The amyloid concentric ß-barrel hypothesis: Models of amyloid beta 42 oligomers and annular protofibrils.

Amyloid beta (Aß) peptides are a major contributor to Alzheimer's disease. They occur in differing lengths, each of which forms a multitude of assembly types. The most toxic soluble oligomers are formed by Aß42; some of which have antiparallel ß-sheets. Previously, our group proposed molecular models of Aß42 hexamers in which the C-terminus third of the peptide (S3) forms an antiparallel 6-stranded ß-barrel that is surrounded by an antiparallel barrel formed by the more polar N-terminus (S1) and middle (S2) portions. These hexamers were proposed to act as seeds from which dodecamers, octadecamers, both smooth annular protofibrils (sAPFs) and beaded annular protofibrils (bAPFs), and transmembrane channels form. Since then, numerous aspects of our models have been supported by experimental findings. Recently, NMR-based structures have been proposed for Aß42 tetramers and octamers, and NMR studies have been reported for oligomers composed of ~32 monomers. Here we propose a range of concentric ß-barrel models and compare their dimensions to image-averaged electron micrographs of both bAPFs and sAPFs of Aß42. The smaller oligomers have 6, 8, 12, 16, and 18 monomers. These beads string together to form necklace-like bAPFs. These bAPRs gradually morph into sAPFs in which a S3 ß-barrel is shielded on one or both sides by ß-barrels formed from S1 and S2 segments.

The amyloid concentric ß-barrel hypothesis: Models of synuclein oligomers, annular protofibrils, lipoproteins, and transmembrane channels.

Amyloid beta (Aß of Alzheimer's disease) and α-synuclein (α-Syn of Parkinson's disease) form large fibrils. Evidence is increasing however that much smaller oligomers are more toxic and that these oligomers can form transmembrane ion channels. We have proposed previously that Aß42 oligomers, annular protofibrils, and ion channels adopt concentric ß-barrel molecular structures. Here we extend that hypothesis to the superfamily of α, ß, and γ-synucleins. Our models of numerous synuclein oligomers, annular protofibrils, tubular protofibrils, lipoproteins, and ion channels were developed to be consistent with sizes, shapes, molecular weights, and secondary structures of assemblies as determined by electron microscopy and other studies. The models have the following features: (1) all subunits have identical structures and interactions; (2) they are consistent with conventional ß-barrel theory; (3) the distance between walls of adjacent ß-barrels is between 0.6 and 1.2 nm; (4) hydrogen bonds, salt bridges, interactions among aromatic side-chains, burial and tight packing of hydrophobic side-chains, and aqueous solvent exposure of hydrophilic side-chains are relatively optimal; and (5) residues that are identical among distantly related homologous proteins cluster in the interior of most oligomers whereas residues that are hypervariable are exposed on protein surfaces. Atomic scale models of some assemblies were developed.

Stability tests on known and misfolded structures with discrete and all atom molecular dynamics simulations.

The brevity of molecular dynamics simulations often limits their utility in developing and evaluating structural models of proteins. The duration of simulations can be increased greatly using discrete molecular dynamics (DMD). However, the trade off is that coarse graining, implicit solvent, and other time-saving procedures reduce the accuracy of DMD simulations. Here we address some of these issues by comparing results of DMD and conventional all atom MD simulations on proteins of known structure and misfolded proteins. DMD simulations were performed at a range of temperatures to identify a 'physiological' temperature for DMD that mimicked molecular motions of conventional MD simulations at 310K. We also compared results obtained with a new implicit solvent model developed here based on Miyazawa-Jernigan interaction pair potential to those obtained with a previously used model based on Kyte-Doolittle hydropathy scale. We compared DMD and all atom molecular dynamics with explicit water by simulating both correctly and incorrectly folded structures, and monomeric and dimeric α ß-barrel structures to analyze the ability of these procedures to distinguish between good and bad models. Deviations from the correct structures were substantially greater with DMD, as would be expected from coarse-graining and longer simulation time. Deviations were smallest for ß-strands and greatest for coiled loops. Structures of the incorrectly folded models were very poorly preserved during the DMD simulations; but both methods were able to distinguish between the correct and the incorrect structures based on differences in the magnitudes of the root mean squared deviation (RMSD) from the starting conformation.

Analysis of the stabilities of hexameric amyloid-ß(1-42) models using discrete molecular dynamics simulations.

Amyloid-ß (Aß) oligomers appear to play a pivotal role in Alzheimer's disease. A 42 residue long alloform, Aß42, is closely related to etiology of the disease. In vitro results show evidences of hexamers; however structures of these hexamers have not been resolved experimentally. Here, we use discrete molecular dynamics (DMD) to analyze long duration stabilities of Aß42 hexamer models developed previously in our lab. The hydrophobic core of these models is a six-stranded ß-barrel with 3-fold radial symmetry formed by residues 30-40. This core is shielded from water by residues 1-28. The nine models we analyzed differ by the relative positions of the core ß-strands, and whether the other segments surrounding the core contain α helices or ß-strands. A model of an annular protofibril composed of 36 Aß peptides was also simulated. Results of these model simulations were compared with results of aggregation simulations that started from six well separated random coils of Aß42 and with simulations of two known ß-barrel structures. These results can be categorized into three groups: stable models with properties similar or superior to those of experimentally determined ß-barrel proteins, aggregation-prone models, and an amorphous aggregate from random coils. Conformations at the end of the simulation for aggregation-prone models have exposed hydrophobic core with dangling ß-strands on the surface. Hydrogen bond patterns within the ß-barrel were a critical factor for stability of the ß-barrel models. Aggregation-prone conformations imply that the association of these hexamers may be possible, which could lead to the formation of larger assemblies.

Structural models of TREK channels and their gating mechanism.

Mechanosensitive TREK channels belong to the family of K2P channels, a family of widely distributed, well modulated channels that uniquely have two similar or identical subunits, each with two TM1-P-TM2 motifs. Our goal is to build viable structural models of TREK channels, as representatives of K2P channels family. The structures available to be used as templates belong to the 2TM channels superfamily. These have low sequence similarity and different structural features: four symmetrically arranged subunits, each having one TM1-P-TM2 motif. Our model building strategy used two subunits of the template (KcsA) to build one subunit of the target (TREK-1). Our models of the Closed channel were adjusted to differ substantially from those of the template, e.g., TM2 of the 2nd repeat is near the axis of the pore whereas TM2 of the 1st repeat is far from the axis. Segments linking the two repeats and immediately following the last TM segment were modeled ab initio as α-helices based on helical periodicities of hydrophobic and hydrophilic residues, highly conserved and poorly conserved residues, and statistically related positions from multiple sequence alignments. The models were further refined by two-fold symmetry-constrained MD simulations using a protocol we developed previously. We also built models of the Open state and suggest a possible tension-activated gating mechanism characterized by helical motion with two-fold symmetry. Our models are consistent with deletion/truncation mutagenesis and thermodynamic analysis of gating described in the accompanying paper.

Analyses of gating thermodynamics and effects of deletions in the mechanosensitive channel TREK-1: comparisons with structural models.

TREK-1, a mechanosensitive K channel from the two-pore family (K(2)P), is involved in protective regulation of the resting potential in CNS neurons and other tissues. The structure of TREK-1 and the basis of its sensitivity to stretch and variety of lipid-soluble factors remain unknown. Using existing K channel structures as modeling templates, TREK-1 was envisioned as a two-fold symmetrical complex with the gate formed primarily by the centrally positioned TM2b helices of the second homologous repeat. Opening was modeled as a conical expansion of the barrel separating TM2b's accompanied by extension of TM2a helices with the cytoplasmic TM2a-TM1b connector. Seeking first experimental support to the models we have accomplished thermodynamic analysis of mouse TREK-1 gating and functional testing of several deletion mutants. The predicted increase of the channel in-plane area (ΔA) of ~5 nm(2) in models was supported by the experimental ΔA of ~4 nm(2) derived from the slope of open probability versus membrane tension in HEK-293T cells and their cytoskeleton-depleted blebs. In response to steps of suction, wild-type channel produced transient currents in cell-attached patches and mostly sustained currents upon patch excision. TREK-1 motifs not present in canonical K channels include divergent cytoplasmic N- and C-termini, and a characteristic 50-residue extracellular loop in the first homologous repeat. Deletion of the extracellular loop (Δ76-124) reduced the average current density in patches, increased spontaneous activity and generated a larger sub-population of high-conductance channels, while activation by tension augmented by arachidonic acid was fully retained. Further deletion of the C-terminal end (Δ76-124/Δ334-411) removed voltage dependency but otherwise produced no additional effect. In an attempt to generate a cysteine-free version of the channel, we mutated two remaining cysteines 159 and 219 in the transmembrane region. C219A did not compromise channel activity, whereas the C159A/S mutants were essentially inactive. Treatment with ß-mercaptoethanol suggested that none of these cysteines form functionally-important disulfides.

Models of membrane-bound Alzheimer's Abeta peptide assemblies.

Although it is clear that amyloid beta (Aß) peptides play a pivotal role in the development of Alzheimer's disease, the precise molecular model of action remains unclear. Aß peptide forms assemble both in aqueous solution and in lipid membranes. It has been proposed that deleterious effects occur when the peptides interact with membranes, possibly by forming Ca(2+) permeant ion channels. In the accompanying manuscript, we propose models in which the C-terminus third of six Aß42 peptides forms a six-stranded ß-barrel in highly toxic soluble oligomers. Here we extend this hypothesis to membrane-bound assemblies. In these Aß models, the hydrophobic ß-barrel of a hexamer may either reside on the surface of the bilayer, or span the bilayer. Transmembrane pores are proposed to form between several hexamers. Once the ß-barrels of six hexamers have spanned the bilayer, they may merge to form a more stable 36-stranded ß-barrel. We favor models in which parallel ß-barrels formed by N-terminus segments comprise the lining of the pores. These types of models explain why the channels are selective for cations and how metal ions, such as Zn(2+) , synthetic peptides that contain histidines, and some small organic cations may block channels or inhibit formation of channels. Our models were developed to be consistent with microscopy studies of Aß assemblies in membranes, one of which is presented here for the first time.

Beta-barrel models of soluble amyloid beta oligomers and annular protofibrils.

Both soluble and membrane-bound prefibrillar assemblies of Abeta (Aß) peptides have been associated with Alzheimer's disease (AD). The size and nature of these assemblies vary greatly and are affected by many factors. Here, we present models of soluble hexameric assemblies of Aß42 and suggest how they can lead to larger assemblies and eventually to fibrils. The common element in most of these assemblies is a six-stranded ß-barrel formed by the last third of Aß42, which is composed of hydrophobic residues and glycines. The hydrophobic core ß-barrels of the hexameric models are shielded from water by the N-terminus and central segments. These more hydrophilic segments were modeled to have either predominantly ß or predominantly α secondary structure. Molecular dynamics simulations were performed to analyze stabilities of the models. The hexameric models were used as starting points from which larger soluble assemblies of 12 and 36 subunits were modeled. These models were developed to be consistent with numerous experimental results.

Polyhistidine peptide inhibitor of the Abeta calcium channel potently blocks the Abeta-induced calcium response in cells. Theoretical modeling suggests a cooperative binding process.

On the basis of the consistent demonstrations that the Abeta peptide of Alzheimer's disease forms calcium permeant channels in artificial membranes, we have proposed that the intracellular calcium increase observed in cells exposed to Abeta is initiated by calcium fluxes through Abeta channels. We have found that a small four-histidine peptide, NAHis04, potently inhibits the Abeta-induced calcium channel currents in artificial lipid membranes. Here we report that NaHis04 also potently blocks the intracellular calcium increase which is observed in cells exposed to Abeta. PC12 cells loaded with Fura-2AM show a rapid increase in fluorescence and a rapid return to baseline after Abeta is added to the medium. This fluorescence change occurs even when the medium contains nitrendipine, a voltage-gated calcium channel blocker, but fails to occur when application of Abeta is preceded by addition of NAHis04. Steep dose-response curves of the percentage of responding cells and cell viability show that NAHis04 inhibits in the micromolar range in an apparently cooperative manner. We have developed numerous models of Abeta pores in which the first part of the Abeta sequence forms a large beta-barrel ending at His 13. We have modeled how up to four NAHis04 peptides may block these types of pores by binding to side chains of Abeta residues Glu 11, His 13, and His 14.

The KtrA and KtrE subunits are required for Na+-dependent K+ uptake by KtrB across the plasma membrane in Synechocystis sp. strain PCC 6803.

The Na+-dependent K+ uptake KtrABE system is essential for the adaptation of Synechocystis to salinity stress and high osmolality. While KtrB forms the K+-translocating pore, the role of the subunits KtrA and KtrE for Ktr function remains elusive. Here, we characterized the role of KtrA and KtrE in Ktr-mediated K+ uptake and in modulating Na+ dependency. Expression of KtrB alone in a K+ uptake-deficient Escherichia coli strain conferred low K+ uptake activity that was not stimulated by Na+. Coexpression of both KtrA and KtrE with KtrB increased the K+ transport activity in a Na+-dependent manner. KtrA and KtrE were found to be localized to the plasma membrane in Synechocystis. Site-directed mutagenesis was used to analyze the role of single charged residues in KtrB for Ktr function. Replacing negatively charged residues facing the extracellular space with residues of the opposite charge increased the apparent Km for K+ in all cases. However, none of the mutations eliminated the Na+ dependency of Ktr-mediated K+ transport. Mutations of residues on the cytoplasmic side had larger effects on K+ uptake activity than those of residues on the extracellular side. Further analysis revealed that replacement of R262, which is well conserved among Ktr/Trk/HKT transporters in the third extracellular loop, by Glu abolished transport activity. The atomic-scale homology model indicated that R262 might interact with E247 and D261. Based on these data, interaction of KtrA and KtrE with KtrB increased the K+ uptake rate and conferred Na+ dependency.

Symmetry-restrained molecular dynamics simulations improve homology models of potassium channels.

Most crystallized homo-oligomeric ion channels are highly symmetric, which dramatically decreases conformational space and facilitates building homology models (HMs). However, in molecular dynamics (MD) simulations channels deviate from ideal symmetry and accumulate thermal defects, which complicate the refinement of HMs using MD. In this work we evaluate the ability of symmetry constrained MD simulations to improve HMs accuracy, using an approach conceptually similar to Critical Assessment of techniques for protein Structure Prediction (CASP) competition: build HMs of channels with known structure and evaluate the efficiency of proposed methods in improving HMs accuracy (measured as deviation from experimental structure). Results indicate that unrestrained MD does not improve the accuracy of HMs, instantaneous symmetrization improves accuracy but not stability of HMs during subsequent unrestrained MD, while gradually imposing symmetry constraints improves both accuracy (by 5-50%) and stability of HMs. Moreover, accuracy and stability are strongly correlated, making stability a reliable criterion in predicting the accuracy of new HMs. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

Pore stability and gating in voltage-activated calcium channels.

Calcium channel family members activate at different membrane potentials, which enables tissue specific calcium entry. Pore mutations affecting this voltage dependence are associated with channelopathies. In this review we analyze the link between voltage sensitivity and corresponding kinetic phenotypes of calcium channel activation. Systematic changes in hydrophobicity in the lower third of S6 segments gradually shift the activation curve thereby determining the voltage sensitivity. Homology modeling suggests that hydrophobic residues that are located in all four S6 segments close to the inner channel mouth might form adhesion points stabilizing the closed gate. Simulation studies support a scenario where voltage sensors and the pore are essentially independent structural units. We speculate that evolution designed the voltage sensing machinery as robust "all-or-non" device while the varietys of voltage sensitivities of different channel types was accomplished by shaping pore stability.

Molecular dynamics and mutational analysis of a channelopathy mutation in the IIS6 helix of Ca V 1.2.

A channelopathy mutation in segment IIS6 of Ca(V)1.4 (I745T) has been shown to cause severe visual impairment by shifting the activation and inactivation curves to more hyperpolarized voltages and slowing activation and inactivation kinetics. A similar gating phenotype is caused by the corresponding mutation, I781T, in Ca(V)1.2 (midpoint of activation curve (V(0.5)) shifted to -37.7 +/- 1.2 mV). We show here that wild-type gating can partially be restored by a helix stabilizing rescue mutation N785A. V(0.5) of I781T/N785A (V(0.5) = -21.5 +/- 0.6 mV) was shifted back towards wild-type (V(0.5) = -9.9 +/- 1.1 mV). Homology models developed in our group (see accompanying article for details) were used to perform Molecular Dynamics-simulations (MD-simulations) on wild-type and mutant channels. Systematic changes in segment IIIS6 (M1187-F1194) and in helix IIS6 (N785-L786) were studied. The simulated structural changes in S6 segments of I781T/N785A were less pronounced than in I781T. A delicate balance between helix flexibility and stability enabling the formation of hydrophobic seals at the inner channel mouth appears to be important for wild-type Ca(V)1.2 gating. Our study illustrates that effects of mutations in the lower part of IIS6 may not be localized to the residue or even segment being mutated, but may affect conformations of interacting segments.

Structural model of the Ca V 1.2 pore.

Understanding the structure and functional mechanisms of voltage-gated calcium channels remains a major task in membrane biophysics. In the absence of three dimensional structures, homology modeling techniques are the method of choice, to address questions concerning the structure of these channels. We have developed models of the open Ca(V)1.2 pore, based on the crystal structure of the mammalian voltage-gated potassium channel K(V)1.2 and a model of the bacterial sodium channel NaChBac. Our models are developed to be consistent with experimental data and modeling criteria. The models highlight major differences between voltage-gated potassium and calcium channels in the P segments, as well as the inner pore helices. Molecular dynamics simulations support the hypothesis of a clockwise domain arrangement and experimental observations of asymmetric calcium channel behavior. In the accompanying paper these models were used to study structural effects of a channelopathy mutation.

Models of voltage-dependent conformational changes in NaChBac channels.

Models of the transmembrane region of the NaChBac channel were developed in two open/inactivated and several closed conformations. Homology models of NaChBac were developed using crystal structures of Kv1.2 and a Kv1.2/2.1 chimera as templates for open conformations, and MlotiK and KcsA channels as templates for closed conformations. Multiple molecular-dynamic simulations were performed to refine and evaluate these models. A striking difference between the S4 structures of the Kv1.2-like open models and MlotiK-like closed models is the secondary structure. In the open model, the first part of S4 forms an alpha-helix, and the last part forms a 3(10) helix, whereas in the closed model, the first part of S4 forms a 3(10) helix, and the last part forms an alpha-helix. A conformational change that involves this type of transition in secondary structure should be voltage-dependent. However, this transition alone is not sufficient to account for the large gating charge movement reported for NaChBac channels and for experimental results in other voltage-gated channels. To increase the magnitude of the motion of S4, we developed another model of an open/inactivated conformation, in which S4 is displaced farther outward, and a number of closed models in which S4 is displaced farther inward. A helical screw motion for the alpha-helical part of S4 and a simple axial translation for the 3(10) portion were used to develop models of these additional conformations. In our models, four positively charged residues of S4 moved outwardly during activation, across a transition barrier formed by highly conserved hydrophobic residues on S1, S2, and S3. The S4 movement was coupled to an opening of the activation gate formed by S6 through interactions with the segment linking S4 to S5. Consistencies of our models with experimental studies of NaChBac and K(v) channels are discussed.

Models of the structure and gating mechanisms of the pore domain of the NaChBac ion channel.

The NaChBac prokaryotic sodium channel appears to be a descendent of an evolutionary link between voltage-gated K(V) and Ca(V) channels. Like K(V) channels, four identical six-transmembrane subunits comprise the NaChBac channel, but its selectivity filter possesses a signature sequence of eukaryotic Ca(V) channels. We developed structural models of the NaChBac channel in closed and open conformations, using K(+)-channel crystal structures as initial templates. Our models were also consistent with numerous experimental results and modeling criteria. This study concerns the pore domain. The major differences between our models and K(+) crystal structures involve the latter portion of the selectivity filter and the bend region in S6 of the open conformation. These NaChBac models may serve as a stepping stone between K(+) channels of known structure and Na(V), Ca(V), and TRP channels of unknown structure.

Probing the outer mouth structure of the HERG channel with peptide toxin footprinting and molecular modeling.

Previous studies have shown that the unusually long S5-P linker lining human ether a-go-go related gene's (hERG's) outer vestibule is critical for its channel function: point mutations at high-impact positions here can interfere with the inactivation process and, in many cases, also reduce the pore's K+ selectivity. Because no data are available on the equivalent region in the available K channel crystal structures to allow for homology modeling, we used alternative approaches to model its three-dimensional structure. The first part of this article describes mutant cycle analysis used to identify residues on hERG's outer vestibule that interact with specific residues on the interaction surface of BeKm-1, a peptide toxin with known NMR structure and a high binding affinity to hERG. The second part describes molecular modeling of hERG's pore domain. The transmembrane region was modeled after the crystal structure of KvAP pore domain. The S5-P linker was docked to the transmembrane region based on data from previous NMR and mutagenesis experiments, as well as a set of modeling criteria. The models were further restrained by contact points between hERG's outer vestibule and the bound BeKm-1 toxin molecule deduced from the mutant cycle analysis. Based on these analyses, we propose a working model for the open conformation of the outer vestibule of the hERG channel, in which the S5-P linkers interact with the pore loops to influence ion flux through the pore.

Role of positively charged amino acids in the M2D transmembrane helix of Ktr/Trk/HKT type cation transporters.

Studies suggest that Ktr/Trk/HKT-type transporters have evolved from multiple gene fusions of simple K(+) channels of the KcsA type into proteins that span the membrane at least eight times. Several positively charged residues are present in the eighth transmembrane segment, M2(D), in the transporters but not K(+) channels. Some models of ion transporters require a barrier to prevent free diffusion of ions down their electrochemical gradient, and it is possible that the positively charged residues within the transporter pore may prevent transporters from being channels. Here we studied the functional role of these positive residues in three Ktr/Trk/HKT-type transporters (Synechocystis KtrB-mediated K(+) uniporter, Arabidopsis AtHKT1-mediated Na(+) uniporter and wheat TaHKT1-mediated K(+)/Na(+) symporter) by examining K(+) uptake rates in E. coli, electrophysiological measurements in oocytes and growth rates of E. coli and yeast. The conserved Arg near the middle of the M2(D) segment was essential for the K(+) transport activity of KtrB and plant HKTs. Combined replacement of several positive residues in TaHKT1 showed that the positive residue at the beginning of the M2(D), which is conserved in many K(+) channels, also contributed to cation transport activity. This positive residue and the conserved Arg both face towards the ion conducting pore side. We introduced an atomic-scale homology model for predicting amino acid interactions. Based on the experimental results and the model, we propose that a salt bridge(s) exists between positive residues in the M2(D) and conserved negative residues in the pore region to reduce electrostatic repulsion against cation permeation caused by the positive residue(s). This salt bridge may help stabilize the transporter configuration, and may also prevent the conformational change that occurs in channels.

Structure-function studies of the outer mouth and voltage sensor domain of hERG.

hERG has uniquely fast inactivation but slow activation processes. We study the structural basis for these unique gating properties using the following approaches: site-specific mutagenesis, the MTS accessibility test, disulfide bond formation, thermodynamic mutant cycle analysis, peptide toxin 'foot-printing', NMR spectroscopy, and molecular modelling. We propose the following: (1) two structural features in hERG's outer mouth contribute to its fast inactivation rate: a lack of 'open mouth'-stabilizing hydrogen bonds and an unusually long extracellular 'S5-P' linker that contains an alpha-helix. During membrane depolarization, four such 'S5-P helices' from the tetramer channel come near each other to occlude the outer mouth. This occurs rapidly due to the dynamic nature of the S5-P helices. (2) Two structural features in hERG's voltage sensor domain contribute to its slow activation rate: hERG's major voltage-sensor, S4, has three (instead of four as in Shaker) positive charges involved in gating charge transfer, and hERG has six (instead of three as in Shaker) negative charges in the other transmembrane segments (S1-S3) of the voltage sensor domain. Thus a less voltage-sensitive S4, in conjunction with more surrounding negative charges (some of which can form salt-bridges with S4's positive charges in the pre-open state), retards channel activation.