Lipid Modulation of a Class B GPCR: Elucidating the Modulatory Role of PI(4,5)P2 Lipids.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) lipids have been shown to stabilize an active conformation of class A G-protein coupled receptors (GPCRs) through a conserved binding site, not present in class B GPCRs. For class B GPCRs, previous molecular dynamics (MD) simulation studies have shown PI(4,5)P2 interacting with the Glucagon receptor (GCGR), which constitutes an important target for diabetes and obesity therapeutics. In this work, we applied MD simulations supported by native mass spectrometry (nMS) to study lipid interactions with GCGR. We demonstrate how tail composition plays a role in modulating the binding of PI(4,5)P2 lipids to GCGR. Specifically, we find the PI(4,5)P2 lipids to have a higher affinity toward the inactive conformation of GCGR. Interestingly, we find that in contrast to class A GPCRs, PI(4,5)P2 appear to stabilize the inactive conformation of GCGR through a binding site conserved across class B GPCRs but absent in class A GPCRs. This suggests differences in the regulatory function of PI(4,5)P2 between class A and class B GPCRs.

Extracellular application of the N-methyl-D-aspartate receptor allosteric modulator rapastinel acts remotely to regulate Ca2+ inactivation at an intracellular locus.

BACKGROUND: A novel N-methyl-D-aspartate receptor (NMDAR) allosteric modulator, rapastinel (RAP, formerly GLYX-13), elicits long-lasting antidepressant-like effects by enhancing long-term potentiation (LTP) of synaptic transmission. RAP elicits these effects by binding to a unique site in the extracellular region of the NMDAR complex, transiently enhancing NMDAR-gated current in pyramidal neurons of both hippocampus and medial prefrontal cortex. METHODS: We compared efficacy of RAP in modulating Schaffer collateral-evoked NMDAR-currents as a function of kinetics of the Ca2+ chelator in the intracellular solution, using whole-cell patch-clamp recordings. The intracellular solution contained either the slow Ca2+ chelator EGTA [3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecane-1,14-dioic acid, 0.5 mmol/l] or the 40-500-fold kinetically faster, more selective Ca2+ chelator BAPTA {2,2',2″,2‴-[ethane-1,2-diylbis(oxy-2,1-phenylenenitrilo)] tetraacetic acid, 5 mmol/l}. NMDAR-gated currents were pharmacologically isolated by bath application of the 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptor antagonist 6-nitro-2,3-dioxo-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (10 µmol/l) plus the GABA receptor blocker bicuculline (20 µmol/l). RESULTS: When the slow Ca2+ chelator EGTA was in the intracellular solution, RAP elicited significant enhancement of NMDAR-gated current at a 1 µmol/l concentration, and significantly reduced current at 10 µmol/l. In contrast, when recording with the 40-500-fold kinetically faster, more selective Ca2+ chelator BAPTA, NMDAR current increased in magnitude by 84% as BAPTA washed into the cell, and the enhancement of NMDAR current by 1 µmol/l RAP was completely blocked. Interestingly, the reduction in NMDAR current from 10 µmol/l RAP was not affected by the presence of BAPTA in the recording pipette, indicating that this effect is mediated by a different mechanism. CONCLUSION: Extracellular binding of RAP to the NMDAR produces a novel, long-range reduction in affinity of the Ca2+ inactivation site on the NMDAR C-terminus accessible to the intracellular space. This action underlies enhancement in NMDAR-gated conductance elicited by RAP.

Effect of cholesterol on the dimerization of C99-A molecular modeling perspective.

C99, a naturally occurring peptide, is a precursor of the amyloid ß-peptide (Aß) and plays an important role in the so-called amyloidogenic pathway of degradation of amyloid precursor protein. While the effect of C99's dimerization is not clearly determined, it has been hypothesized that the dimerization protects C99 from being cleaved further. Cholesterol (CHOL) is known to interact with C99 and its presence in high concentrations has been linked to an increase in the production of Aß; however, to what extent this is correlated, and how, has not yet been determined. In this study, we systematically examine the effect of increasing cholesterol concentration on the homodimerization propensity of C99, combining unbiased atomistic molecular dynamics simulations with biased simulations using a coarse grained resolution. Through the use of umbrella sampling, we show how the presence of high levels of CHOL destabilizes the interaction between two C99 monomers. The interaction pattern between the two C99s has shifted several residues, from the N-terminal end of the transmembrane region toward the corresponding C-terminal in the presence of CHOL. The umbrella sampling shows that the presence of high levels of CHOL led to a decrease of the disassociation energy by approximately 3 kJ/mol. In conclusion, this suggests that increasing CHOL destabilizes the interaction between the two C99 monomers, which may possibly cause an increase in the production of Aß42.

The Effect of Cholesterol on Membrane-Bound Islet Amyloid Polypeptide.

Islet amyloid polypeptide (IAPP) is a proposed cause of the decreased beta-cell mass in patients with type-II diabetes. The molecular composition of the cell-membrane is important for regulating IAPP cytotoxicity and aggregation. Cholesterol is present at high concentrations in the pancreatic beta-cells, and in-vitro experiments have indicated that it affects the amyloid formation of IAPP either by direct interactions or by changing the properties of the membrane. In this study we apply atomistic, unbiased molecular dynamics simulations at a microsecond timescale to investigate the effect of cholesterol on membrane bound IAPP. Simulations were performed with various combinations of cholesterol, phosphatidylcholine (PC) and phosphatidylserine (PS) lipids. In all simulations, the helical structure of monomer IAPP was stabilized by the membrane. We found that cholesterol decreased the insertion depth of IAPP compared to pure phospholipid membranes, while PS lipids counteract the effect of cholesterol. The aggregation propensity has previously been proposed to correlate with the insertion depth of IAPP, which we found to decrease with the increased ordering of the lipids induced by cholesterol. Cholesterol is depleted in the vicinity of IAPP, and thus our results suggest that the effect of cholesterol is indirect.

Effect of palmitoylation on the dimer formation of the human dopamine transporter.

The human dopamine transporter (hDAT) is one in three members of the monoamine transporter family (MAT). hDAT is essential for regulating the dopamine concentration in the synaptic cleft through dopamine reuptake into the presynaptic neuron; thereby controlling hDAT dopamine signaling. Dysfunction of the transporter is linked to several psychiatric disorders. hDAT and the other MATs have been shown to form oligomers in the plasma membrane, but only limited data exists on which dimeric and higher order oligomeric states are accessible and energetically favorable. In this work, we present several probable dimer conformations using computational coarse-grained self-assembly simulations and assess the relative stability of the different dimer conformations using umbrella sampling replica exchange molecular dynamics. Overall, the dimer conformations primarily involve TM9 and/or TM11 and/or TM12 at the interface. Furthermore, we show that a palmitoyl group (palm) attached to hDAT on TM12 modifies the free energy of separation for interfaces involving TM12, suggesting that S-palmitoylation may change the relative abundance of dimers involving TM12 in a biological context. Finally, a comparison of the identified interfaces of hDAT and palmitoylated hDAT to the human serotonin transporter interfaces and the leucine transporter interface, suggests similar dimer conformations across these protein family.

De-risking Drug Discovery of Intracellular Targeting Peptides: Screening Strategies to Eliminate False-Positive Hits.

Nonspecific promiscuous compounds can mislead researchers and waste significant resources. This phenomenon, though well-documented for small molecules, has not been widely explored for the peptide modality. Here we demonstrate that two purported peptide-based KRas inhibitors, SAH-SOS1 A and cyclorasin 9A5, exemplify false-positive molecules-in terms of both their binding affinities and cellular activities. Through multiple gold-standard biophysical techniques, we unambiguously show that both peptides lack specific binding to KRas and instead induce protein unfolding. Although these peptides inhibited cellular proliferation, the activities appeared to be off-target on the basis of a counterscreen with KRas-independent cell lines. We further demonstrate that their cellular activities are derived from membrane disruption. Accordingly, we propose that to de-risk false-positive molecules, orthogonal binding assays and cellular counterscreens are indispensable.

Understanding and modelling the interactions of peptides with membranes: from partitioning to self-assembly.

Atomic detail simulations are starting to reveal how flexible polypeptides interact with fluid lipid bilayers. These insights are transforming our understanding of one of the fundamental processes in biology: membrane protein folding and assembly. Advanced molecular dynamics (MD) simulation techniques enable accurate prediction of protein structure, folding pathways and assembly in microsecond-timescales. Such simulations show how membrane-active peptides self-assemble in cell membranes, revealing their binding, folding, insertion, and aggregation, while at the same time providing atomic resolution details of peptide-lipid interactions. Essential to the impact of simulations are experimental approaches that enable calibration and validation of the computational models and techniques. In this review, we summarize the current development of applying unbiased atomic detail MD simulations and the relation to experimental techniques, to study peptide folding and provide our perspective of the field.

Structure and Dynamics of Cinnamycin-Lipid Complexes: Mechanisms of Selectivity for Phosphatidylethanolamine Lipids.

Cinnamycin is a lantibiotic peptide, which selectively binds to and permeabilizes membranes containing phosphatidylethanolamine (PE) lipids. As PE is a major component of many bacterial cell membranes, cinnamycin has considerable potential for destroying these. In this study, molecular dynamics simulations are used to elucidate the structure of a lipid-cinnamycin complex and the origin of selective lipid binding. The simulations reveal that cinnamycin selectively binds to PE by forming an extensive hydrogen-bonding network involving all three hydrogen atoms of the primary ammonium group of the PE head group. The substitution of a single hydrogen atom with a methyl group on the ammonium nitrogen destabilizes this hydrogen-bonding network. In addition to binding the primary ammonium group, cinnamycin also interacts with the phosphate group of the lipid through a previously uncharacterized phosphate-binding site formed by the backbone Phe10-Abu11-Phe12-Val13 moieties (Abu = 1-α-aminobutyric acid). In addition, hydroxylation of Asp15 at Cß plays a role in selective binding of PE due to its tight interaction with the charged amine of the lipid head group. The simulations reveal that the position and orientation of the peptide in the membrane depend on the type of lipid to which it binds, suggesting a reason for why cinnamycin selectively permeabilizes PE-containing membranes.

Structural basis for endotoxin neutralisation and anti-inflammatory activity of thrombin-derived C-terminal peptides.

Thrombin-derived C-terminal peptides (TCPs) of about 2 kDa are present in wounds, where they exert anti-endotoxic functions. Employing a combination of nuclear magnetic resonance spectroscopy (NMR), biophysical, mass spectrometry and cellular studies combined with in silico multiscale modelling, we here determine the bound conformation of HVF18 (HVFRLKKWIQKVIDQFGE), a TCP generated by neutrophil elastase, in complex with bacterial lipopolysaccharide (LPS) and define a previously undisclosed interaction between TCPs and human CD14. Further, we show that TCPs bind to the LPS-binding hydrophobic pocket of CD14 and identify the peptide region crucial for TCP interaction with LPS and CD14. Taken together, our results demonstrate the role of structural transitions in LPS complex formation and CD14 interaction, providing a molecular explanation for the previously observed therapeutic effects of TCPs in experimental models of bacterial sepsis and endotoxin shock.

A Thermodynamic Funnel Drives Bacterial Lipopolysaccharide Transfer in the TLR4 Pathway.

The Gram-negative bacterial outer membrane contains lipopolysaccharide, which potently stimulates the mammalian innate immune response. This involves a relay of specialized complexes culminating in transfer of lipopolysaccharide from CD14 to Toll-like receptor 4 (TLR4) and its co-receptor MD-2 on the cell surface, leading to activation of downstream inflammatory responses. In this study we develop computational models to trace the TLR4 cascade in near-atomic detail. We demonstrate through rigorous thermodynamic calculations that lipopolysaccharide molecules traversing the receptor cascade fall into a thermodynamic funnel. An affinity gradient for lipopolysaccharide is revealed upon extraction from aggregates or realistic bacterial outer membrane models and transfer through CD14 to the terminal TLR4/MD-2 receptor-co-receptor complex. We subsequently assemble viable CD14/TLR4/MD-2 oligomers at the plasma membrane surface, and observe lipopolysaccharide exchange between CD14 and TLR4/MD-2. Collectively, this work helps to unravel the key structural determinants governing endotoxin recognition in the TLR4 innate immune pathway.

Evidence that TLR4 Is Not a Receptor for Saturated Fatty Acids but Mediates Lipid-Induced Inflammation by Reprogramming Macrophage Metabolism.

Chronic inflammation is a hallmark of obesity and is linked to the development of numerous diseases. The activation of toll-like receptor 4 (TLR4) by long-chain saturated fatty acids (lcSFAs) is an important process in understanding how obesity initiates inflammation. While experimental evidence supports an important role for TLR4 in obesity-induced inflammation in vivo, via a mechanism thought to involve direct binding to and activation of TLR4 by lcSFAs, several lines of evidence argue against lcSFAs being direct TLR4 agonists. Using multiple orthogonal approaches, we herein provide evidence that while loss-of-function models confirm that TLR4 does, indeed, regulate lcSFA-induced inflammation, TLR4 is not a receptor for lcSFAs. Rather, we show that TLR4-dependent priming alters cellular metabolism, gene expression, lipid metabolic pathways, and membrane lipid composition, changes that are necessary for lcSFA-induced inflammation. These results reconcile previous discordant observations and challenge the prevailing view of TLR4's role in initiating obesity-induced inflammation.

Activation of Toll-like receptors nucleates assembly of the MyDDosome signaling hub.

Infection and tissue damage induces assembly of supramolecular organizing centres (SMOCs)), such as the Toll-like receptor (TLR) MyDDosome, to co-ordinate inflammatory signaling. SMOC assembly is thought to drive digital all-or-none responses, yet TLR activation by diverse microbes induces anything from mild to severe inflammation. Using single-molecule imaging of TLR4-MyDDosome signaling in living macrophages, we find that MyDDosomes assemble within minutes of TLR4 stimulation. TLR4/MD2 activation leads only to formation of TLR4/MD2 heterotetramers, but not oligomers, suggesting a stoichiometric mismatch between activated receptors and MyDDosomes. The strength of TLR4 signalling depends not only on the number and size of MyDDosomes formed but also how quickly these structures assemble. Activated TLR4, therefore, acts transiently nucleating assembly of MyDDosomes, a process that is uncoupled from receptor activation. These data explain how the oncogenic mutation of MyD88 (L265P) assembles MyDDosomes in the absence of receptor activation to cause constitutive activation of pro-survival NF-κB signalling.

The role of protein-protein interactions in Toll-like receptor function.

As part of the innate immune system, the Toll-like receptors (TLRs) represent key players in the first line of defense against invading foreign pathogens, and are also major targets for therapeutic immunomodulation. TLRs are type I transmembrane proteins composed of an ectodomain responsible for ligand binding, a single-pass transmembrane domain, and a cytoplasmic Toll/Interleukin-1 receptor (TIR) signaling domain. The ectodomains of TLRs are specialized for recognizing a wide variety of pathogen-associated molecular patterns, ranging from lipids and lipopeptides to proteins and nucleic acid fragments. The members of the TLR family are highly conserved and their ectodomains are composed of characteristic, solenoidal leucine-rich repeats (LRRs). Upon ligand binding, these rigid LRR scaffolds dimerize (or re-organize in the case of pre-formed dimers) to bring together their carboxy-terminal transmembrane and TIR domains. The latter are proposed to act as a platform for recruitment of adaptor proteins and formation of higher-order complexes, resulting in propagation of downstream signaling cascades. In this review, we discuss the protein-protein interactions critical for formation and stability of productive, ligand-bound TLR complexes. In particular, we focus on the large body of high-resolution crystallographic data now available for the ectodomains of homo- and heterodimeric TLR complexes, as well as inhibitory TLR-like receptors, and also consider computational approaches that can facilitate our understanding of the ligand-induced conformational changes associated with TLR function. We also briefly consider what is known about the protein-protein interactions involved in both TLR transmembrane domain assembly and TIR-mediated signaling complex formation in light of recent structural and biochemical data.

Interaction of the antimicrobial peptide polymyxin B1 with both membranes of E. coli: a molecular dynamics study.

Antimicrobial peptides are small, cationic proteins that can induce lysis of bacterial cells through interaction with their membranes. Different mechanisms for cell lysis have been proposed, but these models tend to neglect the role of the chemical composition of the membrane, which differs between bacterial species and can be heterogeneous even within a single cell. Moreover, the cell envelope of Gram-negative bacteria such as E. coli contains two membranes with differing compositions. To this end, we report the first molecular dynamics simulation study of the interaction of the antimicrobial peptide, polymyxin B1 with complex models of both the inner and outer membranes of E. coli. The results of >16 microseconds of simulation predict that polymyxin B1 is likely to interact with the membranes via distinct mechanisms. The lipopeptides aggregate in the lipopolysaccharide headgroup region of the outer membrane with limited tendency for insertion within the lipid A tails. In contrast, the lipopeptides readily insert into the inner membrane core, and the concomitant increased hydration may be responsible for bilayer destabilization and antimicrobial function. Given the urgent need to develop novel, potent antibiotics, the results presented here reveal key mechanistic details that may be exploited for future rational drug development.

The membranes of Gram-negative bacteria: progress in molecular modelling and simulation.

Molecular modelling and simulations have been employed to study the membranes of Gram-negative bacteria for over 20 years. Proteins native to these membranes, as well as antimicrobial peptides and drug molecules have been studied using molecular dynamics simulations in simple models of membranes, usually only comprising one lipid species. Thus, traditionally, the simulations have reflected the majority of in vitro membrane experimental setups, enabling observations from the latter to be rationalized at the molecular level. In the last few years, the sophistication and complexity of membrane models have improved considerably, such that the heterogeneity of the lipid and protein composition of the membranes can now be considered both at the atomistic and coarse-grain levels of granularity. Importantly this means relevant biology is now being retained in the models, thereby linking the in silico and in vivo scenarios. We discuss recent progress in simulations of proteins in simple lipid bilayers, more complex membrane models and finally describe some efforts to overcome timescale limitations of atomistic molecular dynamics simulations of bacterial membranes.

Family intervention in schizophrenia--impact on family burden and attitude.

BACKGROUND: This study focuses on the effect of psycho-educative family therapy on the self-assessed burden in families in which one member has suffered from relapse of schizophrenia or a schizoaffective syndrome. The impact on the family's self-assessed attitude towards continuing to take care of the patient was also evaluated. Burden and attitude were assessed continuously during a period that contained no further relapse episodes. METHODS: Included were 31 families in which one family member suffered from schizophrenia or a schizoaffective syndrome. Of these, 14 families underwent a psycho-educative intervention programme called BFT (Behavioural Family Therapy). The remaining 17 families, i. e. the contrast group, received conventional family support. The intervention was initiated within 24 h after the patient/family member was admitted to a psychiatric ward due to relapse of the psychotic disorder. The intervention continued until the patient was discharged from hospital. Falloon's Distress Scale and Attitude Scale were used in the families' self-assessments of burden and attitude towards continuing to take care of the patient, respectively. The self-assessments were performed on three occasions: 1) on the day of admission to the ward, or the day after; 2) 4-5 weeks after admission; and 3) on the day of discharge, or the day after. Medication doses were registered upon admission and at the time of discharge. Finally, the rates of re-occurring relapses within 1 year after discharge from hospital were determined, i. e. 1 year after the completion of the family treatment programme. The BFT families had access to the therapist for questions after the programme had been completed, when needed. The patients and families in the contrast group had access to physicians and therapists in the outpatient care. RESULTS: The self-assessed family burden was significantly lower for the BFT families at the time of discharge, compared to the contrast group, and the self-assessed attitude towards continuing to take care of the patient was significantly more positive for the BFT families at the time of discharge, compared to the contrast families. One patient in the BFT group relapsed within 1 year, whereas 13 patients relapsed in the contrast group. The dosages of neuroleptics were significantly lower on discharge than on admission for the patients in the BFT group. CONCLUSIONS: The results suggest that BFT, when provided to schizophrenic patients and their families during a hospitalisation period caused by a psychotic relapse, reduces the feeling of burden in these families. Likewise, the families' attitude towards continuing to take care of the patients was influenced in a positive way.