Palmitoylation of MPP1 (membrane-palmitoylated protein 1)/p55 is crucial for lateral membrane organization in erythroid cells.

S-Acylation of proteins is a ubiquitous post-translational modification and a common signal for membrane association. The major palmitoylated protein in erythrocytes is MPP1, a member of the MAGUK family and an important component of the ternary complex that attaches the spectrin-based skeleton to the plasma membrane. Here we show that DHHC17 is the only acyltransferase present in red blood cells (RBC). Moreover, we give evidence that protein palmitoylation is essential for membrane organization and is crucial for proper RBC morphology, and that the effect is specific for MPP1. Our observations are based on the clinical cases of two related patients whose RBC had no palmitoylation activity, caused by a lack of DHHC17 in the membrane, which resulted in a strong decrease of the amount of detergent-resistant membrane (DRM) material. We confirmed that this loss of detergent-resistant membrane was due to the lack of palmitoylation by treatment of healthy RBC with 2-bromopalmitic acid (2-BrP, common palmitoylation inhibitor). Concomitantly, fluorescence lifetime imaging microscopy (FLIM) analyses of an order-sensing dye revealed a reduction of membrane order after chemical inhibition of palmitoylation in erythrocytes. These data point to a pathophysiological relationship between the loss of MPP1-directed palmitoylation activity and perturbed lateral membrane organization.

Resolution of Maximum Entropy Method-Derived Posterior Conformational Ensembles of a Flexible System Probed by FRET and Molecular Dynamics Simulations.

Maximum entropy methods (MEMs) determine posterior distributions by combining experimental data with prior information. MEMs are frequently used to reconstruct conformational ensembles of molecular systems for experimental information and initial molecular ensembles. We performed time-resolved Förster resonance energy transfer (FRET) experiments to probe the interdye distance distributions of the lipase-specific foldase Lif in the apo state, which likely has highly flexible, disordered, and/or ordered structural elements. Distance distributions estimated from ensembles of molecular dynamics (MD) simulations serve as prior information, and FRET experiments, analyzed within a Bayesian framework to recover distance distributions, are used for optimization. We tested priors obtained by MD with different force fields (FFs) tailored to ordered (FF99SB, FF14SB, and FF19SB) and disordered proteins (IDPSFF and FF99SBdisp). We obtained five substantially different posterior ensembles. As in our FRET experiments the noise is characterized by photon counting statistics, for a validated dye model, MEM can quantify consistencies between experiment and prior or posterior ensembles. However, posterior populations of conformations are uncorrelated to structural similarities for individual structures selected from different prior ensembles. Therefore, we assessed MEM simulating varying priors in synthetic experiments with known target ensembles. We found that (i) the prior and experimental information must be carefully balanced for optimal posterior ensembles to minimize perturbations of populations by overfitting and (ii) only ensemble-integrated quantities like inter-residue distance distributions or density maps can be reliably obtained but not ensembles of atomistic structures. This is because MEM optimizes ensembles but not individual structures. This result for a highly flexible system suggests that structurally varying priors calculated from varying prior ensembles, e.g., generated with different FFs, may serve as an ad hoc estimate for MEM reconstruction robustness.

Fuzzy supertertiary interactions within PSD-95 enable ligand binding.

The scaffold protein PSD-95 links postsynaptic receptors to sites of presynaptic neurotransmitter release. Flexible linkers between folded domains in PSD-95 enable a dynamic supertertiary structure. Interdomain interactions within the PSG supramodule, formed by PDZ3, SH3, and Guanylate Kinase domains, regulate PSD-95 activity. Here we combined discrete molecular dynamics and single molecule Förster resonance energy transfer (FRET) to characterize the PSG supramodule, with time resolution spanning picoseconds to seconds. We used a FRET network to measure distances in full-length PSD-95 and model the conformational ensemble. We found that PDZ3 samples two conformational basins, which we confirmed with disulfide mapping. To understand effects on activity, we measured binding of the synaptic adhesion protein neuroligin. We found that PSD-95 bound neuroligin well at physiological pH while truncated PDZ3 bound poorly. Our hybrid structural models reveal how the supertertiary context of PDZ3 enables recognition of this critical synaptic ligand.

Structural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation.

Folding and cellular localization of many proteins of Gram-negative bacteria rely on a network of chaperones and secretion systems. Among them is the lipase-specific foldase Lif, a membrane-bound steric chaperone that tightly binds (KD = 29 nM) and mediates folding of the lipase LipA, a virulence factor of the pathogenic bacterium P. aeruginosa. Lif consists of five-domains, including a mini domain MD1 essential for LipA folding. However, the molecular mechanism of Lif-assisted LipA folding remains elusive. Here, we show in in vitro experiments using a soluble form of Lif (sLif) that isolated MD1 inhibits sLif-assisted LipA activation. Furthermore, the ability to activate LipA is lost in the variant sLifY99A, in which the evolutionary conserved amino acid Y99 from helix α1 of MD1 is mutated to alanine. This coincides with an approximately three-fold reduced affinity of the variant to LipA together with increased flexibility of sLifY99A in the complex as determined by polarization-resolved fluorescence spectroscopy. We have solved the NMR solution structures of P. aeruginosa MD1 and variant MD1Y99A revealing a similar fold indicating that a structural modification is likely not the reason for the impaired activity of variant sLifY99A. Molecular dynamics simulations of the sLif:LipA complex in connection with rigidity analyses suggest a long-range network of interactions spanning from Y99 of sLif to the active site of LipA, which might be essential for LipA activation. These findings provide important details about the putative mechanism for LipA activation and point to a general mechanism of protein folding by multi-domain steric chaperones.

Integrated NMR, Fluorescence, and Molecular Dynamics Benchmark Study of Protein Mechanics and Hydrodynamics.

Understanding the function of a protein requires not only knowledge of its tertiary structure but also an understanding of its conformational dynamics. Nuclear magnetic resonance (NMR) spectroscopy, polarization-resolved fluorescence spectroscopy and molecular dynamics (MD) simulations are powerful methods to provide detailed insight into protein dynamics on multiple time scales by monitoring global rotational diffusion and local flexibility (order parameters) that are sensitive to inter- and intramolecular interactions, respectively. We present an integrated approach where data from these techniques are analyzed and interpreted within a joint theoretical description of depolarization and diffusion, demonstrating their conceptual similarities. This integrated approach is then applied to the autophagy-related protein GABARAP in its cytosolic form, elucidating its dynamics on the pico- to nanosecond time scale and its rotational and translational diffusion for protein concentrations spanning 9 orders of magnitude. We compare the dynamics of GABARAP as monitored by 15N spin relaxation of the backbone amide groups, fluorescence anisotropy decays and fluorescence correlation spectroscopy of side chains labeled with BODIPY FL, and molecular movies of the protein from MD simulations. The recovered parameters agree very well between the distinct techniques if the different measurement conditions (probe localization, sample concentration) are taken into account. Moreover, we propose a method that compares the order parameters of the backbone and side chains to identify potential hinges for large-scale, functionally relevant intradomain motions, such as residues 27/28 at the interface between the two subdomains of GABARAP. In conclusion, the integrated concept of cross-fertilizing techniques presented here is fundamental to obtaining a comprehensive quantitative picture of multiscale protein dynamics and solvation. The possibility to employ these validated techniques under cellular conditions and combine them with fluorescence imaging opens up the perspective of studying the functional dynamics of GABARAP or other proteins in live cells.

From Enzyme to Whole Blood: Sequential Screening Procedure for Identification and Evaluation of p38 MAPK Inhibitors.

p38 mitogen-activated protein kinase (MAPK) is a pivotal enzyme in the biosynthesis of pro-inflammatory cytokines like IL-1 and TNF. Therefore, the success of anti-cytokine therapy for treatment of inflammatory processes qualified p38-MAPK as a solid target in drug research concerning chronic inflammatory diseases including infectious vascular, neurobiological, and autoimmune disorders. However, the discovery of new kinase inhibitors is limited by the need for a high biological activity combined with restricted activity to the target enzyme or pathway interaction. As a consequence, no p38 MAPK inhibitor has been introduced to the market so far, although several p38 inhibitors have proceeded into clinical trials. The development of novel inhibitor types and optimization of already known structural classes of MAPK inhibitors require appropriate testing systems reaching across these crucial parameters. As a new approach, we describe the sequential arrangement of three testing systems custom-tailored to the requirements of drug discovery programs with focus on p38 inhibition. Integrated analysis of the obtained results enables a concerted step-by-step selection of tested molecules in order to screen a compound library for the most suitable inhibitor. First, evaluation of the inhibitor's activity on the isolated p38 MAPK enzyme via an ELISA assay gives a first idea about the inhibitory potency of the molecule. Moreover, structure-activity relationships can be elucidated when comparing molecules within inhibitor series. Second, screening in living cells via a p38 substrate-specific MK2-EGFP translocation assay supplies further information about efficacy, but provides also a first notion concerning selectivity and toxicity. Third, efficacy is evaluated more specifically in vivo in LPS-stimulated human whole blood with regard to in vivo parameters, e.g., pharmacokinetic characteristics like plasma protein binding and cellular permeability. These three testing systems complement one another synergistically by providing a high overlap and predictability. Clear advantages of all presented systems are their realizability in an academic environment as well as their applicability for high-throughput screenings on a larger scale.

Spatially resolved two-color diffusion measurements in human skin applied to transdermal liposome penetration.

A multiphoton excitation-based fluorescence fluctuation spectroscopy method, Raster image correlation spectroscopy (RICS), was used to measure the local diffusion coefficients of distinct model fluorescent substances in excised human skin. In combination with structural information obtained by multiphoton excitation fluorescence microscopy imaging, the acquired diffusion information was processed to construct spatially resolved diffusion maps at different depths of the stratum corneum (SC). Experiments using amphiphilic and hydrophilic fluorescently labeled molecules show that their diffusion in SC is very heterogeneous on a microscopic scale. This diffusion-based strategy was further exploited to investigate the integrity of liposomes during transdermal penetration. Specifically, the diffusion of dual-color fluorescently labeled liposomes--containing an amphiphilic fluorophore in the lipid bilayer and a hydrophilic fluorophore encapsulated in the liposome lumen--was measured using cross-correlation RICS. This type of experiment allows discrimination between separate (uncorrelated) and joint (correlated) diffusion of the two different fluorescent probes, giving information about liposome integrity. Independent of the liposome composition (phospholipids or transfersomes), our results show a clear lack of cross-correlation below the skin surface, indicating that the penetration of intact liposomes is highly compromised by the skin barrier.

A raft-associated species of phosphatidylethanolamine interacts with cholesterol comparably to sphingomyelin. A Langmuir-Blodgett monolayer study.

BACKGROUND: Specific interactions between sphingomyelin (SM) and cholesterol (Ch) are commonly believed to play a key role in the formation of rafts in the biological membranes. A weakness of this model is the implication that these microdomains are confined to the outer bilayer leaflet. The cytoplasmic leaflet, which contains the bulk of phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI), is thought also to harbour half of the membrane cholesterol. Moreover, SLPE (1-stearoyl-2-linoleoyl-sn-glycero-3-phosphatidyl-ethanolamine) has recently been shown to be enriched in isolated detergent-resistant membranes (DRM), and this enrichment was independent of the method of isolation of DRM. METHODOLOGY/PRINCIPAL FINDINGS: Here we present quantitative evidence coming from Langmuir-Blodgett monolayer experiments that SLPE forms complex with Ch similar to that between SM and Ch. The energies of these interactions as calculated form the monolayer studies are highly negative. FRAP analysis showed that NBD-Ch recovery was similar in liposomes composed of DOPC/Ch SM or SLPE but not DPPE, providing further evidence that SLPE may form an l(o) phase in the presence of high Ch concentration. Experiments on the solubility of DOPC liposomes containing DPPE/Ch (1ratio1), SM/Ch (1ratio1) or SLPE/Ch (1ratio1) showed the presence of Triton X-100 insoluble floating fraction (TIFF) in the case of SM/Ch or SLPE/Ch but not in DPPE/Ch containing liposomes. Quantitative determination of particular lipid species in the TIFF fraction confirms the conclusion that SLPE (or similar PE species) could be an important constituent of the inner leaflet raft. CONCLUSION: Such interactions suggest a possible existence of inner-leaflet nanoscale assemblies composed of cholesterol complexes with SLPE or similar unsaturated PE species.