Alanine Scanning Mutagenesis of the MEDI4893 (Suvratoxumab) Epitope Reduces Alpha Toxin Lytic Activity In Vitro and Staphylococcus aureus Fitness in Infection Models.

Alpha toxin (AT) is a cytolytic pore-forming toxin that plays a key role in Staphylococcus aureus pathogenesis; consequently, extensive research was undertaken to understand the AT mechanism of action and its utility as a target for novel prophylaxis and treatment strategies against S. aureus infections. MEDI4893 (suvratoxumab) is a human anti-AT IgG1 monoclonal antibody (MAb) that targets AT and is currently in phase 2 clinical development. As shown previously, the MEDI4893-binding epitope on AT is comprised of the highly conserved amino acid regions 177 to 200 and 261 to 271, suggesting these amino acids are important for AT function. To test this hypothesis and gain insight into the effect of mutations in the epitope on AT neutralization by MEDI4893, nine MEDI4893 contact residues in AT were individually mutated to alanine. Consistent with our hypothesis, 8 out of 9 mutants exhibited >2-fold loss in lytic activity resulting from a defect in cell binding and pore formation. MEDI4893 binding affinity was reduced >2-fold (2- to 27-fold) for 7 out of 9 mutants, and no binding was detected for the W187A mutant. MEDI4893 effectively neutralized all of the lytic mutants in vitro and in vivo When the defective mutants were introduced into an S. aureus clinical isolate, the mutant-expressing strains exhibited less severe disease in mouse models and were effectively neutralized by MEDI4893. These results indicate the MEDI4893 epitope is highly conserved due in part to its role in AT pore formation and bacterial fitness, thereby decreasing the likelihood for the emergence of MAb-resistant variants.

Pseudomonas aeruginosa PcrV and Psl, the Molecular Targets of Bispecific Antibody MEDI3902, Are Conserved Among Diverse Global Clinical Isolates.

Background: Bispecific antibody MEDI3902, targeting the Pseudomonas aeruginosa type 3 secretion system (PcrV) and Psl exopolysaccharide, is currently in phase 2b development for prevention of nosocomial pneumonia in patients undergoing mechanical ventilation. We surveyed a diverse collection of isolates to study MEDI3902 epitope conservation and protective activity. Methods: P. aeruginosa clinical isolates (n = 913) were collected from diverse patients and geographic locations during 2003-2014. We conducted whole-genome sequencing; performed PcrV and Psl expression analyses via immunoblotting and enzyme-linked immunosorbent assay, respectively; performed crystallography to determine the MEDI3902 PcrV epitope, using anti-PcrV Fab and PcrV components (resolved at 2.8 Å); and evaluated MEDI3902 protective activity against select isolates in vitro and in vivo. Results: Intact psl operon and pcrV genes were present in 94% and 99% of isolates, respectively, and 99.9% of isolates contained at least one of the genetic elements. Anti-Psl binding was confirmed in tested isolates harboring a complete Psl operon or lacking nonessential psl genes. We identified 46 PcrV variant sequences, and MEDI3902-PcrV contact residues were preserved. MEDI3902 maintained potent in vivo activity against various strains, including strains expressing only a single target. Conclusions: Psl and PcrV are highly prevalent in global clinical isolates, suggesting MEDI3902 can mediate broad coverage against P. aeruginosa.

All-atom molecular dynamics simulations of spin labelled double and single-strand DNA for EPR studies.

We report the first application of fully atomistic molecular dynamics (MD) simulations to the prediction of electron paramagnetic resonance (EPR) spectra of spin labelled DNA. Models for two structurally different DNA spin probes with either the rigid or flexible position of the nitroxide group in the base pair, employed in experimental studies previously, have been developed. By the application of the combined MD-EPR simulation methodology we aimed at the following. Firstly, to provide a test bed against a sensitive spectroscopic technique for the recently developed improved version of the parmbsc1 force field for MD modelling of DNA. The predicted EPR spectra show good agreement with the experimental ones available from the literature, thus confirming the accuracy of the currently employed DNA force fields. Secondly, to provide a quantitative interpretation of the motional contributions into the dynamics of spin probes in both duplex and single-strand DNA fragments and to analyse their perturbing effects on the local DNA structure. Finally, a combination of MD and EPR allowed us to test the validity of the application of the Model-Free (M-F) approach coupled with the partial averaging of magnetic tensors to the simulation of EPR spectra of DNA systems by comparing the resultant EPR spectra with those simulated directly from MD trajectories. The advantage of the M-F based EPR simulation approach over the direct propagation techniques is that it requires motional and order parameters that can be calculated from shorter MD trajectories. The reported MD-EPR methodology is transferable to the prediction and interpretation of EPR spectra of higher order DNA structures with novel types of spin labels.

[Sources of electrophysiological and foci of hemodynamic brain activity most relevant for controlling a hybrid brain­Computer interface based on classification of EEG patterns and near-infrared spectrography signals during motor imagery].

The method is described for joint use of electroencephalography and near-infrared spectrography for location of sources of electrophysiological and focuses of hemodynamic brain activity during motor execution and imagination. The sources of electrophysiological and focuses of hemodynamic activity the most relevant for controlling the hybrid brain-computer interface based on motor imagery are revealed and discussed.

Simulation of electron paramagnetic resonance spectra of spin-labeled molecules from replica-exchange molecular dynamics.

We report a general approach for the simulation of the electron paramagnetic resonance (EPR) spectra of spin labels attached to peptides and proteins directly from replica-exchange molecular dynamics (REMD) trajectories. Conventional MD trajectories are generally inadequate for the prediction of EPR line shapes since the label can become trapped in one or more of a set of rotameric states, thus preventing both conformational sampling and accurate estimates of the exchange rates between different rotamers. The advantage of using REMD is its ability to provide both efficient conformational sampling and kinetic information for spin-label dynamics. Our approach is illustrated with spin-labeled peptide. This approach to REMD-EPR simulation paves the way for the wider application of MD modeling to the simulation and interpretation of EPR spectra of spin-labeled molecules.

Electron paramagnetic resonance spectra simulation directly from molecular dynamics trajectories of a liquid crystal with a doped paramagnetic spin probe.

We report simulation of EPR spectra directly and entirely from trajectories generated from molecular dynamics simulations. Results are reported for a model 3beta-DOXYL-5alpha-cholestane spin probe in a coarse-grained solvent representing a 5CB nematic host. The results are in excellent agreement with the experimental spectra. The calculated order parameters associated with the paramagnetic probe show strong correlation with the order parameter of 5CB mesogens and are in agreement with those reported in the literature. Simulation of EPR spectra entirely from molecular dynamics of real structures provides direct correlation between molecular motions and the features observed in the spectra, allowing unambiguous interpretation of the spectra. This method opens the possibility for "computer engineering" of spin-labeled materials with the desired properties, such as spin-labeled proteins, prior to experiment.

Dynamical layer decoupling in a stripe-ordered high-T(c) superconductor.

In the stripe-ordered state of a strongly correlated two-dimensional electronic system, under a set of special circumstances, the superconducting condensate, like the magnetic order, can occur at a nonzero wave vector corresponding to a spatial period double that of the charge order. In this case, the Josephson coupling between near neighbor planes, especially in a crystal with the special structure of La(2-x)Ba(x)CuO(4), vanishes identically. We propose that this is the underlying cause of the dynamical decoupling of the layers recently observed in transport measurements at x = 1/8.

A novel approach to the simulation of nitroxide spin label EPR spectra from a single truncated dynamical trajectory.

A simple effective method for calculation of EPR spectra from a single truncated dynamical trajectory of spin probe orientations is reported. It is shown that an accurate simulation can be achieved from the small initial fraction of a dynamical trajectory until the point when the autocorrelation function of re-orientational motion of spin label has relaxed. This substantially reduces the amount of time for spectra simulation compared to previous approaches, which require multiple full length trajectories (normally of several microseconds) to achieve the desired resolution of EPR spectra. Our method is applicable to trajectories generated from both Brownian dynamics and molecular dynamics (MD) calculations. Simulations of EPR spectra from Brownian dynamical trajectories under a variety of motional conditions including bi-modal dynamics with different hopping rates between the modes are compared to those performed by conventional method. Since the relatively short timescales of spin label motions are realistically accessible by modern MD computational methods, our approach, for the first time, opens the prospect of the simulation of EPR spectra entirely from MD trajectories of real proteins structures.

Analysis of nitroxide spin label motion in a protein-protein complex using multiple frequency EPR spectroscopy.

X- and W-band EPR spectra, at room and low temperatures, are reported for nitroxide spin labels attached to cysteine residues selectively introduced into two proteins, the DNase domain of colicin-E9 and its immunity protein, Im9. The dynamics of each site of attachment on the individual proteins and in the tight DNase-Im9 complex have been analysed by computer simulations of the spectra using a model of Brownian dynamics trajectories for the spin label and protein. Ordering potentials have been introduced to describe mobility of labels restricted by the protein domain. Label mobility varies with position from completely immobilised, to motionally restricted and to freely rotating. Bi-modal dynamics of the spin label have been observed for several sites. We show that W-band spectra are particularly useful for detection of anisotropy of spin label motion. On complex formation significant changes are observed in the dynamics of labels at the binding interface region. This work reveals multi-frequency EPR as a sensitive and valuable tool for detecting conformational changes in protein structure and dynamics especially in protein-protein complexes.

Nodal quasiparticles in stripe ordered superconductors.

We study the properties of a quasi-one-dimensional superconductor which consists of an alternating array of two inequivalent chains. This model is a simple caricature of a striped high temperature superconductor, and is more generally a theoretically controllable system in which the superconducting state emerges from a non-Fermi-liquid normal state. Even in this limit, " d-wave-like" order parameter symmetry is natural, but the superconducting state can either have a complete gap in the quasiparticle spectrum, or gapless "nodal" quasiparticles. We also find circumstances in which antiferromagnetic order (typically incommensurate) coexists with superconductivity.

Angular dependences of perpendicular and parallel mode electron paramagnetic resonance of oxidized beef heart cytochrome c oxidase.

Cytochrome c oxidase catalyzes the reduction of oxygen to water with a concomitant conservation of energy in the form of a transmembrane proton gradient. The enzyme has a catalytic site consisting of a binuclear center of a copper ion and a heme group. The spectroscopic parameters of this center are unusual. The origin of broad electron paramagnetic resonance (EPR) signals in the oxidized state at rather low resonant field, the so-called g' = 12 signal, has been a matter of debate for over 30 years. We have studied the angular dependence of this resonance in both parallel and perpendicular mode X-band EPR in oriented multilayers containing cytochrome c oxidase to resolve the assignment. The "slow" form and compounds formed by the addition of formate and fluoride to the oxidized enzyme display these resonances, which result from transitions between states of an integer-spin multiplet arising from magnetic exchange coupling between the five unpaired electrons of high spin Fe(III) heme a(3) and the single unpaired electron of Cu(B). The first successful simulation of similar signals observed in both perpendicular and parallel mode X-band EPR spectra in frozen aqueous solution of the fluoride compound of the closely related enzyme, quinol oxidase or cytochrome bo(3), has been reported recently (Oganesyan et al., 1998, J. Am. Chem. Soc. 120:4232-4233). This suggested that the exchange interaction between the two metal ions of the binuclear center is very weak (|J| approximately 1 cm(-1)), with the axial zero-field splitting (D approximately 5 cm(-1)) of the high-spin heme dominating the form of the ground state. We show that this model accounts well for the angular dependences of the X-band EPR spectra in both perpendicular and parallel modes of oriented multilayers of cytochrome c oxidase derivatives and that the experimental results are inconsistent with earlier schemes that use exchange coupling parameters of several hundred wavenumbers.

Modulation of redox potential in electron transfer proteins: effects of complex formation on the active site microenvironment of cytochrome b5.

The reduction potential of cytochrome b5 is modulated via the formation of a complex with polylysine at the electrode surface (Rivera et al., Biochemistry, 1998, 37, 1485). This modulation is thought to originate from the neutralization of a solvent exposed heme propionate and from dehydration of the complex interface. Although direct evidence demonstrating that neutralization of the charge on the heme propionate contributes to the modulation of the redox potential of cytochrome b5 has been obtained, evidence demonstrating that water exclusion from the complex interface plays a similar role has not been conclusive. Herein we report the preparation of the V45I/V61I double mutant of rat liver outer mitochondrial membrane (OM) cytochrome b5. This mutant has been engineered with the aim of restricting water accessibility to the exposed heme edge of cytochrome b5. The X-ray crystal structure of the V45I/V61I mutant revealed that the side chain of Ile at positions 45 and 61 restricts water accessibility to the interior of the heme cavity and protects a large section of the heme edge from the aqueous environment. Electrochemical studies performed with the V45I/V61I mutant of cytochrome b5, and with a derivative in which the heme propionates have been converted into the corresponding dimethyl ester groups, clearly demonstrate that dehydration of the heme edge contributes to the modulation of the reduction potential of cytochrome b5. In fact, these studies showed that exclusion of water from the complex interface exerts an effect (approximately 40 mV shift) that is comparable, if not larger, than the one originating from neutralization of the charge on the solvent exposed heme propionate (approximately 30 mV shift).

Determination of zero-field splitting and evidence for the presence of charge-transfer transitions in the Soret region of high-spin ferric hemoproteins obtained from an analysis of low-temperature magnetic circular dichroism.

Theoretical expressions for magnetic circular dichroism (MCD) of the porphyrin pi-->pi*, spin-allowed charge transfer (CT) and spin-forbidden d-d or CT transitions in high-spin ferric heme are derived. The transitions can be discriminated by their MCD to absorption ratio and/or temperature dependence of MCD intensity. An analysis of the Soret MCD of fluoride complexes of myoglobin (Mb), hemoglobin (Hb) and horseradish peroxidase (HRP), recorded at temperatures from 290 down to 2 K, is given. It is shown that the Soret MCD of HRPF can be well described by overlapping of the pi-->pi* transition with one spin-forbidden CT transition of an 6A1-->4E type. In the case of MbF and HbF it is necessary to assume the presence in the Soret region of the second spin-forbidden CT transition, most probably of an 6A1-->4A1 type. The parameters of transitions have been extracted from a non-linear least-squares fitting procedure. The best fit values of parameter D of the zero-field splitting of the ground manifold for HbF (6.1 cm(-1)) and MbF (6.4 cm(-1)) agree well with those obtained by other methods. The D value for HRPF (8.3 cm(-1)) is obtained for the first time.

A 4-term energy level scheme for the high-spin ferrous hemoproteins: evidence for the 5E eta, and 5B2 terms as the ground multiplets in hemoproteins with a histidine and a cysteine protein-derived heme ligand, respectively.

We have carried out analysis of the electronic level scheme of the high-spin ferrous hemoproteins by simultaneous fit of the adjustable parameters of a 4-term theoretical model to low-temperature magnetic circular dichroism (MCD), room temperature absorption spectra and available magnetic susceptibility and or Mössbauer data of myoglobin, horseradish peroxidase and cytochrome P450. The high reliability of the ligand field parameter values obtained for deoxymyoglobin is confirmed by good agreement between the predicted and observed magnetic field dependences of MCD and magnetization not used in the fit procedure. In addition, an energy gap between the ground and first excited singlets, estimated to be 4.2 cm-1, agrees well with the value of approximately 4 cm-1 derived from the far-infrared magnetic resonance. Our computer and explicit theoretical analyses give strong evidence that large distinctions in the shape, intensity and temperature behaviour of the MCD of Mb and HRP from those of cytochrome P450 can be described only if the ground manifold in these proteins is 5E eta and 5B2, respectively. The changes in relative energies of the one-electron 3d-orbitals on substitution of an imidazole of histidine for a sulphur anion of cysteine as a protein-derived heme iron ligand are rationalized by the lower ionization potential of the negatively charged sulphur ligand and the higher pi-orbital overlap of its lone pair orbitals with the iron d pi-orbitals compared to the imidazole ligand.