Maria Musgaard

Mutual adaptation of a membrane protein and its lipid bilayer during conformational changes

The structural elucidation of membrane proteins continues to gather pace, but we know little about their molecular interactions with the lipid environment or how they interact with the surrounding bilayer. Here, with the aid of low-resolution X-ray crystallography, we present direct structural information on membrane interfaces as delineated by lipid phosphate groups surrounding the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) in its phosphorylated and dephosphorylated Ca(2+)-free forms. The protein-lipid interactions are further analysed using molecular dynamics simulations. We find that SERCA adapts to membranes of different hydrophobic thicknesses by inducing local deformations in the lipid bilayers and by undergoing small rearrangements of the amino-acid side chains and helix tilts. These mutually adaptive interactions allow smooth transitions through large conformational changes associated with the transport cycle of SERCA, a strategy that may be of general nature for many membrane proteins.

Binding and Orientation of Tricyclic Antidepressants within the Central Substrate Site of the Human Serotonin Transporter

Tricyclic antidepressants (TCAs) have been used for decades, but their orientation within and molecular interactions with their primary target is yet unsettled. The recent finding of a TCA binding site in the extracellular vestibule of the bacterial leucine transporter 11 Å above the central site has prompted debate about whether this vestibular site in the bacterial transporter is applicable to binding of antidepressants to their relevant physiological target, the human serotonin transporter (hSERT). We present an experimentally validated structural model of imipramine and analogous TCAs in the central substrate binding site of hSERT. Two possible binding modes were observed from induced fit docking calculations. We experimentally validated a single binding mode by combining mutagenesis of hSERT with uptake inhibition studies of different TCA analogs according to the paired mutation ligand analog complementation paradigm. Using this experimental method, we identify a salt bridge between the tertiary aliphatic amine and Asp98. Furthermore, the 7-position of the imipramine ring is found vicinal to Phe335, and the pocket lined by Ala173 and Thr439 is utilized by 3-substituents. These protein-ligand contact points unambiguously orient the TCA within the central binding site and reveal differences between substrate binding and inhibitor binding, giving important clues to the inhibition mechanism. Consonant with the well established competitive inhibition of uptake by TCAs, the resulting binding site for TCAs in hSERT is fully overlapping with the serotonin binding site in hSERT and dissimilar to the low affinity noncompetitive TCA site reported in the leucine transporter (LeuT).

Ion Pathways in the Sarcoplasmic Reticulum Ca2+-ATPase

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a transmembrane ion transporter belonging to the PII-type ATPase family. It performs the vital task of re-sequestering cytoplasmic Ca2+ to the sarco/endoplasmic reticulum store, thereby also terminating Ca2+-induced signaling such as in muscle contraction. This minireview focuses on the transport pathways of Ca2+ and H+ ions across the lipid bilayer through SERCA. The ion-binding sites of SERCA are accessible from either the cytoplasm or the sarco/endoplasmic reticulum lumen, and the Ca2+ entry and exit channels are both formed mainly by rearrangements of four N-terminal transmembrane α-helices. Recent improvements in the resolution of the crystal structures of rabbit SERCA1a have revealed a hydrated pathway in the C-terminal transmembrane region leading from the ion-binding sites to the cytosol. A comparison of different SERCA conformations reveals that this C-terminal pathway is exclusive to Ca2+-free E2 states, suggesting that it may play a functional role in proton release from the ion-binding sites. This is in agreement with molecular dynamics simulations and mutational studies and is in striking analogy to a similar pathway recently described for the related sodium pump. We therefore suggest a model for the ion exchange mechanism in PII-ATPases including not one, but two cytoplasmic pathways working in concert.

Protonation States of Important Acidic Residues in the Central Ca2+ Ion Binding Sites of the Ca2+-ATPase: A Molecular Modeling Study

The P-type ATPases are responsible for the transport of cations across cell membranes. The sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) transports two Ca²⁺ ions from the cytoplasm to the lumen of the sarco(endo)plasmic reticulum and countertransports two or three protons per catalytic cycle. Two binding sites for Ca²⁺ ions have been located via protein crystallography, including four acidic amino acid residues that are essential to the ion coordination. In this study, we present molecular dynamics (MD) simulations examining the protonation states of these amino acid residues in a Ca²⁺-free conformation of SERCA. Such knowledge will be important for an improved understanding of atomistic details of the transport mechanism of protons and Ca²⁺ ions. Eight combinations of the protonation of four central acidic residues, Glu309, Glu771, Asp800, and Glu908, are tested from 10 ns MD simulations with respect to protein stability and ability to maintain a structure similar to the crystal structure. The trajectories for the most prospective combinations of protonation states were elongated to 50 ns and subjected to more detailed analysis, including prediction of pK(a) values of the four acidic residues over the trajectories. From the simulations we find that the combination leaving only Asp800 as charged is most likely. The results are compared to available experimental data and explain the observed destabilization upon full deprotonation, resulting in the entry of cytoplasmic K⁺ ions into the Ca²⁺ binding sites during the simulation in which Ca²⁺ ions are absent. Furthermore, a hypothesis for the exchange of protons from the central binding cavity is proposed.

Distinct Structural Pathways Coordinate the Activation of AMPA Receptor-Auxiliary Subunit Complexes

Summary Neurotransmitter-gated ion channels adopt different gating modes to fine-tune signaling at central synapses. At glutamatergic synapses, high and low activity of AMPA receptors (AMPARs) is observed when pore-forming subunits coassemble with or without auxiliary subunits, respectively. Whether a common structural pathway accounts for these different gating modes is unclear. Here, we identify two structural motifs that determine the time course of AMPAR channel activation. A network of electrostatic interactions at the apex of the AMPAR ligand-binding domain (LBD) is essential for gating by pore-forming subunits, whereas a conserved motif on the lower, D2 lobe of the LBD prolongs channel activity when auxiliary subunits are present. Accordingly, channel activity is almost entirely abolished by elimination of the electrostatic network but restored via auxiliary protein interactions at the D2 lobe. In summary, we propose that activation of native AMPAR complexes is coordinated by distinct structural pathways, favored by the association/dissociation of auxiliary subunits.

Defining the structural relationship between kainate-receptor deactivation and desensitization

Desensitization is an important mechanism curtailing the activity of ligand-gated ion channels (LGICs). Although the structural basis of desensitization is not fully resolved, it is thought to be governed by physicochemical properties of bound ligands. Here, we show the importance of an allosteric cation-binding pocket in controlling transitions between activated and desensitized states of rat kainate-type (KAR) ionotropic glutamate receptors (iGluRs). Tethering a positive charge to this pocket sustains KAR activation, preventing desensitization, whereas mutations that disrupt cation binding eliminate channel gating. These different outcomes explain the structural distinction between deactivation and desensitization. Deactivation occurs when the ligand unbinds before the cation, whereas desensitization proceeds if a ligand is bound without cation pocket occupancy. This sequence of events is absent from AMPA-type iGluRs; thus, cations are identified as gatekeepers of KAR gating, a role unique among even closely related LGICs.

Tracing Cytoplasmic Ca2+ Ion and Water Access Points in the Ca2+-ATPase

Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) transports two Ca(2+) ions across the membrane of the sarco(endo)plasmic reticulum against the concentration gradient, harvesting the required energy by hydrolyzing one ATP molecule during each transport cycle. Although SERCA is one of the best structurally characterized membrane transporters, it is still largely unknown how the transported Ca(2+) ions reach their transmembrane binding sites in SERCA from the cytoplasmic side. Here, we performed extended all-atom molecular dynamics simulations of SERCA. The calculated electrostatic potential of the protein reveals a putative mechanism by which cations may be attracted to and bind to the Ca(2+)-free state of the transporter. Additional molecular dynamics simulations performed on a Ca(2+)-bound state of SERCA reveal a water-filled pathway that may be used by the Ca(2+) ions to reach their buried binding sites from the cytoplasm. Finally, several residues that are involved in attracting and guiding the cations toward the possible entry channel are identified. The results point to a single Ca(2+) entry site close to the kinked part of the first transmembrane helix, in a region loaded with negatively charged residues. From this point, a water pathway outlines a putative Ca(2+) translocation pathway toward the transmembrane ion-binding sites.

Steered Molecular Dynamics Simulations Predict Conformational Stability of Glutamate Receptors

The stability of protein-protein interfaces can be essential for protein function. For ionotropic glutamate receptors, a family of ligand-gated ion channels vital for normal function of the central nervous system, such an interface exists between the extracellular ligand binding domains (LBDs). In the full-length protein, the LBDs are arranged as a dimer of dimers. Agonist binding to the LBDs opens the ion channel, and briefly after activation the receptor desensitizes. Several residues at the LBD dimer interface are known to modulate desensitization, and conformational changes around these residues are believed to be involved in the state transition. The general hypothesis is that the interface is disrupted upon desensitization, and structural evidence suggests that the disruption might be substantial. However, when cross-linking the central part of this interface, functional data suggest that the receptor can still undergo desensitization, contradicting the hypothesis of major interface disruption. Here, we illustrate how opening the dimer interface using steered molecular dynamics (SMD) simulations, and analyzing the work values required, provides a quantitative measure for interface stability. For one subtype of glutamate receptors, which is regulated by ion binding to the dimer interface, we show that opening the interface without ions bound requires less work than with ions present, suggesting that ion binding indeed stabilizes the interface. Likewise, for interface mutants with longer-lived active states, the interface is more stable, while the work required to open the interface is reduced for less active mutants. Moreover, a cross-linked mutant can still undergo initial interface opening motions similar to the native receptor and at similar energetic cost. Thus, our results support that interface opening is involved in desensitization. Furthermore, they provide reconciliation of apparently opposing data and demonstrate that SMD simulations can give relevant biological insight into longer time scale processes without the need for expensive calculations.

The dynamics of camphor in the cytochrome P450 CYP101D2.

The recent crystal structures of CYP101D2, a cytochrome P450 protein from the oligotrophic bacterium Novosphingobium aromaticivorans DSM12444 revealed that both the native (substrate‐free) and camphor‐soaked forms have open conformations. Furthermore, two other potential camphor‐binding sites were also identified from electron densities in the camphor‐soaked structure, one being located in the access channel and the other in a cavity on the surface near the F‐helix side of the F‐G loop termed the substrate recognition site. These latter sites may be key intermediate positions on the pathway for substrate access to or product egress from the active site. Here, we show via the use of unbiased atomistic molecular dynamics simulations that despite the open conformation of the native and camphor‐bound crystal structures, the underlying dynamics of CYP101D2 appear to be very similar to other CYP proteins. Simulations of the native structure demonstrated that the protein is capable of sampling many different conformational substates. At the same time, simulations with the camphor positioned at various locations within the access channel or recognition site show that movement towards the active site or towards bulk solvent can readily occur on a short timescale, thus confirming many previously reported in silico studies using steered molecular dynamics. The simulations also demonstrate how the fluctuations of an aromatic gate appear to control access to the active site. Finally, comparison of camphor‐bound simulations with the native simulations suggests that the fluctuations can be of similar level and thus are more representative of the conformational selection model rather than induced fit.

Actions of agonists, fipronil and ivermectin on the predominant in vivo splice and edit variant (RDLbd, I/V) of the Drosophila GABA receptor expressed in Xenopus laevis oocytes.

Ionotropic GABA receptors are the targets for several classes of insecticides. One of the most widely-studied insect GABA receptors is RDL (resistance to dieldrin), originally isolated from Drosophila melanogaster. RDL undergoes alternative splicing and RNA editing, which influence the potency of GABA. Most work has focussed on minority isoforms. Here, we report the first characterisation of the predominant native splice variant and RNA edit, combining functional characterisation with molecular modelling of the agonist-binding region. The relative order of agonist potency is GABA> muscimol> TACA> β-alanine. The I/V edit does not alter the potency of GABA compared to RDLbd. Docking calculations suggest that these agonists bind and activate RDLbdI/V through a similar binding mode. TACA and β-alanine are predicted to bind with lower affinity than GABA, potentially explaining their lower potency, whereas the lower potency of muscimol and isoguvacine cannot be explained structurally from the docking calculations. The A301S (resistance to dieldrin) mutation reduced the potency of antagonists picrotoxin, fipronil and pyrafluprole but the I/V edit had no measurable effect. Ivermectin suppressed responses to GABA of RDLbdI/V, RDLbd and RDLbdI/VA301S. The dieldrin resistant variant also showed reduced sensitivity to Ivermectin. This study of a highly abundant insect GABA receptor isoform will help the design of new insecticides.