Isothermal titration calorimetry of membrane protein interactions: FNR and the cytochrome b6f complex.

Ferredoxin-NADP+ reductase (FNR) was previously inferred to bind to the cytochrome b6f complex in the electron transport chain of oxygenic photosynthesis. In the present study, this inference has been examined through analysis of the thermodynamics of the interaction between FNR and the b6f complex. Isothermal titration calorimetry (ITC) was used to characterize the physical interaction of FNR with b6f complex derived from two plant sources (Spinacia oleracea and Zea maize). ITC did not detect a significant interaction of FNR with the b6f complex in detergent solution nor with the complex reconstituted in liposomes. A previous inference of a small amplitude but defined FNR-b6f interaction is explained by FNR interaction with micelles of the undecyl ß-D maltoside (UDM) detergent micelles used to purify b6f. Circular dichroism, employed to analyze the effect of detergent on the FNR structure, did not reveal significant changes in secondary or tertiary structures of FNR domains in the presence of UDM detergent. However, thermodynamic analysis implied a significant decrease in an interaction between the N-terminal FAD-binding and C-terminal NADP+-binding domains of FNR caused by detergent. The enthalpy, ΔHo, and the entropy, ΔSo, associated with FNR unfolding decreased four-fold in the presence of 1 mM UDM at pH 6.5. In addition to the conclusion regarding the absence of a binding interaction of significant amplitude between FNR and the b6f complex, these studies provide a precedent for consideration of significant background protein-detergent interactions in ITC analyses involving integral membrane proteins.

An evolutionarily conserved iron-sulfur cluster underlies redox sensory function of the Chloroplast Sensor Kinase.

Photosynthetic efficiency depends on equal light energy conversion by two spectrally distinct, serially-connected photosystems. The redox state of the plastoquinone pool, located between the two photosystems, is a key regulatory signal that initiates acclimatory changes in the relative abundance of photosystems. The Chloroplast Sensor Kinase (CSK) links the plastoquinone redox signal with photosystem gene expression but the mechanism by which it monitors the plastoquinone redox state is unclear. Here we show that the purified Arabidopsis and Phaeodactylum CSK and the cyanobacterial CSK homologue, Histidine kinase 2 (Hik2), are iron-sulfur proteins. The Fe-S cluster of CSK is further revealed to be a high potential redox-responsive [3Fe-4S] center. CSK responds to redox agents with reduced plastoquinone suppressing its autokinase activity. Redox changes within the CSK iron-sulfur cluster translate into conformational changes in the protein fold. These results provide key insights into redox signal perception and propagation by the CSK-based chloroplast two-component system.

Structural insight into the role of the Ton complex in energy transduction.

In Gram-negative bacteria, outer membrane transporters import nutrients by coupling to an inner membrane protein complex called the Ton complex. The Ton complex consists of TonB, ExbB, and ExbD, and uses the proton motive force at the inner membrane to transduce energy to the outer membrane via TonB. Here, we structurally characterize the Ton complex from Escherichia coli using X-ray crystallography, electron microscopy, double electron-electron resonance (DEER) spectroscopy, and crosslinking. Our results reveal a stoichiometry consisting of a pentamer of ExbB, a dimer of ExbD, and at least one TonB. Electrophysiology studies show that the Ton subcomplex forms pH-sensitive cation-selective channels and provide insight into the mechanism by which it may harness the proton motive force to produce energy.

Trans-membrane Signaling in Photosynthetic State Transitions: REDOX- AND STRUCTURE-DEPENDENT INTERACTION IN VITRO BETWEEN STT7 KINASE AND THE CYTOCHROME b6f COMPLEX.

Trans-membrane signaling involving a serine/threonine kinase (Stt7 in Chlamydomonas reinhardtii) directs light energy distribution between the two photosystems of oxygenic photosynthesis. Oxidation of plastoquinol mediated by the cytochrome b6f complex on the electrochemically positive side of the thylakoid membrane activates the kinase domain of Stt7 on the trans (negative) side, leading to phosphorylation and redistribution ("state transition") of the light-harvesting chlorophyll proteins between the two photosystems. The molecular description of the Stt7 kinase and its interaction with the cytochrome b6f complex are unknown or unclear. In this study, Stt7 kinase has been cloned, expressed, and purified in a heterologous host. Stt7 kinase is shown to be active in vitro in the presence of reductant and purified as a tetramer, as determined by analytical ultracentrifugation, electron microscopy, and electrospray ionization mass spectrometry, with a molecular weight of 332 kDa, consisting of an 83.41-kDa monomer. Far-UV circular dichroism spectra show Stt7 to be mostly α-helical and document a physical interaction with the b6f complex through increased thermal stability of Stt7 secondary structure. The activity of wild-type Stt7 and its Cys-Ser mutant at positions 68 and 73 in the presence of a reductant suggest that the enzyme does not require a disulfide bridge for its activity as suggested elsewhere. Kinase activation in vivo could result from direct interaction between Stt7 and the b6f complex or long-range reduction of Stt7 by superoxide, known to be generated in the b6f complex by quinol oxidation.

The Colicin E1 TolC-Binding Conformer: Pillar or Pore Function of TolC in Colicin Import?

The mechanism by which the drug export protein TolC is utilized for import of the cytotoxin colicin E1 across the outer membrane and periplasmic space is addressed. Studies of the initial binding of colicin E1 with TolC, occlusion of membrane-incorporated TolC ion channels, and the structure underlying the colicin-TolC complex were based on the interactions with TolC of individual colicin translocation domain (T-domain) peptides from a set of 19 that span different segments of the T-domain. These studies led to identification of a short 20-residue segment 101-120, a "TolC box", located near the center of the colicin T-domain, which is necessary for binding of colicin to TolC. Omission of this segment eliminated the ability of the T-domain to occlude TolC channels and to co-elute with TolC on a size-exclusion column. Far-ultraviolet circular dichroism spectral and thermal stability analysis of the structure of T-domain peptides implies (i) a helical hairpin conformation of the T-domain, (ii) the overlap of the TolC-binding site with a hinge of the helical hairpin, and (iii) a TolC-dependent stage of colicin import in which a central segment of the T-domain in a helical hairpin conformation binds to the TolC entry port following initial binding to the BtuB receptor. These studies provide the first structure-based information about the interaction of colicin E1 with the unique TolC protein. The model inferred for binding of the T-domain to TolC implies reservations about the traditional model for colicin import in which TolC functions to provide a channel for translocation of the colicin in an unfolded state across the bacterial outer membrane and a large part of the periplasmic space.

A map of dielectric heterogeneity in a membrane protein: the hetero-oligomeric cytochrome b6f complex.

The cytochrome b6f complex, a member of the cytochrome bc family that mediates energy transduction in photosynthetic and respiratory membranes, is a hetero-oligomeric complex that utilizes two pairs of b-hemes in a symmetric dimer to accomplish trans-membrane electron transfer, quinone oxidation-reduction, and generation of a proton electrochemical potential. Analysis of electron storage in this pathway, utilizing simultaneous measurement of heme reduction, and of circular dichroism (CD) spectra, to assay heme-heme interactions, implies a heterogeneous distribution of the dielectric constants that mediate electrostatic interactions between the four hemes in the complex. Crystallographic information was used to determine the identity of the interacting hemes. The Soret band CD signal is dominated by excitonic interaction between the intramonomer b-hemes, bn and bp, on the electrochemically negative and positive sides of the complex. Kinetic data imply that the most probable pathway for transfer of the two electrons needed for quinone oxidation-reduction utilizes this intramonomer heme pair, contradicting the expectation based on heme redox potentials and thermodynamics, that the two higher potential hemes bn on different monomers would be preferentially reduced. Energetically preferred intramonomer electron storage of electrons on the intramonomer b-hemes is found to require heterogeneity of interheme dielectric constants. Relative to the medium separating the two higher potential hemes bn, a relatively large dielectric constant must exist between the intramonomer b-hemes, allowing a smaller electrostatic repulsion between the reduced hemes. Heterogeneity of dielectric constants is an additional structure-function parameter of membrane protein complexes.

Self-oscillating Water Chemiluminescence Modes and Reactive Oxygen Species Generation Induced by Laser Irradiation; Effect of the Exclusion Zone Created by Nafion.

Samples of water inside and outside an exclusion zone (EZ), created by Nafion swollen in water, were irradiated at the wavelength l = 1264 nm, which stimulates the electronic transition of dissolved oxygen from the triplet state to the excited singlet state. This irradiation induces, after a long latent period, chemiluminescence self-oscillations in the visible and near UV spectral range, which last many hours. It occurs that this effect is EZ-specific: the chemiluminescence intensity is twice lower than that from the bulk water, while the latent period is longer for the EZ. Laser irradiation causes accumulation of H2O2, which is also EZ-specific: its concentration inside the EZ is less than that in the bulk water. These phenomena can be interpreted in terms of a model of decreasing O2 content in the EZ due to increased chemical activity of bisulfite anions (HSO3-), arisen as the result of dissociation of terminal sulfonate groups of the Nafion. The wavelet transform analysis of the chemiluminescence intensity from the EZ and the bulk water gives, that self-oscillations regimes occurring in the liquid after the latent period are the determinate processes. It occurred that the chemiluminescence dynamics in case of EZ is characterized by a single-frequency self-oscillating regime, whereas in case of the bulk water, the self-oscillation spectrum consists of three spectral bands.

Methods for studying interactions of detergents and lipids with α-helical and ß-barrel integral membrane proteins.

Methods for studying interactions of protein with lipids and detergents are described for representatives of two major classes of membrane proteins: (1) the α-helical hetero-oligomeric integral cytochrome b6 f complex of oxygenic photosynthesis from cyanobacteria, and (2) the outer membrane ß-barrel proteins BtuB and OmpF from Gram-negative Escherichia coli bacteria. Details are presented on the use of detergents for purification and crystallization of the b6 f complex as well as a method for lipid exchange. The positions of detergent and lipid molecules, which define eight potential lipid-binding sites in the b6 f complex, are described. Differences in detergent strategies for isolation and crystallization of ß-barrel proteins relative to those for oligomeric helical membrane proteins are discussed, and purification and assessment of protein quality by circular dichroism (CD) is presented.

Pathways of colicin import: utilization of BtuB, OmpF porin and the TolC drug-export protein.

Pathway I. Group A nuclease colicins parasitize and bind tightly (Kd ≤ 10(-9) M) to the vitamin B12 receptor on which they diffuse laterally in the OM (outer membrane) and use their long (≥100 Å; 1 Å=0.1 nm) receptor-binding domain as a 'fishing pole' to locate the OmpF porin channel for translocation. Crystal structures of OmpF imply that a disordered N-terminal segment of the colicin T-domain initiates insertion. Pathway II. Colicin N does not possess a 'fishing pole' receptor-binding domain. Instead, it uses OmpF as the Omp (outer membrane protein) for reception and translocation, processes in which LPS (lipopolysaccharide) may also serve. Keio collection experiments defined the LPS core that is used. Pathway III. Colicin E1 utilizes the drug-export protein TolC for import. CD spectra and thermal-melting analysis predict: (i) N-terminal translocation (T) and central receptor (BtuB) -binding (R) domains are predominantly α-helical; and (ii) helical coiled-coil conformation of the R-domain is similar to that of colicins E3 and Ia. Recombinant colicin peptides spanning the N-terminal translocation domain defined TolC-binding site(s). The N-terminal 40-residue segment lacks the ordered secondary structure. Peptide 41-190 is helical (78%), co-elutes with TolC and occluded TolC channels. Driven by a trans-negative potential, peptides 82-140 and 141-190 occluded TolC channels. The use of TolC for colicin E1 import implies that the interaction of this colicin with the other Tol proteins does not occur in the periplasmic space, but rather through Tol domains in the cytoplasmic membrane, thus explaining colicin E1 cytotoxicity towards a strain in which a 234 residue periplasmic TolA segment is deleted.

Oxygen-dependent auto-oscillations of water luminescence triggered by the 1264 nm radiation.

A 5-min exposure of air-saturated bidistilled water to low-intensity laser infrared radiation at the wavelength of the electronic transition of dissolved oxygen to the singlet state ((3)∑(g)(-)→ (1)Δ(g)) induces, after a long latent period, auto-oscillations of water luminescence in the blue-green region, which last many hours. Laser irradiation causes the accumulation of hydrogen peroxide, which depends on the concentration of dissolved oxygen. The auto-oscillations do not arise if water is irradiated beyond the oxygen absorption band and if the oxygen is removed from water. The wavelet transform analysis of luminescence records indicates that there are two characteristic periods of pulsations of about 300 and 1150 s. The results obtained suggest that auto-oscillations are triggered by photoinduced singlet oxygen (1)Δ(g), and this phenomenon is closely related to formation of hydrogen peroxide.

Primary events in the colicin translocon: FRET analysis of colicin unfolding initiated by binding to BtuB and OmpF.

Cellular import of colicin E3 is initiated by high affinity binding of the colicin receptor-binding (R) domain to the vitamin B(12) (BtuB) receptor in the Escherichia coli outer membrane. The BtuB binding site, at the apex of its extended coiled-coil R-domain, is distant from the C-terminal nuclease domain that must be imported for expression of cytotoxicity. Based on genetic analysis and previously determined crystal structures of the R-domain bound to BtuB, and of an N-terminal disordered segment of the translocation (T) domain inserted into the OmpF porin, a translocon model for colicin import has been inferred. Implicit in the model is the requirement for unfolding of the colicin segments inserted into OmpF. FRET analysis was employed to study colicin unfolding upon interaction with BtuB and OmpF. A novel method of Cys-specific dual labeling of a native polypeptide, which allows precise placement of donor and acceptor fluorescent dyes on the same polypeptide chain, was developed. A decrease in FRET efficiency between the translocation and cytotoxic domains of the colicin E3 was observed upon colicin binding in vitro to BtuB or OmpF. The two events were independent and additive. The colicin interactions with BtuB and OmpF have a major electrostatic component. The R-domain Arg399 is responsible for electrostatic interaction with BtuB. It is concluded that free energy for colicin unfolding is provided by binding of the R- domain to BtuB and binding/insertion of the T-domain to/into OmpF.

Crystal structures of the OmpF porin: function in a colicin translocon.

The OmpF porin in the Escherichia coli outer membrane (OM) is required for the cytotoxic action of group A colicins, which are proposed to insert their translocation and active domains through OmpF pores. A crystal structure was sought of OmpF with an inserted colicin segment. A 1.6 A OmpF structure, obtained from crystals formed in 1 M Mg2+, has one Mg2+ bound in the selectivity filter between Asp113 and Glu117 of loop 3. Co-crystallization of OmpF with the unfolded 83 residue glycine-rich N-terminal segment of colicin E3 (T83) that occludes OmpF ion channels yielded a 3.0 A structure with inserted T83, which was obtained without Mg2+ as was T83 binding to OmpF. The incremental electron density could be modelled as an extended poly-glycine peptide of at least seven residues. It overlapped the Mg2+ binding site obtained without T83, explaining the absence of peptide binding in the presence of Mg2+. Involvement of OmpF in colicin passage through the OM was further documented by immuno-extraction of an OM complex, the colicin translocon, consisting of colicin E3, BtuB and OmpF.

Helical alpha-synuclein forms highly conductive ion channels.

Alpha-synuclein (alphaS) is a cytosolic protein involved in the etiology of Parkinson's disease (PD). Disordered in an aqueous environment, alphaS develops a highly helical conformation when bound to membranes having a negatively charged surface and a large curvature. It exhibits a membrane-permeabilizing activity that has been attributed to oligomeric protofibrillar forms. In this study, monomeric wild-type alphaS and two mutants associated with familial PD, E46K and A53T, formed ion channels with well-defined conductance states in membranes containing 25-50% anionic lipid and 50% phosphatidylethanolamine (PE) in the presence of a trans-negative potential. Another familial mutant, A30P, known to have a lower membrane affinity, did not form ion channels. Ca2+ prevented channel formation when added to membranes before alphaS and decreased channel conductance when added to preformed channels. In contrast to the monomer, membrane permeabilization by oligomeric alphaS was not characterized by formation of discrete channels, a requirement for PE lipid, or a membrane potential. Channel activity, alpha-helical content, thermal stability of membrane-bound alphaS determined by far-UV CD, and lateral mobility of alphaS bound to planar membranes measured by fluorescence correlation spectroscopy were correlated. It was inferred that discrete ion channels with well-defined conductance states were formed in the presence of a membrane potential by one or several molecules of monomeric alphaS in an alpha-helical conformation and that such channels may have a role in the normal function and/or pathophysiology of the protein.

Structure of the complex of the colicin E2 R-domain and its BtuB receptor. The outer membrane colicin translocon.

The crystal structure of the complex of the BtuB receptor and the 135-residue coiled-coil receptor-binding R-domain of colicin E3 (E3R135) suggested a novel mechanism for import of colicin proteins across the outer membrane. It was proposed that one function of the R-domain, which extends along the outer membrane surface, is to recruit an additional outer membrane protein(s) to form a translocon for passage colicin activity domain. A 3.5-A crystal structure of the complex of E2R135 and BtuB (E2R135-BtuB) was obtained, which revealed E2R135 bound to BtuB in an oblique orientation identical to that previously found for E3R135. The only significant difference between the two structures was that the bound coiled-coil R-domain of colicin E2, compared with that of colicin E3, was extended by two and five residues at the N and C termini, respectively. There was no detectable displacement of the BtuB plug domain in either structure, implying that colicin is not imported through the outer membrane by BtuB alone. It was concluded that the oblique orientation of the R-domain of the nuclease E colicins has a function in the recruitment of another member(s) of an outer membrane translocon. Screening of porin knock-out mutants showed that either OmpF or OmpC can function in such a translocon. Arg(452) at the R/C-domain interface in colicin E2 was found have an essential role at a putative site of protease cleavage, which would liberate the C-terminal activity domain for passage through the outer membrane translocon.

The colicin E3 outer membrane translocon: immunity protein release allows interaction of the cytotoxic domain with OmpF porin.

The crystal structure previously obtained for the complex of BtuB and the receptor binding domain of colicin E3 forms a basis for further analysis of the mechanism of colicin import through the bacterial outer membrane. Together with genetic analysis and studies on colicin occlusion of OmpF channels, this implied a colicin translocon consisting of BtuB and OmpF that would transfer the C-terminal cytotoxic domain (C96) of colicin E3 through the Escherichia coli outer membrane. This model does not, however, explain how the colicin attains the unfolded conformation necessary for transfer. Such a conformation change would require removal of the immunity (Imm) protein, which is bound tightly in a complex with the folded colicin E3. In the present study, it was possible to obtain reversible removal of Imm in vitro in a single column chromatography step without colicin denaturation. This resulted in a mostly unordered secondary structure of the cytotoxic domain and a large decrease in stability, which was also found in the receptor binding domain. These structure changes were documented by near- and far-UV circular dichroism and intrinsic tryptophan fluorescence. Reconstitution of Imm in a complex with C96 or colicin E3 restored the native structure. C96 depleted of Imm, in contrast to the native complex with Imm, efficiently occluded OmpF channels, implying that the presence of tightly bound Imm prevents its unfolding and utilization of the OmpF porin for subsequent import of the cytotoxic domain.

Lipid dependence of the channel properties of a colicin E1-lipid toroidal pore.

Colicin E1 belongs to a group of bacteriocins whose cytotoxicity toward Escherichia coli is exerted through formation of ion channels that depolarize the cytoplasmic membrane. The lipid dependence of colicin single-channel conductance demonstrated intimate involvement of lipid in the structure of this channel. The colicin formed "small" conductance 60-picosiemens (pS) channels, with properties similar to those previously characterized, in 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (C20) or thinner membranes, whereas it formed a novel "large" conductance 600-pS state in thicker 1,2-dierucoyl-sn-glycero-3-phosphocholine (C22) bilayers. Both channel states were anion-selective and voltage-gated and displayed a requirement for acidic pH. Lipids having negative spontaneous curvature inhibited the formation of both channels but increased the ratio of open 600 pS to 60 pS conductance states. Different diameters of small and large channels, 12 and 16 A, were determined from the dependence of single-channel conductance on the size of nonelectrolyte solute probes. Colicin-induced lipid "flip-flop" and the decrease in anion selectivity of the channel in the presence of negatively charged lipids implied a significant contribution of lipid to the structure of the channel, most readily described as toroidal organization of lipid and protein to form the channel pore.

On the role of lipid in colicin pore formation.

Insights into the protein-membrane interactions by which the C-terminal pore-forming domain of colicins inserts into membranes and forms voltage-gated channels, and the nature of the colicin channel, are provided by data on: (i) the flexible helix-elongated state of the colicin pore-forming domain in the fluid anionic membrane interfacial layer, the optimum anionic surface charge for channel formation, and voltage-gated translocation of charged regions of the colicin domain across the membrane; (ii) structure-function data on the voltage-gated K(+) channel showing translocation of an arginine-rich helical segment through the membrane; (iii) toroidal channels formed by small peptides that involve local participation of anionic lipids in an inverted phase. It is proposed that translocation of the colicin across the membrane occurs through minimization of the Born charging energy for translocation of positively charged basic residues across the lipid bilayer by neutralization with anionic lipid head groups. The resulting pore structure may consist of somewhat short, ca. 16 residues, trans-membrane helices, in a locally thinned membrane, together with surface elements of inverted phase lipid micelles.

Colicin occlusion of OmpF and TolC channels: outer membrane translocons for colicin import.

The interaction of colicins with target cells is a paradigm for protein import. To enter cells, bactericidal colicins parasitize Escherichia coli outer membrane receptors whose physiological purpose is the import of essential metabolites. Colicins E1 and E3 initially bind to the BtuB receptor, whose beta-barrel pore is occluded by an N-terminal globular "plug". The x-ray structure of a complex of BtuB with the coiled-coil BtuB-binding domain of colicin E3 did not reveal displacement of the BtuB plug that would allow passage of the colicin (Kurisu, G., S. D. Zakharov, M. V. Zhalnina, S. Bano, V. Y. Eroukova, T. I. Rokitskaya, Y. N. Antonenko, M. C. Wiener, and W. A. Cramer. 2003. Nat. Struct. Biol. 10:948-954). This correlates with the inability of BtuB to form ion channels in planar bilayers, shown in this work, suggesting that an additional outer membrane protein(s) is required for colicin import across the outer membrane. The identity and interaction properties of this OMP were analyzed in planar bilayer experiments.OmpF and TolC channels in planar bilayers were occluded by colicins E3 and E1, respectively, from the trans-side of the membrane. Occlusion was dependent upon a cis-negative transmembrane potential. A positive potential reversibly opened OmpF and TolC channels. Colicin N, which uses only OmpF for entry, occludes OmpF in planar bilayers with the same orientation constraints as colicins E1 and E3. The OmpF recognition sites of colicins E3 and N, and the TolC recognition site of colicin E1, were found to reside in the N-terminal translocation domains. These data are considered in the context of a two-receptor translocon model for colicin entry into cells.

Effect of lipids with different spontaneous curvature on the channel activity of colicin E1: evidence in favor of a toroidal pore.

The channel activity of colicin E1 was studied in planar lipid bilayers and liposomes. Colicin E1 pore-forming activity was found to depend on the curvature of the lipid bilayer, as judged by the effect on channel activity of curvature-modulating agents. In particular, the colicin-induced trans-membrane current was augmented by lysophosphatidylcholine and reduced by oleic acid, agents promoting positive and negative membrane curvature, respectively. The data obtained imply direct involvement of lipids in the formation of colicin E1-induced pore walls. It is inferred that the toroidal pore model previously validated for small antimicrobial peptides is applicable to colicin E1, a large protein that contains ten alpha-helices in its pore-forming domain.

On the mechanism and pathway of colicin import across the E. Coli outer membrane.

Colicins and phages parasitize outer membrane receptors whose physiological purpose is the transport of metabolites, metals, vitamins, and sugars. From mutagenesis studies, it is known that several colicins require the function of two outer membrane protein (Omp) receptors for cytotoxicity. A formidable list of problems associated with an understanding of a two receptor mechanism for colicin translocation includes the definition of the sites of initial binding and interactions of the colicin with the OM translocator protein, the working lumenal aperture of the translocator, the question of whether the colicin must be unfolded for translocation, the source of energy for unfolding and translocation, the order of colicin translocation, and the sites and mechanism of interaction of the colicins with the Tol-Pal proteins on the periplasmic side of the outer membrane. 3D crystal structures recently obtained of the cobalamin (vitamin B12) receptor (BtuB), and of the complex of BtuB with the 135 residue receptor binding domain (R135) of colicin E3, have provided some new insights on the interactions between two Omp receptors that are necessary for translocation of colicins. Together with spectroscopic data on the R135-BtuB interaction and electrophysiological data on the colicin E3-OmpF interaction, this has led to a proposal for the utilization of two receptors, BtuB-OmpF, in an outer membrane translocon for colicin E3.