Molecular basis of PIP2-dependent conformational switching of phosphorylated CD44 in binding FERM.

Association of the cellular adhesive protein CD44 and the N-terminal (FERM) domain of cytoskeleton adaptors is critical for cell proliferation, migration, and signaling. Phosphorylation of the cytoplasmic domain (CTD) of CD44 acts as an important regulator of the protein association, but the structural transformation and dynamics mechanism remain enigmatic. In this study, extensive coarse-grained simulations were employed to explore the molecular details in the formation of CD44-FERM complex under S291 and S325 phosphorylation, a modification path known to exert reciprocal effects on the protein association. We find that phosphorylation of S291 inhibits complexation by causing the CTD of CD44 to adopt a more closed structure. In contrast, S325 phosphorylation liberates the CD44-CTD from the membrane surface and promotes the linkage with FERM. The phosphorylation-driven transformation is found to occur in a PIP2-dependent manner, with PIP2 effecting the relative stability of the closed and open conformation, and a replacement of PIP2 by POPS greatly abrogates this effect. The revealed interdependent regulation mechanism by phosphorylation and PIP2 in the association of CD44 and FERM further strengthens our understanding of the molecular basis of cellular signaling and migration.

Bottom-up fabrication of a proteasome-nanopore that unravels and processes single proteins.

The precise assembly and engineering of molecular machines capable of handling biomolecules play crucial roles in most single-molecule methods. In this work we use components from all three domains of life to fabricate an integrated multiprotein complex that controls the unfolding and threading of individual proteins across a nanopore. This 900 kDa multicomponent device was made in two steps. First, we designed a stable and low-noise ß-barrel nanopore sensor by linking the transmembrane region of bacterial protective antigen to a mammalian proteasome activator. An archaeal 20S proteasome was then built into the artificial nanopore to control the unfolding and linearized transport of proteins across the nanopore. This multicomponent molecular machine opens the door to two approaches in single-molecule protein analysis, in which selected substrate proteins are unfolded, fed to into the proteasomal chamber and then addressed either as fragmented peptides or intact polypeptides.

Thermostable D-amino acid decarboxylases derived from Thermotoga maritima diaminopimelate decarboxylase.

Diaminopimelate decarboxylases (DAPDCs) are highly selective enzymes that catalyze the common final step in different lysine biosynthetic pathways, i.e. the conversion of meso-diaminopimelate (DAP) to L-lysine. We examined the modification of the substrate specificity of the thermostable decarboxylase from Thermotoga maritima with the aim to introduce activity with 2-aminopimelic acid (2-APA) since its decarboxylation leads to 6-aminocaproic acid (6-ACA), a building block for the synthesis of nylon-6. Structure-based mutagenesis of the distal carboxylate binding site resulted in a set of enzyme variants with new activities toward different D-amino acids. One of the mutants (E315T) had lost most of its activity toward DAP and primarily acted as a 2-APA decarboxylase. We next used computational modeling to explain the observed shift in catalytic activities of the mutants. The results suggest that predictive computational protocols can support the redesign of the catalytic properties of this class of decarboxylating PLP-dependent enzymes.

Adaption to glucose limitation is modulated by the pleotropic regulator CcpA, independent of selection pressure strength.

BACKGROUND: A central theme in (micro)biology is understanding the molecular basis of fitness i.e. which strategies are successful under which conditions; how do organisms implement such strategies at the molecular level; and which constraints shape the trade-offs between alternative strategies. Highly standardized microbial laboratory evolution experiments are ideally suited to approach these questions. For example, prolonged chemostats provide a constant environment in which the growth rate can be set, and the adaptive process of the organism to such environment can be subsequently characterized. RESULTS: We performed parallel laboratory evolution of Lactococcus lactis in chemostats varying the quantitative value of the selective pressure by imposing two different growth rates. A mutation in one specific amino acid residue of the global transcriptional regulator of carbon metabolism, CcpA, was selected in all of the evolution experiments performed. We subsequently showed that this mutation confers predictable fitness improvements at other glucose-limited growth rates as well. In silico protein structural analysis of wild type and evolved CcpA, as well as biochemical and phenotypic assays, provided the underpinning molecular mechanisms that resulted in the specific reprogramming favored in constant environments. CONCLUSION: This study provides a comprehensive understanding of a case of microbial evolution and hints at the wide dynamic range that a single fitness-enhancing mutation may display. It demonstrates how the modulation of a pleiotropic regulator can be used by cells to improve one trait while simultaneously work around other limiting constraints, by fine-tuning the expression of a wide range of cellular processes.

Role of Charge and Hydrophobicity in Liprotide Formation: A Molecular Dynamics Study with Experimental Constraints.

Bovine α-lactalbumin (aLA) and oleate (OA) form a complex that has been intensively studied for its tumoricidal activity. Small-angle X-ray scattering (SAXS) has revealed that this complex consists of a lipid core surrounded by partially unfolded protein. We call this type of complex a liprotide. Little is known of the molecular interactions between OA and aLA, and no technique has so far provided any high-resolution structure of a liprotide. Here we have used coarse-grained (CG) molecular dynamics (MD) simulations, isothermal titration calorimetry (ITC) and SAXS to investigate the interactions between aLA and OA during the process of liprotide formation. With ITC we found that the strongest enthalpic interactions occurred at a molar ratio of 12.0±1.4:1 OA/aLA. Liprotides formed between OA and aLA at several OA/aLA ratios in silico were stable both in CG and in all-atom simulations. From the simulated structures we calculated SAXS spectra that show good agreement with experimentally measured patterns of matching liprotides. The simulations showed that aLA assumes a molten globular (MG) state, exposing several hydrophobic patches involved in interactions with OA. Initial binding of aLA to OA occurs in an area of aLA in which a high amount of positive charge is located, and only later do hydrophobic interactions become important. The results reveal how unfolding of aLA to expose hydrophobic residues is important for complex formation between aLA and OA. Our findings suggest a general mechanism for liprotide formation and might explain the ability of a large number of proteins to form liprotides with OA.

Altered secondary structure of Dynorphin A associates with loss of opioid signalling and NMDA-mediated excitotoxicity in SCA23.

Spinocerebellar ataxia type 23 (SCA23) is caused by missense mutations in prodynorphin, encoding the precursor protein for the opioid neuropeptides α-neoendorphin, Dynorphin (Dyn) A and Dyn B, leading to neurotoxic elevated mutant Dyn A levels. Dyn A acts on opioid receptors to reduce pain in the spinal cord, but its cerebellar function remains largely unknown. Increased concentration of or prolonged exposure to Dyn A is neurotoxic and these deleterious effects are very likely caused by an N-methyl-d-aspartate-mediated non-opioid mechanism as Dyn A peptides were shown to bind NMDA receptors and potentiate their glutamate-evoked currents. In the present study, we investigated the cellular mechanisms underlying SCA23-mutant Dyn A neurotoxicity. We show that SCA23 mutations in the Dyn A-coding region disrupted peptide secondary structure leading to a loss of the N-terminal α-helix associated with decreased κ-opioid receptor affinity. Additionally, the altered secondary structure led to increased peptide stability of R6W and R9C Dyn A, as these peptides showed marked degradation resistance, which coincided with decreased peptide solubility. Notably, L5S Dyn A displayed increased degradation and no aggregation. R6W and wt Dyn A peptides were most toxic to primary cerebellar neurons. For R6W Dyn A, this is likely because of a switch from opioid to NMDA- receptor signalling, while for wt Dyn A, this switch was not observed. We propose that the pathology of SCA23 results from converging mechanisms of loss of opioid-mediated neuroprotection and NMDA-mediated excitotoxicity.

Hsc70-4 Deforms Membranes to Promote Synaptic Protein Turnover by Endosomal Microautophagy.

Synapses are often far from their cell bodies and must largely independently cope with dysfunctional proteins resulting from synaptic activity and stress. To identify membrane-associated machines that can engulf synaptic targets destined for degradation, we performed a large-scale in vitro liposome-based screen followed by functional studies. We identified a presynaptically enriched chaperone Hsc70-4 that bends membranes based on its ability to oligomerize. This activity promotes endosomal microautophagy and the turnover of specific synaptic proteins. Loss of microautophagy slows down neurotransmission while gain of microautophagy increases neurotransmission. Interestingly, Sgt, a cochaperone of Hsc70-4, is able to switch the activity of Hsc70-4 from synaptic endosomal microautophagy toward chaperone activity. Hence, Hsc70-4 controls rejuvenation of the synaptic protein pool in a dual way: either by refolding proteins together with Sgt, or by targeting them for degradation by facilitating endosomal microautophagy based on its membrane deforming activity.

Dual action of BPC194: a membrane active peptide killing bacterial cells.

Membrane active peptides can perturb the lipid bilayer in several ways, such as poration and fusion of the target cell membrane, and thereby efficiently kill bacterial cells. We probe here the mechanistic basis of membrane poration and fusion caused by membrane-active, antimicrobial peptides. We show that the cyclic antimicrobial peptide, BPC194, inhibits growth of Gram-negative bacteria and ruptures the outer and inner membrane at the onset of killing, suggesting that not just poration is taking place at the cell envelope. To simplify the system and to better understand the mechanism of action, we performed Förster resonance energy transfer and cryogenic transmission electron microscopy studies in model membranes and show that the BPC194 causes fusion of vesicles. The fusogenic action is accompanied by leakage as probed by dual-color fluorescence burst analysis at a single liposome level. Atomistic molecular dynamics simulations reveal how the peptides are able to simultaneously perturb the membrane towards porated and fused states. We show that the cyclic antimicrobial peptides trigger both fusion and pore formation and that such large membrane perturbations have a similar mechanistic basis.

Semiconducting single-walled carbon nanotubes on demand by polymer wrapping.

Efficient selection of semiconducting SWCNTs of large diameter range (0.8-1.6 nm) on demand is demonstrated. Different diameters of SWCNT are systematically selected by tuning the alkyl side-chain lengths of the wrapping polymers of similar backbone. The exceptional quality and high concentration of the SWCNTs is validated by the outstanding optical properties and the highly performing random network ambipolar field-effect transistors.

The Martini coarse-grained force field.

The Martini force field is a coarse-grained force field suited for molecular dynamics simulations of biomolecular systems. The force field has been parameterized in a systematic way, based on the reproduction of partitioning free energies between polar and apolar phases of a large number of chemical compounds. In this chapter the methodology underlying the force field is presented together with details of its parameterization and limitations. Then currently available topologies are described with a short overview of the key elements of their parameterization. These include the new polarizable Martini water model. A set of three selected ongoing studies using the Martini force field is presented. Finally the latest lines of development are discussed.

Line-tension controlled mechanism for influenza fusion.

Our molecular simulations reveal that wild-type influenza fusion peptides are able to stabilize a highly fusogenic pre-fusion structure, i.e. a peptide bundle formed by four or more trans-membrane arranged fusion peptides. We rationalize that the lipid rim around such bundle has a non-vanishing rim energy (line-tension), which is essential to (i) stabilize the initial contact point between the fusing bilayers, i.e. the stalk, and (ii) drive its subsequent evolution. Such line-tension controlled fusion event does not proceed along the hypothesized standard stalk-hemifusion pathway. In modeled influenza fusion, single point mutations in the influenza fusion peptide either completely inhibit fusion (mutants G1V and W14A) or, intriguingly, specifically arrest fusion at a hemifusion state (mutant G1S). Our simulations demonstrate that, within a line-tension controlled fusion mechanism, these known point mutations either completely inhibit fusion by impairing the peptide's ability to stabilize the required peptide bundle (G1V and W14A) or stabilize a persistent bundle that leads to a kinetically trapped hemifusion state (G1S). In addition, our results further suggest that the recently discovered leaky fusion mutant G13A, which is known to facilitate a pronounced leakage of the target membrane prior to lipid mixing, reduces the membrane integrity by forming a 'super' bundle. Our simulations offer a new interpretation for a number of experimentally observed features of the fusion reaction mediated by the prototypical fusion protein, influenza hemagglutinin, and might bring new insights into mechanisms of other viral fusion reactions.

Rhodopsin forms a dimer with cytoplasmic helix 8 contacts in native membranes.

G protein-coupled receptors form dimers and higher-order oligomers in membranes, but the precise mode of receptor-receptor interaction remains unknown. To probe the intradimeric proximity of helix 8 (H8), we conducted chemical cross-linking of endogenous cysteines in rhodopsin in disk membranes. We identified a Cys316-Cys316 cross-link using partial proteolysis and liquid chromatography with mass spectrometry. These results show that a symmetric dimer interface mediated by H1 and H8 contacts is present in native membranes.

Sphere-to-rod transitions of nonionic surfactant micelles in aqueous solution modeled by molecular dynamics simulations.

Control of the size and agglomeration of micellar systems is important for pharmaceutical applications such as drug delivery. Although shape-related transitions in surfactant solutions are studied experimentally, their molecular mechanisms are still not well understood. In this study, we use coarse-grained molecular dynamics simulations to describe micellar assemblies of pentaethylene glycol monododecyl ether (C(12)E(5)) in aqueous solution at different concentrations. The obtained size and aggregation numbers of the aggregates formed are in very good agreement with the available experimental data. Importantly, increase of the concentration leads to a second critical micelle concentration where a transition to rod-like aggregates is observed. This transition is quantified in terms of shape anisotropy, together with a detailed structural analysis of the micelles as a function of aggregation number.

Curvature-dependent elastic properties of liquid-ordered domains result in inverted domain sorting on uniaxially compressed vesicles.

Using a coarse-grained molecular model we study the spatial distribution of lipid domains on a 20-nm-sized vesicle. The lipid mixture laterally phase separates into a raftlike, liquid-ordered (l(o)) phase and a liquid-disordered phase. As we uniaxially compress the mixed vesicle keeping the enclosed volume constant, we impart tension onto the membrane. The vesicle adopts a barrel shape, which is composed of two flat contact zones and a curved edge. The l(o) domain, which exhibits a higher bending rigidity, segregates to the highly curved edge. This inverted domain sorting switches to normal domain sorting, where the l(o) domain prefers the flat contact zone, when we release the contents of the vesicle. We rationalize this domain sorting by a pronounced reduction of the bending rigidity and area compressibility of the l(o) phase upon bending.

A tool for the morphological analysis of mixtures of lipids and water in computer simulations.

When analyzing computer simulations of mixtures of lipids and water, the questions to be answered are often of a morphological nature. They can deal with global properties, like the kind of phase that is adopted or the presence or absence of certain key features like a pore or stalk, or with local properties, like the local curvature present at a particular part of the lipid/water interface. While in principle all of the information relating to the global and local morphological properties of a system can be obtained from the set of atomic coordinates generated by a computer simulation, the extraction of this information is a tedious task that usually involves using a visualization program and performing the analysis by eye. Here we present a tool that employs the technique of morphological image analysis (MIA) to automatically extract the global morphology--as given by Minkowski functionals--from a set of atomic coordinates, and creates an image of the system onto which the local curvatures are mapped as a color code.

Role of lipids in spheroidal high density lipoproteins.

We study the structure and dynamics of spherical high density lipoprotein (HDL) particles through coarse-grained multi-microsecond molecular dynamics simulations. We simulate both a lipid droplet without the apolipoprotein A-I (apoA-I) and the full HDL particle including two apoA-I molecules surrounding the lipid compartment. The present models are the first ones among computational studies where the size and lipid composition of HDL are realistic, corresponding to human serum HDL. We focus on the role of lipids in HDL structure and dynamics. Particular attention is paid to the assembly of lipids and the influence of lipid-protein interactions on HDL properties. We find that the properties of lipids depend significantly on their location in the particle (core, intermediate region, surface). Unlike the hydrophobic core, the intermediate and surface regions are characterized by prominent conformational lipid order. Yet, not only the conformations but also the dynamics of lipids are found to be distinctly different in the different regions of HDL, highlighting the importance of dynamics in considering the functionalization of HDL. The structure of the lipid droplet close to the HDL-water interface is altered by the presence of apoA-Is, with most prominent changes being observed for cholesterol and polar lipids. For cholesterol, slow trafficking between the surface layer and the regimes underneath is observed. The lipid-protein interactions are strongest for cholesterol, in particular its interaction with hydrophobic residues of apoA-I. Our results reveal that not only hydrophobicity but also conformational entropy of the molecules are the driving forces in the formation of HDL structure. The results provide the first detailed structural model for HDL and its dynamics with and without apoA-I, and indicate how the interplay and competition between entropy and detailed interactions may be used in nanoparticle and drug design through self-assembly.

Immobilization of the plug domain inside the SecY channel allows unrestricted protein translocation.

The SecYEG complex forms a protein-conducting channel in the inner membrane of Escherichia coli to support the translocation of secretory proteins in their unfolded state. The SecY channel is closed at the periplasmic face of the membrane by a small re-entrance loop that connects transmembrane segment 1 with 2b. This helical domain 2a is termed the plug domain. By the introduction of pairs of cysteines and crosslinkers, the plug domain was immobilized inside the channel and connected to transmembrane segment 10. Translocation was inhibited to various degrees depending on the position and crosslinker spacer length. With one of the crosslinked mutants translocation occurred unrestricted. Biochemical characterization of this mutant as well as molecular dynamics simulations suggest that only a limited movement of the plug domain suffices for translocation.

Solvent-exposed tails as prestalk transition states for membrane fusion at low hydration.

Membrane fusion is a key step in intracellular trafficking and viral infection. The underlying molecular mechanism is poorly understood. We have used molecular dynamics simulations in conjunction with a coarse grained model to study early metastable and transition states during the fusion of two planar palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayers separated by five waters per lipid in the cis leaflets at zero tension. This system mimics the contact area between two vesicles with large diameters compared to the membrane thickness at conditions where fusion may start in the core of the contact area. At elevated temperatures, the two proximal leaflets become connected via multiple lipid molecules and form a stalklike structure. At room temperature, this structure has a free energy of 3k(B)T and is separated from the unconnected state by a significant free energy barrier of 20k(B)T. Stalk formation is initiated by the establishment of a localized hydrophobic contact between the bilayers. This contact is either formed by two partially splayed lipids or a single fully splayed one leading to the formation of a (metastable) splayed lipid bond intermediate. These findings indicate that, for low hydration, early membrane fusion kinetics is not determined by the stalk energy but by the energy of prestalk transition states involving solvent-exposed lipid tails.

Concerted diffusion of lipids in raft-like membranes.

Currently, there is no comprehensive model for the dynamics of cellular membranes. The understanding of even the basic dynamic processes, such as lateral diffusion of lipids, is still quite limited. Recent studies of one-component membrane systems have shown that instead of single-particle motions, the lateral diffusion is driven by a more complex, concerted mechanism for lipid diffusion (E. Falck et al., J. Am. Chem. Soc., 2008, 130, 44-45), where a lipid and its neighbors move in unison in terms of loosely defined clusters. In this work, we extend the previous study by considering the concerted lipid diffusion phenomena in many-component raft-like membranes. This nature of diffusion phenomena emerge in all the cases we have considered, including both atom-scale simulations of lateral diffusion within rafts and coarse-grained MARTINI simulations of diffusion in membranes characterized by coexistence of raft and non-raft domains. The data allows us to identify characteristic time scales for the concerted lipid motions, which turn out to range from hundreds of nanoseconds to several microseconds. Further, we characterize typical length scales associated with the correlated lipid diffusion patterns and find them to be about 10 nm, or even larger if weak correlations are taken into account. Finally, the concerted nature of lipid motions is also found in dissipative particle dynamics simulations of lipid membranes, clarifying the role of hydrodynamics (local momentum conservation) in membrane diffusion phenomena.

Simulations of the c-subunit of ATP-synthase reveal helix rearrangements.

The c-subunit of the enzyme, ATP synthase couples the proton movement through the a-subunit with its own rotation and subsequent rotation of the F1 ring to drive ATP synthesis. Here, we perform mus time-scale coarse-grained molecular dynamics simulations of the c-subunit to characterize its structure and dynamics. Two different helix-helix interfaces, albeit with similar interfacial characteristics, are sampled in the simulations. The helix-2 of the c-subunit monomer rotates around the axis of helix-1 bringing about a change in the interface. Previous models have also proposed such a change in the helix interface but postulated that helix-2 swivels around its own axis. Such large-scale changes in helix packing motifs have not been observed before. The helix-swirling persists even in the c-subunit ring but the dynamics is much slower. The cooperative behavior in the ring appears to stabilize a conformation less-populated in the monomer. Analyzing the stability of the c-subunit ring, it was found that six lipid molecules are necessary to fill the central cavity of the ring. These lipid molecules were not aligned with the surrounding bilayer but protruded towards the periplasmic side. The characterization of the monomer and ring presented in this work sheds light into the structural dynamics of the c-subunit and its functional relevance.