Solid-state NMR reveals structural differences between fibrils of wild-type and disease-related A53T mutant alpha-synuclein

Fibrils from the Parkinson's-disease-related A53T mutant of α-synuclein were investigated by solid-state NMR spectroscopy, electron microscopy, and atomic force microscopy. Sequential solid-state NMR resonance assignments were obtained for a large fraction of the fibril core. Experiments conducted above and below the freezing point suggest that the fibrils contain regions with increased mobility and structural elements different from β-strand character, in addition to the rigid β-sheet-rich core region. As in earlier studies on wild-type α-synuclein, the C-terminus was found to be flexible and unfolded, whereas the main core region was highly rigid and rich in β-sheets. Compared to fibrils from wild-type α-synuclein, the well-ordered β-sheet region extends to at least L38 and L100. These results demonstrate that a disease-related mutant of α-synuclein differs in both aggregation kinetics and fibril structure.     

The interaction of capping protein with the barbed end of the actin filament

The interaction of capping protein (CP) with actin filaments is an essential element of actin assembly and actin-based motility in nearly all eukaryotes. The dendritic nucleation model for Arp2/3-based lamellipodial assembly features capping of barbed ends by CP, and the formation of filopodia is proposed to involve inhibition of capping by formins and other proteins. To understand the molecular basis for how CP binds the barbed end of the actin filament, we have used a combination of computational and experimental approaches, primarily involving molecular docking and site-directed mutagenesis. We arrive at a model that supports all of our biochemical data and agrees very well with a cryo-electron microscopy structure of the capped filament. CP interacts with both actin protomers at the barbed end of the filament, and the amphipathic helix at the C-terminus of the β-subunit binds to the hydrophobic cleft on actin, in a manner similar to that of WH2 domains. These studies provide us with new molecular insight into how CP binds to the actin filament.     

Conformations of NhaA, the Na/H Exchanger from Escherichia coli, in the pH-Activated and Ion-Translocating States

NhaA, the main sodium-proton exchanger in the inner membrane of Escherichia coli, regulates the cytosolic concentrations of H and Na. It is inactive at acidic pH, becomes active between pH 6 and pH 7, and reaches maximum activity at pH 8. By cryo-electron microscopy of two-dimensional crystals grown at pH 4 and incubated at higher pH, we identified two sequential conformational changes in the protein in response to pH or substrate ions. The first change is induced by a rise in pH from 6 to 7 and marks the transition from the inactive state to the pH-activated state. pH activation, which precedes the ion-induced conformational change, is accompanied by an overall expansion of the NhaA monomer and a local ordering of the N-terminus. The second conformational change is induced by the substrate ions Na and Li at pH above 7 and involves a 7-Å displacement of helix IVp. This movement would cause a charge imbalance at the ion-binding site that may trigger the release of the substrate ion and open a periplasmic exit channel.     

Dipolar assisted rotational resonance NMR of tryptophan and tyrosine in rhodopsin

Two dimensional (2D) solid-state (13)C.(13)C dipolar recoupling experiments are performed on a series of model compounds and on the visual pigment rhodopsin to establish the most effective method for long range distance measurements in reconstituted membrane proteins. The effects of uniform labeling, inhomogeneous B(1) fields, relaxation and dipolar truncation on cross peak intensity are investigated through NMR measurements of simple amino acid and peptide model compounds. We first show that dipolar assisted rotational resonance (DARR) is more effective than RFDR in recoupling long-range dipolar interactions in these model systems. We then use DARR to establish (13)C-(13)C correlations in rhodopsin. In rhodopsin containing 4'-(13)C-Tyr and 8,19-(13)C retinal, we observe two distinct tyrosine-to-retinal correlations in the DARR spectrum. The most intense cross peak arises from a correlation between Tyr268 and the retinal 19-(13)CH(3), which are 4.8 A apart in the rhodopsin crystal structure. A second cross peak arises from a correlation between Tyr191 and the retinal 19-(13)CH(3), which are 5.5 A apart in the crystal structure. These data demonstrate that long range (13)C em leader (13)C correlations can be obtained in non-crystalline integral membrane proteins reconstituted into lipid membranes containing less than 150 nmoles of protein. In rhodopsin containing 2-(13)C Gly121 and U-(13)C Trp265, we do not observe a Trp-Gly cross peak in the DARR spectrum despite their close proximity (3.6 A) in the crystal structure. Based on model compounds, the absence of a (13)C em leader (13)C cross peak is due to loss of intensity in the diagonal Trp resonances rather than to dipolar truncation.     

Erratum to “Conformations of NhaA, the Na/H Exchanger from Escherichia coli, in the pH-Activated and Ion-Translocating States” [J. Mol. Biol. 386 (2009) 351-385]: Conformations of NhaA, the Na/H Exchanger from Escherichia coli, in the pH-Activated and Ion-Translocating States

NhaA, the main sodium-proton exchanger in the inner membrane of Escherichia coli, regulates the cytosolic concentrations of H⁺ and Na⁺. It is inactive at acidic pH, becomes active between pH 6 and pH 7, and reaches maximum activity at pH 8. By cryo-electron microscopy of two-dimensional crystals grown at pH 4 and incubated at higher pH, we identified two sequential conformational changes in the protein in response to pH or substrate ions. The first change is induced by a rise in pH from 6 to 7 and marks the transition from the inactive state to the pH-activated state. pH activation, which precedes the ion-induced conformational change, is accompanied by an overall expansion of the NhaA monomer and a local ordering of the N-terminus. The second conformational change is induced by the substrate ions Na⁺ and Li⁺ at pH above 7 and involves a 7-Å displacement of helix IVp. This movement would cause a charge imbalance at the ion-binding site that may trigger the release of the substrate ion and open a periplasmic exit channel.     

Characterization of the sodium binding sites in microcrystalline ATP by Na-solid-state NMR and ab initio calculations

Stepwise conformational transition of disodium adenosine 5′-triphosphate (Na₂ATP) crystals as a function of relative humidity (r.H.), was examined by means of high resolution ²³Na, ³¹P solid state NMR spectroscopy and quantum mechanics calculations. ²³Na-MQMAS solid state NMR spectra of hydrated disodium salts of adenosine 5,5′-triphosphate were measured to characterize the individual sodium sites. Quadrupolar coupling constants (C_Q), and the asymmetry parameters of the electric field gradient tensors (η) of sodium-23 determined by quantum mechanical calculations (gaussian03) using larger clusters than in previous studies were compared to the experimental ²³Na-MQMAS spectra.     

Structured regions of α-synuclein fibrils include the early-onset Parkinson's disease mutation sites

α-Synuclein (AS) fibrils are the major component of Lewy bodies, the pathological hallmark of Parkinson's disease (PD). Here, we use results from an extensive investigation employing solid-state NMR to present a detailed structural characterization and conformational dynamics quantification of full-length AS fibrils. Our results show that the core extends with a repeated structural motif. This result disagrees with the previously proposed fold of AS fibrils obtained with limited solid-state NMR data. Additionally, our results demonstrate that the three single point mutations associated with early-onset PD—A30P, E46K and A53T—are located in structured regions. We find that E46K and A53T mutations, located in rigid β-strands of the wild-type fibrils, are associated with major and minor structural perturbations, respectively.     

Structure and dynamics of photosynthetic membrane-bound proteins in Rhodobacter Sphaeroides,studied with solid-state NMR spectroscopy

The photosynthetic purple bacteria such as Rb. sphaeroides possesses an intracytoplasmic membrane (ICM) and a variety of pigment-binding membrane proteins located in the ICM, acting as photoreceptor. Such photosynthetic apparatus is concentrated in the ICM. It is composed of three multimeric membrane-bound proteins; light-harvesting complexes (LH 1, LH 2), a reaction center (RC) and a cytochrome b/c1 complex. We have purified these membranes, which are called chromatophores, and characterized the structure and dynamics of the photosynthetic membrane-bound proteins by means of multi-nuclear solid state NMR. First, the isotropic chemical shift of carbonyl carbons in natural abundance and [1-¹³C] Phe labeled chromatophores indicates that the membrane-bound proteins take mainly the helical conformation. Second, the chemical shifts of side-chain resonances of uniformly ¹⁵N-labeled chromatophores indicate the side-chain histidine residue is mainly hydrogen bonded, whereas structural heterogeneity of arginine and lysine side-chains are probed by those wide distribution of ¹⁵N shifts. Thirdly, the [β-²H₃]Ala and [ε-²H₂]Tyr labeling of the chromatophores are performed and dynamics of the [β-²H]Ala and the [ε-²H₂]Tyr labeled chromatophores are studied by means of ²H solid state NMR. The dynamics of [β-²H₃]Ala is found to be a 10⁸Hz three-site jump motion with 10° liberation along the Cα-Cβ bond axis. The ²H-NMR powder pattern spectrum of [ε-²H₂] Tyr labeled chromatophores was interpreted with an averaged correlation time of 5×10⁵ Hz with 180° two-fold flips, the result of the averaging of two kinds of split spectra in terms of motional time scale. This revised version was published online in June 2006 with corrections to the Cover Date.     

The interaction of cofilin with the actin filament

Cofilin is a key actin-binding protein that is critical for controlling the assembly of actin within the cell. Here, we present the results of molecular docking and dynamics studies using a muscle actin filament and human cofilin I. Guided by extensive mutagenesis results and other biophysical and structural studies, we arrive at a model for cofilin bound to the actin filament. This predicted structure agrees very well with electron microscopy results for cofilin-decorated filaments, provides molecular insight into how the known F- and G-actin sites on cofilin interact with the filament, and also suggests new interaction sites that may play a role in cofilin binding. The resulting atomic-scale model also helps us understand the molecular function and regulation of cofilin and provides testable data for future experimental and simulation work.     

Peptide antibiotics in action: Investigation of polypeptide chains in insoluble environments by rotational-echo double resonance

Rotational-echo double resonance (REDOR) is a solid-state NMR technique that has the capability of providing intra- and intermolecular distance and orientational restraints in non-crystallizable, poorly soluble heterogeneous molecular systems such as cell membranes and cell walls. In this review, we will present two applications of REDOR: the investigation of a magainin-related antimicrobial peptide in lipid bilayers and the study of a vancomycin-like glycopeptide in the cell walls of Staphylococcus aureus.     

Structural constraints on the transmembrane and juxtamembrane regions of the phospholamban pentamer in membrane bilayers: Gln29 and Leu52

The Ca²⁺-ATPase of cardiac muscle cells transports Ca²⁺ ions against a concentration gradient into the sarcoplasmic reticulum and is regulated by phospholamban, a 52-residue integral membrane protein. It is known that phospholamban inhibits the Ca²⁺ pump during muscle contraction and that inhibition is removed by phosphorylation of the protein during muscle relaxation. Phospholamban forms a pentameric complex with a central pore. The solid-state magic angle spinning (MAS) NMR measurements presented here address the structure of the phospholamban pentamer in the region of Gln22-Gln29. Rotational echo double resonance (REDOR) NMR measurements show that the side chain amide groups of Gln29 are in close proximity, consistent with a hydrogen-bonded network within the central pore. ¹³C MAS NMR measurements are also presented on phospholamban that is 1-¹³C-labeled at Leu52, the last residue of the protein. pH titration of the C-terminal carboxyl group suggests that it forms a ring of negative charge on the lumenal side of the sarcoplasmic reticulum membrane. The structural constraints on the phospholamban pentamer described in this study are discussed in the context of a multifaceted mechanism for Ca²⁺ regulation that may involve phospholamban as both an inhibitor of the Ca²⁺ ATPase and as an ion channel.     

Solid-State NMR Studies of Full-Length BamA in Lipid Bilayers Suggest Limited Overall POTRA Mobility

The outer membrane protein BamA is the key player in β-barrel assembly in Gram-negative bacteria. Despite the availability of high-resolution crystal structures, the dynamic behavior of the transmembrane domain and the large periplasmic extension consisting of five POTRA (POlypeptide-TRansport-Associated) domains remains unclear. We demonstrate reconstitution of full-length BamA in proteoliposomes at low lipid-to-protein ratio, leading to high sensitivity and resolution in solid-state NMR (ssNMR) experiments. We detect POTRA domains in ssNMR experiments probing rigid protein segments in our preparations. These results suggest that the periplasmic region of BamA is firmly attached to the β-barrel and does not experience fast global motion around the angle between POTRA 2 and 3. We show that this behavior holds at lower protein concentrations and elevated temperatures. Chemical shift variations observed after reconstitution in lipids with different chain lengths and saturation levels are compatible with conformational plasticity of BamA's transmembrane domain. Electron microscopy of the ssNMR samples shows that BamA can cause local disruptions of the lipid bilayer in proteoliposomes. The observed interplay between protein–protein and protein–lipid interactions may be critical for BamA-mediated insertion of substrates into the outer membrane.     

Comparative Analysis of Membrane-Associated Fusion Peptide Secondary Structure and Lipid Mixing Function of HIV gp41 Constructs that Model the Early Pre-Hairpin Intermediate and Final Hairpin Conformations

Fusion between viral and host cell membranes is the initial step of human immunodeficiency virus infection and is mediated by the gp41 protein, which is embedded in the viral membrane. The ∼ 20-residue N-terminal fusion peptide (FP) region of gp41 binds to the host cell membrane and plays a critical role in fusion catalysis. Key gp41 fusion conformations include an early pre-hairpin intermediate (PHI) characterized by extended coiled-coil structure in the region C-terminal of the FP and a final hairpin state with compact six-helix bundle structure. The large “N70” (gp41 1-70) and “FP-Hairpin” constructs of the present study contained the FP and respectively modeled the PHI and hairpin conformations. Comparison was also made to the shorter “FP34” (gp41 1-34) fragment. Studies were done in membranes with physiologically relevant cholesterol content and in membranes without cholesterol. In either membrane type, there were large differences in fusion function among the constructs with little fusion induced by FP-Hairpin, moderate fusion for FP34, and very rapid fusion for N70. Overall, our findings support acceleration of gp41-induced membrane fusion by early PHI conformation and fusion arrest after folding to the final six-helix bundle structure. FP secondary structure at Leu7 of the membrane-associated constructs was probed by solid-state nuclear magnetic resonance and showed populations of molecules with either β-sheet or helical structure with greater β-sheet population observed for FP34 than for N70 or FP-Hairpin. The large differences in fusion function among the constructs were not obviously correlated with FP secondary structure. Observation of cholesterol-dependent FP structure for fusogenic FP34 and N70 and cholesterol-independent structure for non-fusogenic FP-Hairpin was consistent with membrane insertion of the FP for FP34 and N70 and with lack of insertion for FP-Hairpin. Membrane insertion of the FP may therefore be associated with the early PHI conformation and FP withdrawal with the final hairpin conformation.     

Solid-state NMR and molecular dynamics simulations reveal the oligomeric ion-channels of TM2-GABAA stabilized by intermolecular hydrogen bonding

The second transmembrane (TM2) domain of GABA_A receptor forms the inner-lining surface of chloride ion-channel and plays important roles in the function of the receptor protein. In this study, we report the first structure of TM2 in lipid bilayers determined using solid-state NMR and MD simulations. The interatomic ¹³C-¹⁵N distances measured from REDOR magic angle spinning experiments on multilamellar vesicles, containing a TM2 peptide site specifically labeled with ¹³C′ and ¹⁵N isotopes, were used to determine the secondary structure of the peptide. The ¹⁵N chemical shift and ¹H-¹⁵N dipolar coupling parameters measured from PISEMA experiments on mechanically aligned phospholipid bilayers, containing a TM2 peptide site specifically labeled with ¹⁵N isotopes, under static conditions were used to determine the membrane orientation of the peptide. Our results reveal that the TM2 peptide forms an alpha helical conformation with a tilted transmembrane orientation, which is unstable as a monomer but stable as pentameric oligomers as indicated by MD simulations. Even though the peptide consists of a number of hydrophilic residues, the transmembrane folding of the peptide is stabilized by intermolecular hydrogen bondings between the side chains of Ser and Thr residues as revealed by MD simulations. The results also suggest that peptide-peptide interactions in the tilted transmembrane orientation overcome the hydrophobic mismatch between the peptide and bilayer thickness.     

Structural Insights into Yeast DNA Polymerase δ by Small Angle X-ray Scattering

DNA polymerase δ (Polδ) is a multisubunit polymerase that plays an indispensable role in replication from yeast to humans. Polδ from Saccharomyces cerevisiae is composed of three subunits: Pol3, Pol31, and Pol32. Despite the elucidation of the structures and models of the individual subunits (or portions, thereof), the nature of their assembly remains unclear. We present here a small-angle X-ray scattering analysis of a yeast Polδ complex (Polδ_T) composed of Pol3, Pol31, and Pol32N (amino acids 1-103 of Pol32). From the small angle X-ray scattering global parameters and reconstructed envelopes, we show that Polδ_T adopts an elongated conformation with a radius of gyration (R_g) of ∼ 52 Å and a maximal dimension of ∼ 190 Å. We also propose an orientation for the accessory Pol31-Pol32N subunits relative to the Pol3 catalytic core that best agrees with the experimental scattering profile. The analysis also points to significant conformational variability that may allow Polδ to better coordinate its action with other proteins at the replication fork.     

Steric zipper of the amyloid fibrils formed by residues 109-122 of the Syrian hamster prion protein

We report the results of atomic force microscopy, Fourier-transform infrared spectroscopy, solid-state nuclear magnetic resonance, and molecular dynamics (MD) calculations for amyloid fibrils formed by residues 109-122 of the Syrian hamster prion protein (H1). Our data reveal that H1 fibrils contain no more than two β-sheet layers. The peptide strands of H1 fibrils are antiparallel with the A117 residues aligned to form a linear chain in the direction of the fibril axis. The molecular structure of the H1 fibrils, which adopts the motif of steric zipper, is highly uniform in the region of the palindrome sequence AGAAAAGA. The closest distance between the two adjacent β-sheet layers is found to be about 5 Å. The structural features of the molecular model of H1 fibrils obtained by MD simulations are consistent with the experimental results. Overall, our solid-state NMR and MD simulation data indicate that a steric zipper, which was first observed in the crystals of fibril-forming peptides, can be formed in H1 fibrils near the region of the palindrome sequence.     

Lipid domains in bacterial membranes and the action of antimicrobial agents

There has been increasing interest in recent years in describing the lateral organization of membranes and the formation of membrane domains. Much of the focus in this area has been on the formation of cholesterol-rich domains in mammalian membranes. However, it is likely that there are domains in all biological membranes. One of the challenges has been to define the chemical composition, lifetime and size of these domains. There is evidence that bacteria have domains that are enriched in cardiolipin. In addition, the formation of lipid domains can be induced in bacteria by clustering negatively charged lipids with polycationic substances. Many antimicrobial compounds have multiple positive charges. Such polycationic compounds can sequester anionic lipids to induce lipid phase separation. The molecular interactions among lipids and their lateral packing density will be different in a domain from its environment. This will lead to phase boundary defects that will lower the permeability barrier between the cell and its surroundings. The formation of these clusters of anionic lipids may also alter the stability or composition of existing membrane domains that may affect bacterial function. Interestingly many antimicrobial agents are polycationic and therefore likely have some effect in promoting lipid phase segregation between anionic and zwitterionic lipids. However, this mechanism is expected to be most important for substances with sequential positive charges contained within a flexible molecule that can adapt to the arrangement of charged groups on the surface of the bacterial cell. When this mechanism is dominant it can allow the prediction of the bacterial species that will be most affected by the agent as a consequence of the nature of the lipid composition of the bacterial membrane.     

Backbone conformational flexibility of the lipid modified membrane anchor of the human N-Ras protein investigated by solid-state NMR and molecular dynamics simulation

The lipid modified human N-Ras protein, implicated in human cancer development, is of particular interest due to its membrane anchor that determines the activity and subcellular location of the protein. Previous solid-state NMR investigations indicated that this membrane anchor is highly dynamic, which may be indicative of backbone conformational flexibility. This article aims to address if a dynamic exchange between three structural models exist that had been determined previously. We applied a combination of solid-state nuclear magnetic resonance (NMR) methods and replica exchange molecular dynamics (MD) simulations using a Ras peptide that represents the terminal seven amino acids of the human N-Ras protein. Analysis of correlations between the conformations of individual amino acids revealed that Cys 181 and Met 182 undergo collective conformational exchange. Two major structures constituting about 60% of all conformations could be identified. The two conformations found in the simulation are in rapid exchange, which gives rise to low backbone order parameters and nuclear spin relaxation as measured by experimental NMR methods. These parameters were also determined from two 300 ns conventional MD simulations, providing very good agreement with the experimental data.     

A molecular dynamics study of the interaction of D-peptide amyloid inhibitors with their target sequence reveals a potential inhibitory pharmacophore conformation

The self-assembly of soluble proteins and peptides into β-sheet-rich oligomeric structures and insoluble fibrils is a hallmark of a large number of human diseases known as amyloid diseases. Drugs that are able to interfere with these processes may be able to prevent and/or cure these diseases. Experimental difficulties in the characterization of the intermediates involved in the amyloid formation process have seriously hampered the application of rational drug design approaches to the inhibition of amyloid formation and growth. Recently, short model peptide systems have proved useful in understanding the relationship between amino acid sequence and amyloid formation using both experimental and theoretical approaches. Moreover, short d-peptide sequences have been shown to specifically interfere with those short amyloid stretches in proteins, blocking oligomer formation or disassembling mature fibrils. With the aim of rationalizing which interactions drive the binding of inhibitors to nascent β-sheet oligomers, in this study, we have carried out extensive molecular dynamics simulations of the interaction of selected d-peptide sequences with oligomers of the target model sequence STVIIE. Structural analysis of the simulations helped to identify the molecular determinants of an inhibitory core whose conformational and physicochemical properties are actually shared by nonpeptidic small-molecule inhibitors of amyloidogenesis. Selection of one of these small molecules and experimental validation against our model system proved that it was indeed an effective inhibitor of fibril formation by the STVIIE sequence, supporting theoretical predictions. We propose that the inhibitory determinants derived from this work be used as structural templates in the development of pharmacophore models for the identification of novel nonpeptidic inhibitors of aggregation.