Site-directed Spin Labeling Studies Research Articles

Secondary structure and transmembrane topology analysis of the N-terminal domain of the inner membrane protein EccE1 from M. smegmatis using site-directed spin labeling EPR

Protein EccE1 is an essential component of the mycobacterial ESX-1 secretion system, which plays a crucial part in the process of virulence factors secretion, especially for pathogenic mycobacteria such as Mycobacterium tuberculosis. While EccE1 was previously postulated to be the inner membrane pore-forming unit of a membrane complex through which substrates are transported, the structural properties of EccE1 remains to be explored. In the present study, systematic Site-Directed Spin Labeling (SDSL) and Electron Paramagnetic Resonance (EPR) spectroscopic studies was carried out to reveal the secondary structure and transmembrane topology of the N-terminal Domain of EccE1 protein (EccE1-NTD) from M. smegmatis in detergent micelles. EPR-based mobility and accessibility analysis of the R1 side chain for 64 residue positions of EccE1-NTD indicates that the transmembrane domain adopts two α-helices spanning Phe7-Cys30 and Leu36-Ile54. A tentative structural topology model of EccE1-NTD embedded in membrane is also suggested based on EPR spectroscopic data in this study, which will provide further insights into this protein and the ESX secretion systems of mycobacteria.

Synthetic and Biophysical Studies on the Toxic Conformer in Amyloid β with the E22Δ Mutation in Alzheimer Pathology.

The toxic conformer of the 40- or 42-mer-amyloid β-proteins (Aβ) (Aβ40, Aβ42) with a turn at positions 22 and 23 plays a role in oligomer formation, leading to neurotoxicity as part of the pathogenesis of Alzheimer's disease (AD). A deletion mutant at Glu22 (E22Δ) of Aβ, known as an Osaka mutation, accelerates oligomerization. Although E22Δ-Aβ has not been found to be toxic to cultured neuronal cells and is instead synaptotoxic in long-term potentiation, there is no information on the toxic conformer of E22Δ-Aβ in AD. The site-directed spin labeling study of E22Δ-Aβ40 by continuous wave-electron spin resonance (CW-ESR) spectroscopy in part showed the spatial proximity between positions 10 and 35, which are characteristic of the toxic conformation of Aβ, indicating the existence of a toxic conformer of Aβ with the E22Δ mutation. To obtain structural insight, E22Δ-Aβ42 substitutes with proline (F20P, A21P, D23P, and V24P), in which proline is known as a turn inducer but is a β-sheet breaker, were synthesized. An enzyme immunoassay using the 24B3 antibody recognizing toxic conformer of Aβ was carried out. 24B3 reacted with these substitutes of E22Δ-Aβ42 as well as E22Δ-Aβ42 in a similar manner to WT-Aβ42. Notably, only A21P-E22Δ-Aβ42 exhibited strong neurotoxicity in rat primary neurons after 8 days of incubation, with potent high-order oligomerization compared with E22Δ-Aβ42. These results suggest that E22Δ-Aβ42 could enhance neurotoxicity by generating a toxic oligomer conformation with a turn near position 21.

Continuous Wave Electron Paramagnetic Resonance Spectroscopy Reveals the Structural Topology and Dynamic Properties of Active Pinholin S2168 in a Lipid Bilayer.

Pinholin S2168 is an essential part of the phage Φ21 lytic protein system to release the virus progeny at the end of the infection cycle. It is known as the simplest natural timing system for its precise control of hole formation in the inner cytoplasmic membrane. Pinholin S2168 is a 68 amino acid integral membrane protein consisting of two transmembrane domains (TMDs) called TMD1 and TMD2. Despite its biological importance, structural and dynamic information of the S2168 protein in a membrane environment is not well understood. Systematic site-directed spin labeling and continuous wave electron paramagnetic resonance (CW-EPR) spectroscopic studies of pinholin S2168 in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) proteoliposomes are used to reveal the structural topology and dynamic properties in a native-like environment. CW-EPR spectral line-shape analysis of the R1 side chain for 39 residue positions of S2168 indicates that the TMDs have more restricted mobility when compared to the N- and C-termini. CW-EPR power saturation data indicate that TMD1 partially externalizes from the lipid bilayer and interacts with the membrane surface, whereas TMD2 remains buried in the lipid bilayer in the active conformation of pinholin S2168. A tentative structural topology model of pinholin S2168 is also suggested based on EPR spectroscopic data reported in this study.

Improved signal fidelity in 4-pulse DEER with Gaussian pulses

The introduction of arbitrary waveform generator (AWG) technology and the availability of high power microwave amplifiers mark a “new era” in pulse EPR due to significant sensitivity improvements and the possibility to perform novel types of experiments. We present an optimized 4-pulse DEER setup that uses Gaussian observer pulses (GaussDEER) in connection with a Gaussian/shaped pump pulse. Gaussian pulses allow to experimentally remove the “2+1” pulse train ESE signal which is intrinsically present in any DEER experiment performed with rectangular pulses. Further signal improvements are obtained with shaped pump pulses, which can significantly increase the modulation depth of the DEER experiment due to their tailored excitation bandwidth. Although sequences like CP (Carr-Purcell) DEER offer advantages such as a prolongation of the dipolar evolution time, they suffer from post-processing of the time-domain data to remove artifacts. Therefore, it is worth having a 4-pulse DEER experiment free of residual “2+1” signal since this is still the main dipolar spectroscopic technique used in structural biology. In this work we focus on nitroxides, which are the spin probes primarily used in site-directed spin labeling studies of biomolecules, however, the advantages introduced by Gaussian pulses can be extended to any spin type.

Combined approaches of EPR and NMR illustrate only one transmembrane helix in the human IFITM3

Interferon-inducible transmembrane protein IFITM3 was known to restrict the entry of a wide spectrum of viruses to the cytosol of the host. The mechanism used by the protein to restrict viral entry is unclear given the unavailability of the membrane topology and structures of the IFITM family proteins. Systematic site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) studies of IFITM3 in detergent micelles identified a single, long transmembrane helix in the C-terminus and an intramembrane segment in the N-terminal hydrophobic region. Solution NMR studies of the same sample verified the secondary structure distribution and demonstrated two rigid regions interacting with the micellar surface. The resulting membrane topology of IFITM3 supports the mechanism of an enhanced restricted membrane hemi-fusion.

CONDENSED-MATTER SPECTROSCOPY SPECTRAL METHODS FOR STUDY OF THE G-PROTEIN-COUPLED RECEPTOR RHODOPSIN. II. MAGNETIC RESONANCE METHODS.

This article continues our review of spectroscopic studies of G-protein-coupled receptors. Magnetic resonance methods including electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) provide specific structural and dynamical data for the protein in conjunction with optical methods (vibrational, electronic spectroscopy) as discussed in the accompanying article. An additional advantage is the opportunity to explore the receptor proteins in the natural membrane lipid environment. Solid-state 2H and 13C NMR methods yield information about the both local structure and dynamics of the cofactor bound to the protein and its light induced changes. Complementary site-directed spin labeling studies monitor the structural alterations over larger distances and correspondingly longer time scales. A multi-scale reaction mechanism describes how local changes of the retinal cofactor unlock the receptor to initiate large-scale conformational changes of rhodopsin. Activation of the G-protein-coupled receptor involves an ensemble of conformational substates within the rhodopsin manifold that characterize the dynamically active receptor.

Antiparallel Triple-strand Architecture for Prefibrillar Aβ42 Oligomers

Aβ42 oligomers play key roles in the pathogenesis of Alzheimer disease, but their structures remain elusive partly due to their transient nature. Here, we show that Aβ42 in a fusion construct can be trapped in a stable oligomer state, which recapitulates characteristics of prefibrillar Aβ42 oligomers and enables us to establish their detailed structures. Site-directed spin labeling and electron paramagnetic resonance studies provide structural restraints in terms of side chain mobility and intermolecular distances at all 42 residue positions. Using these restraints and other biophysical data, we present a novel atomic-level oligomer model. In our model, each Aβ42 protein forms a single β-sheet with three β-strands in an antiparallel arrangement. Each β-sheet consists of four Aβ42 molecules in a head-to-tail arrangement. Four β-sheets are packed together in a face-to-back fashion. The stacking of identical segments between different β-sheets within an oligomer suggests that prefibrillar oligomers may interconvert with fibrils via strand rotation, wherein β-strands undergo an ∼90° rotation along the strand direction. This work provides insights into rational design of therapeutics targeting the process of interconversion between toxic oligomers and non-toxic fibrils.

A versatile approach for site-directed spin labeling and structural EPR studies of RNAs

Site-directed spin labeling (SDSL) is widely applied for structural studies of biopolymers by electron paramagnetic resonance (EPR). However, SDSL of long RNA sequences still remains a challenging task. Here, we propose a novel SDSL approach potentially suitable for long natural RNAs, which is based on the attachment of a linker containing an aliphatic amino group to the target nucleotide residue followed by selective coupling of a spin label to this amino group. Such a linker can be attached to the desired RNA residue via a sequence-specific reaction with the derivatives of oligodeoxyribonucleotides. To verify this approach, we applied it to model RNA duplex with known structure and expected distance between corresponding residues. A new 2,5-bis(spirocyclohexane)-substituted spin label with advanced stability and relaxation properties has been used, and the distance distribution measured using Q-band (34 GHz) pulsed double electron-electron resonance corresponds well to the expected one. We have additionally validated the obtained results by studying a similar RNA duplex, where the linker with the aliphatic amino group was introduced via solid-phase synthesis. Although this novel SDSL approach does not provide an advantage in precision of molecular distance measurements, we believe that its applicability to long RNAs is a crucial benefit for future structural studies using pulse EPR.

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

Ligand binding at the extracellular domain of pentameric ligand-gated ion channels initiates a relay of conformational changes that culminates at the gate within the transmembrane domain. The interface between the two domains is a key structural entity that governs gating. Molecular events in signal transduction at the interface are poorly defined because of its intrinsically dynamic nature combined with functional modulation by membrane lipid and water vestibules. Here we used electron paramagnetic resonance spectroscopy to delineate protein motions underlying Gloeobacter violaceus ligand-gated ion channel gating in a membrane environment and report the interface conformation in the closed and the desensitized states. Extensive intrasubunit interactions were observed in the closed state that are weakened upon desensitization and replaced by newer intersubunit contacts. Gating involves major rearrangements of the interfacial loops, accompanied by reorganization of the protein-lipid-water interface. These structural changes may serve as targets for modulation of gating by lipids, alcohols, and amphipathic drug molecules.

A Single-Stranded Junction Modulates Nanosecond Motional Ordering of the Substrate Recognition Duplex of a Group I Ribozyme

Rigid spinning: Site-directed spin-labeling studies using a rigid nitroxide spin label (Ç) reveal that both length and sequence of a single-stranded junction (J1/2) modulate nanosecond motional ordering of the substrate-recognition duplex (P1) of the 120 kD group I ribozyme. The studies demonstrate an approach for experimental measurements of nanosecond dynamics in high-molecular-weight RNA complexes.

Acidic pH-Induced Membrane Insertion of Colicin A into E. coli Natural Lipids Probed by Site-Directed Spin Labeling

Colicin A is a pore-forming toxin that forms a voltage-gated channel in the inner membrane of the target bacteria. The structures of the closed and open channel states of membrane-bound colicin A are not resolved. In the present site-directed spin-labeling study, the insertion-competent state of colicin A is provoked by an acidic pH jump prior to the insertion into liposomes prepared from Escherichia coli natural lipids. The membrane-bound colicin A is able to open a voltage-dependent channel as demonstrated by the efflux of tempophosphate spin label from the lumen of liposomes. The EPR spectra of spin-labeled colicin A variants in the membrane-bound closed channel state reveal a conformational equilibrium with resolved interhelical tertiary contacts. The spin label accessibility and polarity profiles suggest the amphipathic helices (H1–H7 and H10) to be located in the membrane close to the membrane–water interface and the hydrophobic hairpin (H8 and H9) to be immersed more deeply in the membrane.

Functional Transcriptional Regulatory Sequence (TRS) RNA Binding and Helix Destabilizing Determinants of Murine Hepatitis Virus (MHV) Nucleocapsid (N) Protein

Coronavirus (CoV) nucleocapsid (N) protein contains two structurally independent RNA binding domains. These are denoted N-terminal domain (NTD) and C-terminal domain and are joined by a charged linker region rich in serine and arginine residues (SR linker). In mouse hepatitis virus (MHV), the NTD binds the transcriptional regulatory sequence (TRS) RNA, a conserved hexanucleotide sequence required for subgenomic RNA synthesis. The NTD is also capable of disrupting a short RNA duplex. We show here that three residues on the β3 (Arg-125 and Tyr-127) and β5 (Tyr-190) strands play key roles in TRS RNA binding and helix destabilization with Ala substitutions of these residues lethal to the virus. NMR studies of the MHV NTD·TRS complex revealed that this region defines a major RNA binding interface in MHV with site-directed spin labeling studies consistent with a model in which the adenosine-rich 3'-region of TRS is anchored by Arg-125, Tyr-127, and Tyr-190 in a way that is critical for efficient subgenomic RNA synthesis in MHV. Characterization of CoV N NTDs from infectious bronchitis virus and from severe acute respiratory syndrome CoV revealed that, although detailed NTD-TRS determinants are distinct from those of MHV NTD, rapid helix destabilization activity of CoV N NTDs is most strongly correlated with CoV function and virus viability.

Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension

Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a flavine mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme’s N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme’s active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.

RNA dynamics: perspectives from spin labels

Dynamics are important and indispensible physical attributes that play essential roles in RNA function. RNA dynamics are complex, spanning vast timescales, and encompassing a large number of physical modes. The technique of site-directed spin labeling (SDSL), which derives information on local structural and dynamic features of a macromolecule by monitoring a chemically stable nitroxide radical using electron paramagnetic resonance spectroscopy, has been applied to monitor intrinsic dynamics at defined structural states as well as to probe conformational transition dynamics of RNAs. The current state of SDSL studies of RNA dynamics is summarized here. Further development and application of SDSL promise to open up many more opportunities for probing RNA dynamics and connecting dynamics to structure and function.

Site-Directed Spin-Labeling Studies: Structure of the AnkD34-Cdb3 Complex

The adaptor protein ankyrin-R interacts via its membrane binding domain with the cytoplasmic domain of the anion exchange protein (AE1) and via its spectrin binding domain with the spectrin based membrane skeleton in human erythrocytes. This set of interactions provides a bridge between the lipid bilayer and the membrane skeleton thereby stabilizing the membrane. Atomic resolution structures for the dimeric cytoplasmic domain of AE1 (cdb3) and for a twelve ankyrin repeat segment (repeats 13-24) from the membrane binding domain of ankyrin-R (AnkD34) have been reported. However, structural data on how these proteins assemble to form a stable complex have not been reported. In the current studies, site directed spin labeling, in combination with electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER), have been utilized to map the binding interfaces of the two proteins in the complex and to obtain inter-protein distance constraints. These data have been utilized to construct a family of structural models that are consistent with the full range of experimental data. These models indicate that an extensive area on the peripheral domain of cdb3 binds to ankyrin repeats 18-20 on the top convex surface of AnkD34 along the flexible linker away from the ankyrin groove primarily through hydrophobic interactions. This is a previously structurally uncharacterized surface for binding of cdb3 to AnkD34. Since a second dimer of cdb3 is known to bind to ankyrin repeats 7-12 of the membrane binding domain of ankyrin-R, the current models have significant implications regarding the structural nature of a tetrameric form of AE1 that is hypothesized to be involved in binding to Ankyrin-R in the erythrocyte membrane. Supported by NIH P01 GM080513.

Interactions of anthrax lethal factor with protective antigen defined by site-directed spin labeling

The protective antigen (PA) moiety of anthrax toxin forms oligomeric pores that translocate the enzymatic moieties of the toxin--lethal factor (LF) and edema factor (EF)--across the endosomal membrane of mammalian cells. Here we describe site-directed spin-labeling studies that identify interactions of LF with the prepore and pore conformations of PA. Our results reveal a direct interaction between the extreme N terminus of LF (residues 2-5) and the Φ-clamp, a structure within the lumen of the pore that catalyzes translocation. Also, consistent with a recent crystallographic model, we find that, upon binding of the translocation substrate to PA, LF helix α1 separates from helices α2 and α3 and binds in the α-clamp of PA. These interactions, together with the binding of the globular part of the N-terminal domain of LF to domain 1' of PA, indicate that LF interacts with the PA pore at three distinct sites. Our findings elucidate the state from which translocation of LF and EF proceeds through the PA pore.

Structural characteristics of the hydrophobic patch of azurin and its interaction with p53: a site-directed spin labeling study

Site-directed spin labeling (SDSL) is a powerful tool for monitoring protein structure, dynamics and conformational changes. In this study, the domain-specific properties of azurin and its interaction with p53 were studied using this technique. Mutations of six residues, that are located in the hydrophobic patch of azurin, were prepared and spin labeled. Spectra of the six azurin mutants in solution showed that spin labeled residues 45 and 63 are in a very restricted environment, residues 59 and 65 are in a spacious environment and have free movement, and residues 49 and 51 are located in a relatively closed pocket. Polarity experiments confirmed these results. The changes observed in the spectra of spin labeled azurin upon interaction with p53 indicate that the hydrophobic patch is involved in this interaction. Our results provide valuable insight into the topographic structure of the hydrophobic domain of azurin, as well as direct evidence of its interaction with p53 in solution via the hydrophobic patch. Cytotoxicity studies of azurin mutants showed that residues along the hydrophobic patch are important for its cytotoxicity.

Electron Spin-Echo Envelope Modulation (ESEEM) Reveals Water and Phosphate Interactions with the KcsA Potassium Channel,

Electron spin-echo envelope modulation (ESEEM) spectroscopy is a well-established technique for the study of naturally occurring paramagnetic metal centers. The technique has been used to study copper complexes, hemes, enzyme mechanisms, micellar water content, and water permeation profiles in membranes, among other applications. In the present study, we combine ESEEM spectroscopy with site-directed spin labeling (SDSL) and X-ray crystallography in order to evaluate the technique's potential as a structural tool to describe the native environment of membrane proteins. Using the KcsA potassium channel as a model system, we demonstrate that deuterium ESEEM can detect water permeation along the lipid-exposed surface of the KcsA outer helix. We further demonstrate that (31)P ESEEM is able to identify channel residues that interact with the phosphate headgroup of the lipid bilayer. In combination with X-ray crystallography, the (31)P data may be used to define the phosphate interaction surface of the protein. The results presented here establish ESEEM as a highly informative technique for SDSL studies of membrane proteins.

The Conformational Transition Pathway of ATP Binding Cassette Transporter MsbA Revealed by Atomistic Simulations

ATP binding cassette transporters are integral membrane proteins that use the energy released from ATP hydrolysis at the two nucleotide binding domains (NBDs) to translocate a wide variety of substrates through a channel at the two transmembrane domains (TMDs) across the cell membranes. MsbA from Gram-negative bacteria is a lipid and multidrug resistance ATP binding cassette exporter that can undergo large scale conformational changes between the outward-facing and the inward-facing conformations revealed by crystal structures in different states. Here, we use targeted molecular dynamics simulation methods to explore the atomic details of the conformational transition from the outward-facing to the inward-facing states of MsbA. The molecular dynamics trajectories revealed a clear spatiotemporal order of the conformational movements. The disruption of the nucleotide binding sites at the NBD dimer interface is the very first event that initiates the following conformational changes, verifying the assumption that the conformational conversion is triggered by ATP hydrolysis. The conserved x-loops of the NBDs were identified to participate in the interaction network that stabilizes the cytoplasmic tetrahelix bundle of the TMDs and play an important role in mediating the cross-talk between the NBD and TMD. The movement of the NBD dimer is transmitted through x-loops to break the tetrahelix bundle, inducing the packing rearrangements of the transmembrane helices at the cytoplasmic side and the periplasmic side sequentially. The packing rearrangement within each periplasmic wing of TMD that results in exposure of the substrate binding sites occurred at the end stage of the trajectory, preventing the wrong timing of the binding site accessibility.

Structure of the cdb3-ankD34 Complex from Site Directed Spin Labeling Studies

The spectin-based membrane skeleton is responsible for the remarkable mechanical stability and the unique viscoelastisity of the erythrocyte membrane, which are both essential for the survival of red blood cells in the circulatory system. One of the major junctional sites that links the membrane skeleton to the plasma membrane is a protein complex formed by the cytoplasmic domain of band3 (cdb3) and ankyrinR. In this study, site directed spin labeling (SDSL) has been utilized to investigate the global structure of the complex formed between cdb3 and ankD34 (ankyrin repeats 13-24 of full length ankyrinR). We first characterized physicochemical properties of the complex using gel permeation chromatography and sucrose-gradient sedimentation and determined the stoichiometry of the complex to be one cdb3 dimer bound to two ankD34s in vitro. For a series of surface sites residing on the binding interface of cdb3, spin label R1 side chain mobility and solvent accessibility were scanned using cw-EPR and the power saturation technique. Both parameters show significant changes at multiple sites which are widely scattered over the peripheral domain of cdb3 indicating that the binding interface involves multiple discontinuous patches rather than the single binding motif revealed by previous mutation deletion studies. The global structure of the complex has been investigated by determining multiple inter-molecular distance constraints using DEER (double electron electron resonance) between selected sites on the peripheral domain of cdb3 and surface sites on the backbone region of ankD34. The measured distances are not consistent with the previous docking model reported in the literature (Michaely et al., EMBO J. 21(23):6387-96, 2002). The EPR and DEER data are now being utilized in concert with molecular modeling approaches to construct a new structural model for the cdb3•ankD34 complex. Supported by: NIH P01 GM080513.