Molecular Insights into the Dynamics of Amyloid Fibril Growth: Elongation and Lateral Assembly of GNNQQNY Protofibrils.

The self-assembly of peptides and proteins into ß-sheet rich amyloid fibrils is linked to both functional and pathological states. In this study, the growth of fibrillar structures of the short peptide GNNQQNY, a fragment from the yeast prion Sup35 protein, was examined. Molecular dynamics simulations were used to study alternative mechanisms of fibril growth, including elongation through binding of monomers as well as fibril self-assembly into larger, more mature structures. It was found that after binding, monomers diffused along preformed fibrils toward the ends, supporting the mechanism of fibril growth via elongation. Lateral assembly of protofibrils was found to occur readily, suggesting that this could be the key to transitioning from isolated fibrils to mature multilayer structures. Overall, the work provides mechanistic insights into the competitive pathways that govern amyloid fibril growth.

OFraMP: a fragment-based tool to facilitate the parametrization of large molecules.

An Online tool for Fragment-based Molecule Parametrization (OFraMP) is described. OFraMP is a web application for assigning atomic interaction parameters to large molecules by matching sub-fragments within the target molecule to equivalent sub-fragments within the Automated Topology Builder (ATB, atb.uq.edu.au) database. OFraMP identifies and compares alternative molecular fragments from the ATB database, which contains over 890,000 pre-parameterized molecules, using a novel hierarchical matching procedure. Atoms are considered within the context of an extended local environment (buffer region) with the degree of similarity between an atom in the target molecule and that in the proposed match controlled by varying the size of the buffer region. Adjacent matching atoms are combined into progressively larger matched sub-structures. The user then selects the most appropriate match. OFraMP also allows users to manually alter interaction parameters and automates the submission of missing substructures to the ATB in order to generate parameters for atoms in environments not represented in the existing database. The utility of OFraMP is illustrated using the anti-cancer agent paclitaxel and a dendrimer used in organic semiconductor devices. OFraMP applied to paclitaxel (ATB ID 35922).

Understanding the Activated Form of a Class-I Fusion Protein: Modeling the Interaction of the Ebola Virus Glycoprotein 2 with a Lipid Bilayer.

The fusion of the viral and target cell membranes is a key step in the life cycle of all enveloped viruses. Here, a range of structural data is used to generate an evidence-based model of the active conformation of an archetypical type-I fusion protein, the Ebola glycoprotein 2 (GP2). The stability of the trimeric complex is demonstrated using molecular dynamics and validated by simulating the interaction of the complex with a lipid bilayer. In this model, the fusion peptides project away from the central helix bundle parallel to the target membrane. This maximizes contact with the host membrane, enhances lateral stability, and would explain why, when activated, viral fusion proteins are trimeric.

The reliability of molecular dynamics simulations of the multidrug transporter P-glycoprotein in a membrane environment.

Despite decades of research, the mechanism of action of the ABC multidrug transporter P-glycoprotein (P-gp) remains elusive. Due to experimental limitations, many researchers have turned to molecular dynamics simulation studies in order to investigate different aspects of P-gp function. However, such studies are challenging and caution is required when interpreting the results. P-gp is highly flexible and the time scale on which it can be simulated is limited. There is also uncertainty regarding the accuracy of the various crystal structures available, let alone the structure of the protein in a physiologically relevant environment. In this study, three alternative structural models of mouse P-gp (3G5U, 4KSB, 4M1M), all resolved to 3.8 Å, were used to initiate sets of simulations of P-gp in a membrane environment in order to determine: a) the sensitivity of the results to differences in the starting configuration; and b) the extent to which converged results could be expected on the times scales commonly simulated for this system. The simulations suggest that the arrangement of the nucleotide binding domains (NBDs) observed in the crystal structures is not stable in a membrane environment. In all simulations, the NBDs rapidly associated (within 10 ns) and changes within the transmembrane helices were observed. The secondary structure within the transmembrane domain was best preserved in the 4M1M model under the simulation conditions used. However, the extent to which replicate simulations diverged on a 100 to 200 ns timescale meant that it was not possible to draw definitive conclusions as to which structure overall was most stable, or to obtain converged and reliable results for any of the properties examined. The work brings into question the reliability of conclusions made in regard to the nature of specific interactions inferred from previous simulation studies on this system involving similar sampling times. It also highlights the need to demonstrate the statistical significance of any results obtained in simulations of large flexible proteins, especially where the initial structure is uncertain.

Membrane-binding properties of gating modifier and pore-blocking toxins: Membrane interaction is not a prerequisite for modification of channel gating.

Many venom peptides are potent and selective inhibitors of voltage-gated ion channels, including channels that are validated therapeutic targets for treatment of a wide range of human diseases. However, the development of novel venom-peptide-based therapeutics requires an understanding of their mechanism of action. In the case of voltage-gated ion channels, venom peptides act either as pore blockers that bind to the extracellular side of the channel pore or gating modifiers that bind to one or more of the membrane-embedded voltage sensor domains. In the case of gating modifiers, it has been debated whether the peptide must partition into the membrane to reach its binding site. In this study, we used surface plasmon resonance, fluorescence spectroscopy and molecular dynamics to directly compare the lipid-binding properties of two gating modifiers (µ-TRTX-Hd1a and ProTx-I) and two pore blockers (ShK and KIIIA). Only ProTx-I was found to bind to model membranes. Our results provide further evidence that the ability to insert into the lipid bilayer is not a requirement to be a gating modifier. In addition, we characterised the surface of ProTx-I that mediates its interaction with neutral and anionic phospholipid membranes and show that it preferentially interacts with anionic lipids.

Molecular dynamics and functional studies define a hot spot of crystal contacts essential for PcTx1 inhibition of acid-sensing ion channel 1a.

BACKGROUND AND PURPOSE: The spider-venom peptide PcTx1 is the most potent and selective inhibitor of acid-sensing ion channel (ASIC) 1a. It has centrally acting analgesic activity and is neuroprotective in rodent models of ischaemic stroke. Understanding the molecular details of the PcTx1 : ASIC1a interaction should facilitate development of therapeutically useful ASIC1a modulators. Previously, we showed that several key pharmacophore residues of PcTx1 reside in a dynamic ß-hairpin loop; conclusions confirmed by recent crystal structures of the complex formed between PcTx1 and chicken ASIC1 (cASIC1). Numerous peptide : channel contacts were observed in these crystal structures, but it remains unclear which of these are functionally important. EXPERIMENTAL APPROACH: We combined molecular dynamics (MD) simulations of the PcTx1 : cASIC1 complex with mutagenesis of PcTx1 and rat ASIC1a. KEY RESULTS: Crystal structures of the PcTx1 : cASIC1 complex indicated that 15 PcTx1 residues form a total of 57 pairwise intermolecular contacts (<5 Å) with 32 channel residues. MD simulations, however, suggested that about half of these interactions do not persist in solution. Mutation to alanine of only eight of 15 PcTx1 contact residues substantially altered ASIC1a inhibition by PcTx1. Our data reveal that many of the peptide-channel interactions observed in the PcTx1 : cASIC1 crystal structures are not important for PcTx1 inhibition of rat ASIC1a. CONCLUSIONS AND IMPLICATIONS: We identified the atomic interactions that are critical for PcTx1 inhibition of ASIC1a. Our data highlight the value of combining structural information, MD and functional experiments to obtain detailed insight into the molecular basis of protein : protein interactions.

Identification of Possible Binding Sites for Morphine and Nicardipine on the Multidrug Transporter P-Glycoprotein Using Umbrella Sampling Techniques.

The multidrug transporter P-glycoprotein (P-gp) is central to the development of multidrug resistance in cancer. While residues essential for transport and binding have been identified, the location, composition, and specificity of potential drug binding sites are uncertain. Here molecular dynamics simulations are used to calculate the free energy profile for the binding of morphine and nicardipine to P-gp. We show that morphine and nicardipine primarily interact with key residues implicated in binding and transport from mutational studies, binding at different but overlapping sites within the transmembrane pore. Their permeation pathways were distinct but involved overlapping sets of residues. The results indicate that the binding location and permeation pathways of morphine and nicardipine are not well separated and cannot be considered as unique. This has important implications for our understanding of substrate uptake and transport by P-gp. Our results are independent of the choice of starting structure and consistent with a range of experimental studies.

Determining the structure of interfacial peptide films: comparing neutron reflectometry and molecular dynamics simulations.

The peptides AM1 and Lac21E self-organize into switchable films at an air-water interface. In an earlier study, it was proposed that both AM1 and Lac21E formed monolayers of α-helical peptides based on consistency with neutron reflectivity data. In this article, molecular dynamics simulations of assemblies of helical and nonhelical AM1 and Lac21E at an air-water interface suggest some tendency for the peptides to spontaneously adopt an α-helical conformation. However, irrespective of the structure of the peptides, the simulations reproduced not only the structural properties of the films (thickness and distribution of the hydrophobic and hydrophilic amino acids) but also the experimental neutron reflectivity measurements at different contrast variations. This suggests that neutron reflectometry alone cannot be used to determine the structure of the peptides in this case. However, together with molecular dynamics simulations, it is possible to obtain a detailed understanding of peptide films at an atomic level.

Mechanism of activation of protein kinase JAK2 by the growth hormone receptor.

Signaling from JAK (Janus kinase) protein kinases to STAT (signal transducers and activators of transcription) transcription factors is key to many aspects of biology and medicine, yet the mechanism by which cytokine receptors initiate signaling is enigmatic. We present a complete mechanistic model for activation of receptor-bound JAK2, based on an archetypal cytokine receptor, the growth hormone receptor. For this, we used fluorescence resonance energy transfer to monitor positioning of the JAK2 binding motif in the receptor dimer, substitution of the receptor extracellular domains with Jun zippers to control the position of its transmembrane (TM) helices, atomistic modeling of TM helix movements, and docking of the crystal structures of the JAK2 kinase and its inhibitory pseudokinase domain with an opposing kinase-pseudokinase domain pair. Activation of the receptor dimer induced a separation of its JAK2 binding motifs, driven by a ligand-induced transition from a parallel TM helix pair to a left-handed crossover arrangement. This separation leads to removal of the pseudokinase domain from the kinase domain of the partner JAK2 and pairing of the two kinase domains, facilitating trans-activation. This model may well generalize to other class I cytokine receptors.

Activation of the epidermal growth factor receptor: a series of twists and turns.

The cell surface epidermal growth factor receptor (EGFR) plays a critical role in cell development and oncogenesis. The binding of growth factors to the EGFR results in a mechanical signal being transmitted through the plasma membrane. In this study, atomistic molecular dynamics simulations have been used to investigate the conformational changes associated with the binding of the epidermal growth factor (EGF) and transforming growth factor α (TGFα) to the EGFR. In the simulations, the removal of the EGF and TGFα from the extracellular domain of the EGFR homodimer led to a relative rotation of the protomers of 16-35° about the dimerization axis. The three N-terminal domains that make up the extracellular region of the receptor undergo essentially rigid-body motion. The dimerization interface itself was found to be largely unaffected by the removal of the ligand. In most simulations, the rotation within the dimer was associated with an opening of the cytokine-binding sites. On the basis of these simulations, a simple mechanical model that explains the coupling between the binding of ligand and the motions in the extracellular domains is proposed.

The recognition of membrane-bound PtdIns3P by PX domains.

Phox-homology (PX) domains target proteins to the organelles of the secretary and endocytic systems by binding to phosphatidylinositol phospholipids (PIPs). Among all the structures of PX domains that have been solved, only three have been solved in a complex with the main physiological ligand: PtdIns3P. In this work, molecular dynamic simulations have been used to explore the structure and dynamics of the p40(phox) -PX domain and the SNX17-PX domain and their interaction with membrane-bound PtdIns3P. In the simulations, both PX domains associated spontaneously with the membrane-bound PtdIns3P and formed stable complexes. The interaction between the p40(phox) -PX domain and PtdIns3P in the membrane was found to be similar to the crystal structure of the p40(phox) -PX-PtdIns3P complex that is available. The interaction between the SNX17-PX domain and PtdIns3P was similar to that observed in the p40(phox) -PX-PtdIns3P complex; however, some residues adopted different orientations. The simulations also showed that nonspecific interactions between the ß1-ß2 loop and the membrane play an important role in the interaction of membrane bound PtdIns3P and different PX domains. The behaviour of unbound PtdIns3P within a 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) membrane environment was also examined and compared to the available experimental data and simulation studies of related molecules.

Structural characterization of two metastable ATP-bound states of P-glycoprotein.

ATP Binding Cassette (ABC) transporters couple the binding and hydrolysis of ATP to the transport of substrate molecules across the membrane. The mechanism by which ATP binding and/or hydrolysis drives the conformational changes associated with substrate transport has not yet been characterized fully. Here, changes in the conformation of the ABC export protein P-glycoprotein on ATP binding are examined in a series of molecular dynamics simulations. When one molecule of ATP is placed at the ATP binding site associated with each of the two nucleotide binding domains (NBDs), the membrane-embedded P-glycoprotein crystal structure adopts two distinct metastable conformations. In one, each ATP molecule interacts primarily with the Walker A motif of the corresponding NBD. In the other, the ATP molecules interacts with both Walker A motif of one NBD and the Signature motif of the opposite NBD inducing the partial dimerization of the NBDs. This interaction is more extensive in one of the two ATP binding site, leading to an asymmetric structure. The overall conformation of the transmembrane domains is not altered in either of these metastable states, indicating that the conformational changes associated with ATP binding observed in the simulations in the absence of substrate do not lead to the outward-facing conformation and thus would be insufficient in themselves to drive transport. Nevertheless, the metastable intermediate ATP-bound conformations observed are compatible with a wide range of experimental cross-linking data demonstrating the simulations do capture physiologically important conformations. Analysis of the interaction between ATP and its cofactor Mg(2+) with each NBD indicates that the coordination of ATP and Mg(2+) differs between the two NBDs. The role structural asymmetry may play in ATP binding and hydrolysis is discussed. Furthermore, we demonstrate that our results are not heavily influenced by the crystal structure chosen for initiation of the simulations.

Charge group partitioning in biomolecular simulation.

Molecular simulation techniques are increasingly being used to study biomolecular systems at an atomic level. Such simulations rely on empirical force fields to represent the intermolecular interactions. There are many different force fields available--each based on a different set of assumptions and thus requiring different parametrization procedures. Recently, efforts have been made to fully automate the assignment of force-field parameters, including atomic partial charges, for novel molecules. In this work, we focus on a problem arising in the automated parametrization of molecules for use in combination with the GROMOS family of force fields: namely, the assignment of atoms to charge groups such that for every charge group the sum of the partial charges is ideally equal to its formal charge. In addition, charge groups are required to have size at most k. We show NP-hardness and give an exact algorithm that solves practical problem instances to provable optimality in a fraction of a second.

A dynamic pharmacophore drives the interaction between Psalmotoxin-1 and the putative drug target acid-sensing ion channel 1a.

Acid-sensing ion channel 1a (ASIC1a) is a primary acid sensor in the peripheral and central nervous system. It has been implicated as a novel therapeutic target for a broad range of pathophysiological conditions including pain, ischemic stroke, depression, and autoimmune diseases such as multiple sclerosis. The only known selective blocker of ASIC1a is π-TRTX-Pc1a (PcTx1), a disulfide-rich 40-residue peptide isolated from spider venom. π-TRTX-Pc1a is an effective analgesic in rodent models of acute pain and it provides neuroprotection in a mouse model of ischemic stroke. Thus, understanding the molecular basis of the π-TRTX-Pc1a-ASIC1a interaction should facilitate development of therapeutically useful ASIC1a blockers. We therefore developed an efficient bacterial expression system to produce a panel of π-TRTX-Pc1a mutants for probing structure-activity relationships as well as isotopically labeled toxin for determination of its solution structure and dynamics. We demonstrate that the toxin pharmacophore resides in a ß-hairpin loop that was revealed to be mobile over a wide range of time scales using molecular dynamics simulations in combination with NMR spin relaxation and relaxation dispersion measurements. The toxin-receptor interaction was modeled by in silico docking of the toxin structure onto a homology model of rat ASIC1a in a restraints-driven approach that was designed to take account of the dynamics of the toxin pharmacophore and the consequent remodeling of side-chain conformations upon receptor binding. The resulting model reveals new insights into the mechanism of action of π-TRTX-Pc1a and provides an experimentally validated template for the rational design of therapeutically useful π-TRTX-Pc1a mimetics.

Effect of poly(ethylene glycol) (PEG) spacers on the conformational properties of small peptides: a molecular dynamics study.

Poly(ethylene glycol) (PEG) is used as an inert spacer in a wide range of biotechnological applications such as to display peptides and proteins on surfaces for diagnostic purposes. In such applications it is critical that the peptide is accessible to solvent and that the PEG does not affect the conformational properties of the peptide to which it is attached. Using molecular dynamics (MD) simulation techniques, we have investigated the influence of a commonly used PEG spacer on the conformation properties of a series of five peptides with differing physical-chemical properties (YGSLPQ, VFVVFV, GSGGSG, EEGEEG, and KKGKKG). The conformational properties of the peptides were compared (a) free in solution, (b) attached to a PEG-11 spacer in solution, and (c) constrained to a two-dimensional lattice via a (PEG-11)(3) spacer, mimicking a peptide displayed on a surface as used in microarray techniques. The simulations suggest that the PEG spacer has little effect on the conformational properties of small neutral peptides but has a significant effect on the conformational properties of small highly charged peptides. When constrained to a two-dimensional surface at peptide densities similar to those used experimentally, it was found that the peptides, in particular the polar and nonpolar peptides, aggregated strongly. The peptides also partitioned into the PEG layer. Potentially, this means that at high packing densities only a small fraction of the peptide attached to the surface would in fact be accessible to a potential interaction partner.

The Cys3-Cys4 loop of the hydrophobin EAS is not required for rodlet formation and surface activity.

Class I hydrophobins are fungal proteins that self-assemble into robust amphipathic rodlet monolayers on the surface of aerial structures such as spores and fruiting bodies. These layers share many structural characteristics with amyloid fibrils and belong to the growing family of functional amyloid-like materials produced by microorganisms. Although the three-dimensional structure of the soluble monomeric form of a class I hydrophobin has been determined, little is known about the molecular structure of the rodlets or their assembly mechanism. Several models have been proposed, some of which suggest that the Cys3-Cys4 loop has a critical role in the initiation of assembly or in the polymeric structure. In order to provide insight into the relationship between hydrophobin sequence and rodlet assembly, we investigated the role of the Cys3-Cys4 loop in EAS, a class I hydrophobin from Neurospora crassa. Remarkably, deletion of up to 15 residues from this 25-residue loop does not impair rodlet formation or reduce the surface activity of the protein, and the physicochemical properties of rodlets formed by this mutant are indistinguishable from those of its full-length counterpart. In addition, the core structure of the truncation mutant is essentially unchanged. Molecular dynamics simulations carried out on the full-length protein and this truncation mutant binding to an air-water interface show that, although it is hydrophobic, the loop does not play a role in positioning the protein at the surface. These results demonstrate that the Cys3-Cys4 loop does not have an integral role in the formation or structure of the rodlets and that the major determinant of the unique properties of these proteins is the amphipathic core structure, which is likely to be preserved in all hydrophobins despite the high degree of sequence variation across the family.

The structure of a two-disulfide intermediate assists in elucidating the oxidative folding pathway of a cyclic cystine knot protein.

We have determined the three-dimensional structure of a two-disulfide intermediate (Cys(8)-Cys(20), Cys(14)-Cys(26)) on the oxidative folding pathway of the cyclotide MCoTI-II. Cyclotides have a range of bioactivities and, because of their exceptional stability, have been proposed as potential molecular scaffolds for drug design applications. The three-dimensional structure of the stable two-disulfide intermediate shows for the most part identical secondary and tertiary structure to the native state. The only exception is a flexible loop, which is collapsed onto the protein core in the native state, whereas in the intermediate it is more loosely associated with the remainder of the protein. The results suggest that the native fold of the peptide does not represent the free energy minimum in the absence of the Cys(1)-Cys(18) disulfide bridge and that although there is not a large energy barrier, the peptide must transiently adopt an energetically unfavorable state before the final disulfide can form.

Histidine protonation and the activation of viral fusion proteins.

Many viral fusion proteins only become activated under mildly acidic condition (pH 4.5-6.5) close to the pK(a) of histidine side-chain protonation. Analysis of the sequences and structures of influenza HA (haemagglutinin) and flaviviral envelope glycoproteins has led to the identification of a number of histidine residues that are not only fully conserved themselves but have local environments that are also highly conserved [Kampmann, Mueller, Mark, Young and Kobe (2006) Structure 14, 1481-1487]. Here, we summarize studies aimed at determining the role, if any, that protonation of these potential switch histidine residues plays in the low-pH-dependent conformational changes associated with fusion activation of a flaviviral envelope protein. Specifically, we report on MD (Molecular Dynamics) simulations of the DEN2 (dengue virus type 2) envelope protein ectodomain sE (soluble E) performed under varied pH conditions designed to test the histidine switch hypothesis of Kampmann et al. (2006).

The conformation of the extracellular binding domain of Death Receptor 5 in the presence and absence of the activating ligand TRAIL: a molecular dynamics study.

The Death Receptor 5 (DR5), a member of tumor necrosis factor receptor (TNFR) superfamily of receptors, triggers apoptosis (programmed cell death) when stimulated by its tridentate ligand TRAIL. Until recently it was generally assumed that the activation of DR5 resulted from the recruitment of three independent receptor units, leading to the trimerization of intracellular domains. However, there is mounting evidence to suggest that, in the absence of ligand, such cytokine receptors primarily reside as preformed complexes. In this work, molecular dynamics simulations of the TRAIL-DR5 complex, the unbound receptor trimer and individual receptor monomers are compared to gain insight in the mechanism of activation. The results suggest that, in the absence of TRAIL, DR5 has a strong propensity to self-associate and that this is primarily mediated through interactions of the membrane proximal domains. The association of the free receptors leads to a loss of the threefold symmetry found within the receptor-ligand complex. The simulations suggest that the primary role of TRAIL is to induce threefold-symmetry within the DR5 complex and to constrain the receptor to a specific conformation. The implications of this in terms of the mechanism by which the receptor switches from an inactive to an active state are discussed.

Convergence and sampling efficiency in replica exchange simulations of peptide folding in explicit solvent.

Replica exchange methods (REMs) are increasingly used to improve sampling in molecular dynamics (MD) simulations of biomolecular systems. However, despite having been shown to be very effective on model systems, the application of REM in complex systems such as for the simulation of protein and peptide folding in explicit solvent has not been objectively tested in detail. Here we present a comparison of conventional MD and temperature replica exchange MD (T-REMD) simulations of a beta-heptapeptide in explicit solvent. This system has previously been shown to undergo reversible folding on the time scales accessible to MD simulation and thus allows a direct one-to-one comparison of efficiency. The primary properties compared are the free energy of folding and the relative populations of different conformers as a function of temperature. It is found that to achieve a similar degree of precision T-REMD simulations starting from a random set of initial configurations were approximately an order of magnitude more computationally efficient than a single 800 ns conventional MD simulation for this system at the lowest temperature investigated (275 K). However, whereas it was found that T-REMD simulations are more than four times more efficient than multiple independent MD simulations at one temperature (300 K) the actual increase in conformation sampling was only twofold. The overall gain in efficiency using REMD resulted primarily from the ordering of different conformational states over temperature, as opposed to a large increase of conformational sampling. It is also shown that in this system exchanges are accepted primarily based on (random) fluctuations within the solvent and are not strongly correlated with the instantaneous peptide conformation raising questions in regard to the efficiency of T-REMD in larger systems.