces PLB-PLB interactions. The spin label MTSSL, attached to Cys11 on PLB by a disulfide bond, was stable at position 11 in the absence of SERCA. In the presence of SERCA, the spin label was released and a covalent bond was formed between PLB and SERCA, indicating direct interaction of one or more SERCA cysteine residues with Cys11 on PLB. The accessibility of the PLB-bound spin label IPSL to paramagnetic agents, localized in different phases of the membrane, indicates that SERCA greatly reduces the level of interaction of the spin label with the membrane surface. We propose that the cytoplasmic domain of PLB associates with the lipid surface, and that association with SERCA induces a major conformational change in PLB in which the cytoplasmic domain is drawn away from the lipid surface by SERCA.     

Effect of Membrane Thickness on Conformational Sampling of Phospholamban from Computer Simulations

The conformational sampling of monomeric, membrane-bound phospholamban is described from computer simulations. Phospholamban (PLB) plays a key role as a regulator of sarcoplasmic reticulum calcium ATPase. An implicit membrane model is used in conjunction with replica exchange molecular dynamics simulations to reach μs-ms timescales. The implicit membrane model was also used to study the effect of different membrane thicknesses by scaling the low-dielectric region. The conformational sampling with the membrane model mimicking dipalmitoylphosphatidylcholine bilayers is in good agreement overall with experimental measurements, but consists of a wide variety of different conformations including structures not described previously. The conformational ensemble shifts significantly in the presence of thinner or thicker membranes. This has implications for the structure and dynamics of PLB in physiological membranes and offers what we believe to be a new interpretation of previous experimental measurements of PLB in detergents and microsomal membrane.     

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.     

Rotational dynamics of phospholamban determined by multifrequency electron paramagnetic resonance

We have used multifrequency electron paramagnetic resonance to define the multistate structural dynamics of an integral membrane protein, phospholamban (PLB), in a lipid bilayer. PLB is a key regulator of cardiac calcium transport, and its function requires transitions between distinct states of intramolecular dynamics. Monomeric PLB was synthesized with the TOAC spin label at positions 11 (in the cytoplasmic domain) and 46 (in the transmembrane domain) and reconstituted into lipid bilayers. Unlike other protein spin labels, TOAC reports directly the motion of the peptide backbone, so quantitative analysis of its dynamics is worthwhile. Electron paramagnetic resonance spectra at 9.4 GHz (X-band) and 94 GHz (W-band) were analyzed in terms of anisotropic rotational diffusion of the two domains. Motion of the transmembrane domain is highly restricted, while the cytoplasmic domain exhibits two distinct conformations, a major one with moderately restricted nanosecond dynamics (T) and another with nearly unrestricted subnanosecond motion (R). The global analysis of spectra at two frequencies yielded values for the rotational correlation times and order parameters that were much more precisely determined than at either frequency alone. Multifrequency EPR is a powerful approach for analysis of complex rotational dynamics of proteins.     

Synthetic null-cysteine phospholamban analogue and the corresponding transmembrane domain inhibit the Ca-ATPase

Chemical synthesis, functional reconstitution, and electron paramagnetic resonance (EPR) have been used to analyze the structure and function of phospholamban (PLB), a 52-residue integral membrane protein that regulates the calcium pump (Ca-ATPase) in cardiac sarcoplasmic reticulum (SR). PLB exists in equilibrium between monomeric and pentameric forms, as observed by SDS-PAGE, EPR, and fluorescence. It has been proposed that inhibition of the pump is due primarily to the monomeric form, with both pentameric stability and inhibition dependent primarily on the transmembrane (TM) domain. To test these hypotheses, we have studied the physical and functional properties of a synthetic null-cysteine PLB analogue that is entirely monomeric on SDS-PAGE, and compared it with the synthetic null-cysteine TM domain (residues 26-52). The TM domain was found to be primarily oligomeric on SDS-PAGE, and boundary lipid spin label analysis in lipid bilayers verified that the isolated TM domain is more oligomeric than the full-length parent molecule. These results indicate that the stability of the PLB pentamer is due primarily to attractive interactions between hydrophobic TM domains, overcoming the repulsive electrostatic interactions between the cationic cytoplasmic domains (residues 1-25). When reconstituted into liposomes containing the Ca-ATPase, the null-cysteine TM domain had the same inhibitory function as that of the full-length parent molecule. We conclude that the TM domain of PLB is sufficient for inhibitory function, the oligomeric stability of PLB does not determine its inhibitory activity, and the three Cys residues in the TM domain are not required for inhibitory function.     

The alpha-helical propensity of the cytoplasmic domain of phospholamban: a molecular dynamics simulation of the effect of phosphorylation and mutation

We have used molecular dynamics simulations to investigate the effect of phosphorylation and mutation on the cytoplasmic domain of phospholamban (PLB), a 52-residue protein that regulates the calcium pump in cardiac muscle. Simulations were carried out in explicit water systems at 300 K for three peptides spanning the first 25 residues of PLB: wild-type (PLB(1-25)), PLB(1-25) phosphorylated at Ser16 and PLB(1-25) with the R9C mutation, which is known to cause human heart disease. The unphosphorylated peptide maintains a helical conformation from 3 to 15 throughout a 26-ns simulation, in agreement with spectroscopic data. Comparison with simulations of a fourth peptide truncated at Pro21 showed the importance of the region from 17 to 21 in preventing local unfolding of the helix. The results suggest that residues 11-16 are more likely to unfold when specific capping motifs are not present. It is proposed that protein kinase A exploits the intrinsic flexibility of the 11-21 region when binding PLB. In agreement with available CD and NMR data, the simulations show a decrease in the helical content upon phosphorylation. The phosphorylated peptide is characterized by helix spanning residues 3-11, followed by a turn that optimizes the salt-bridge interaction between the side chains of the phosphorylated Ser-16 and Arg-13. Replacing Arg-9 with Cys results in unfolding of the helix from C9 and an overall decrease of the helical conformation. The simulations show that initiation of unfolding is due to increased solvent accessibility of the backbone atoms near the smaller Cys. It is proposed that the loss of inhibitory potency upon Ser-16 phosphorylation or R9C mutation of PLB is due to a similar mechanism, in which the partial unfolding of the cytoplasmic helix of PLB results in a conformation that interacts with the cytoplasmic domain of the calcium pump to relieve its inhibition.     

Molecular dynamics simulations of a bacterial autotransporter: NalP from Neisseria meningitidis

NalP is an autotransporter secretory protein found in the outer membrane of Neisseria meningitidis. The crystal structure of the NalP translocator domain revealed a transmembrane β-barrel containing a central α-helix. The role of this α-helix, and of the conformational dynamics of the β-barrel pore have been studied via atomistic molecular dynamics simulations. Three simulations, each of 10 ns duration, of NalP embedded within a solvated DMPC bilayer were performed. The helix was removed from the barrel interior in one simulation. The conformational stability of the protein is similar to that of other outer membrane proteins, e.g., OmpA, in comparable simulations. The transmembrane β-barrel is stable even in the absence of the α-helix. Removal of the helix results in an influx of water into the pore region, suggesting the helix acts as a ‘plug’. Water molecules entering the resultant pore form hydrogen bonds with the barrel lining that compensate for the loss of helix-barrel hydrogen bonds. The dimensions of the pore fluctuate over the course of the simulation revealing it to be flexible, but only wide enough to allow transport of the passenger domain in an unfolded or extended conformation. The simulations help us to understand the role of the central helix in plugging the pore and in maintaining the width of the barrel, and show that the NalP monomer is sufficient for the transport of the passenger domain in an unfolded or extended conformation.     

Molecular dynamics study of gating in the mechanosensitive channel of small conductance MscS

Mechanosensitive channels are a class of ubiquitous membrane proteins gated by mechanical strain in the cellular membrane. MscS, the mechanosensitive channel of small conductance, is found in the inner membrane of Escherichia coli and its crystallographic structure in an open form has been recently solved. By means of molecular dynamics simulations we studied the stability of the channel conformation suggested by crystallography in a fully solvated lipid (POPC) bilayer, the combined system encompassing 224,340 atoms. When restraining the backbone of the protein, the channel remained in the open form and the simulation revealed intermittent permeation of water molecules through the channel. Abolishing the restraints under constant pressure conditions led to spontaneous closure of the transmembrane channel, whereas abolishing the restraints when surface tension (20 dyn/cm) was applied led to channel widening. The large balloon-shaped cytoplasmic domain of MscS exhibited spontaneous diffusion of ions through its side openings. Interaction between the transmembrane domain and the cytoplasmic domain of MscS was observed and involved formation of salt bridges between residues Asp62 and Arg128; this interaction may be essential for the gating of MscS. K+ and Cl- ions showed distinctively different distributions in and around the channel.     

Transmembrane signaling of chemotaxis receptor tar: insights from molecular dynamics simulation studies

Transmembrane signaling of chemotaxis receptors has long been studied, but how the conformational change induced by ligand binding is transmitted across the bilayer membrane is still elusive at the molecular level. To tackle this problem, we carried out a total of 600-ns comparative molecular dynamics simulations (including model-building simulations) of the chemotaxis aspartate receptor Tar (a part of the periplasmic domain/transmembrane domain/HAMP domain) in explicit lipid bilayers. These simulations reveal valuable insights into the mechanistic picture of Tar transmembrane signaling. The piston-like movement of a transmembrane helix induced by ligand binding on the periplasmic side is transformed into a combination of both longitudinal and transversal movements of the helix on the cytoplasmic side as a result of different protein-lipid interactions in the ligand-off and ligand-on states of the receptor. This conformational change alters the dynamics and conformation of the HAMP domain, which is presumably a mechanism to deliver the signal from the transmembrane domain to the cytoplasmic domain. The current results are consistent with the previously suggested dynamic bundle model in which the HAMP dynamics change is a key to the signaling. The simulations provide further insights into the conformational changes relevant to the HAMP dynamics changes in atomic detail.     

Simulations of a membrane-anchored peptide: structure, dynamics, and influence on bilayer properties

A three-dimensional structure of a model decapeptide is obtained by performing molecular dynamics simulations of the peptide in explicit water. Interactions between an N-myristoylated form of the folded peptide anchored to dipalmitoylphosphatidylcholine fluid phase lipid membranes are studied at different applied surface tensions by molecular dynamics simulations. The lipid membrane environment influences the conformational space explored by the peptide. The overall secondary structure of the anchored peptide is found to deviate at times from its structure in aqueous solution through reversible conformational transitions. The peptide is, despite the anchor, highly mobile at the membrane surface with the peptide motion along the bilayer normal being integrated into the collective modes of the membrane. Peptide anchoring moderately alters the lateral compressibility of the bilayer by changing the equilibrium area of the membrane. Although membrane anchoring moderately affects the elastic properties of the bilayer, the model peptide studied here exhibits conformational flexibility and our results therefore suggest that peptide acylation is a feasible way to reinforce peptide-membrane interactions whereby, e.g., the lifetime of receptor-ligand interactions can be prolonged.     

Characterizing the binding of annexin V to a lipid bilayer using molecular dynamics simulations

Annexins play critical roles in membrane organization, membrane trafficking and vesicle transport. The family members share the ability to bind to membranes with high affinities, but the interactions between annexins and membranes remain unclear. Here, using long‐time molecular dynamics simulations, we provide detailed information for the binding of an annexin V trimer to a POPC/POPS lipid bilayer. Calcium ions function as bridges between several negatively charged residues of annexin V and the oxygen atoms of lipids. The preferred calcium‐bridges are those formed via the carboxyl oxygen atoms of POPS lipids. H‐bonds and hydrophobic interactions formed by several critical residues have also been observed in the annexin‐membrane interface. The annexin‐membrane binding causes small changes of annexin trimer structures, while has significant effects on lipid bilayer structures. The lipid bilayer shows a bent shape and forms a concave region in the annexin‐membrane interaction interface, which provides an atomic‐level evidence to support the view that annexins could disturb the stability of lipids and bend membranes. This study provides insights into the commonly occurring PS‐dependent and calcium‐dependent binding of proteins to membranes. Proteins 2014; 82:312–322. © 2013 Wiley Periodicals, Inc.     

Location of the bioactive pentacyclic triterpene ursolic acid in the membrane. A molecular dynamics study

Ursolic acid (URS), an ursane-representative bioactive pentacyclic triterpene, is a plant secondary metabolite presenting a great number of pharmacological beneficial properties. Due to the prominent hydrophobic character of URS and its high phospholipid/water partition coefficient, some of its possible effects on biological systems might be related to its capacity to interact with and locate into the membrane as well as interact specifically with its components. In this work, we have studied the location and orientation of URS in the membrane by molecular dynamics simulations. At the end of the simulation, URS locates near the surface in vicinity to the phospholipid headgroups but its orientation depends on lipid composition, its final average orientation being a nearly parallel one in POPC but a nearly perpendicular one in POPC/POPE/POPG/PSM/Chol. Furthermore, in the complex lipid system URS seems to interact specifically with POPE, PSM, and Chol excluding POPG from its surroundings, which could lead to phase separation and domain formation. The different disposition of URS in the membrane and its specific interaction with certain lipid types could lead to a significant perturbation of the membrane structure. The important pharmacological activities of URS would rely on the effects it exerts on the membrane structure in general and the existence of specific interactions with specific lipids in particular.     

Conformational heterogeneity of the voltage sensor loop of KvAP in micelles and membranes: A fluorescence approach

KvAP is a tetrameric voltage-gated potassium channel that is composed of a pore domain and a voltage-sensing domain (VSD). The VSD is crucial for sensing transmembrane potential and gating. At 0 mV, the VSD adopts an activated conformation in both n-octylglucoside (OG) micelles and phospholipid membranes. Importantly, gating-modifier toxins that bind at S3b-S4 loop of KvAP-VSD exhibit pronounced differences in binding affinity in these membrane-mimetic systems. However, the conformational heterogeneity of this functionally-important sensor loop in membrane mimetics is poorly understood, and is the focus of this work. In this paper, we establish, using intrinsic fluorescence of the uniquely positioned W70 in KvAP-VSD and environment-sensitive NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl-ethylenediamine) fluorescence of the labelled S3b-S4 loop, that the surface charge of the membrane does not significantly affect the topology and structural dynamics of the sensor loop in membranes. Importantly, the dynamic variability of the sensor loop is preserved in both zwitterionic (POPC) and anionic (POPC/POPG) membranes. Further, the lifetime distribution analysis for the NBD-labelled residues by maximum entropy method (MEM) demonstrates that, in contrast to micelles, the membrane environment not only reduces the relative discrete population of sensor loop conformations, but also broadens the lifetime distribution peaks. Overall, our results strongly suggest that the conformational heterogeneity of the sensor loop is significantly altered in membranes and this correlates well with its environmental heterogeneity. This constitutes the first report demonstrating that MEM-lifetime distribution could be a powerful tool to distinguish changes in conformational heterogeneity in potassium channels with similar architecture and topology.