Spatial distribution of cytoplasmic domains of the Mg(2+)-transporter MgtE, in a solution lacking Mg(2+), revealed by paramagnetic relaxation enhancement

MgtE is a prokaryotic Mg(2+) transporter that controls cellular Mg(2+) concentrations. We previously reported crystal structures of the cytoplasmic region of MgtE, consisting of 2 domains, that is, N and CBS, in the Mg(2+)-free and Mg(2+)-bound forms. The Mg(2+)-binding sites lay at the interface of the 2 domains, making the Mg(2+)-bound form compact and globular. In the Mg(2+)-free structure, however, the domains are far apart, and the Mg(2+)-binding sites are destroyed. Therefore, it is unclear how Mg(2+)-free MgtE changes its conformation to accommodate Mg(2+) ions. Here, we used paramagnetic relaxation enhancement (PRE) to characterize the relative orientation of the N and CBS domains in the absence of Mg(2+) in solution. When the residues on the surface of the CBS domain were labeled with nitroxide tags, significant PRE effects were observed for the residues in the N domain. No single structure satisfied the PRE profiles, suggesting that the N and CBS domains are not fixed in a particular orientation in solution. We then conducted ensemble simulated annealing calculations in order to obtain the atomic probability density and visualize the spatial distribution of the N domain in solution. The results indicate that the N domain tends to occupy the space near its position in the Mg(2+)-bound crystal structure, facilitating efficient capture of Mg(2+) with increased intracellular Mg(2+) concentration, which is necessary to close the gate.     

The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b₆f with the electron transfer proteins plastocyanin and cytochrome c₆. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c₆ from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c₆ were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge–charge interactions pre-orient cytochrome c₆ with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.     

Characterization of conformational and dynamic properties of natively unfolded human and mouse alpha-synuclein ensembles by NMR: implication for aggregation

Conversion of human α-synuclein (aS) from the free soluble state to the insoluble fibrillar state has been implicated in the etiology of Parkinson's disease. Human aS is highly homologous in amino acid sequence to mouse aS, which contains seven substitutions including the A53T that has been linked to familial Parkinson's disease, and including five substitutions in the C-terminal region. It has been shown that the rate of fibrillation is highly dependent on the exact sequence of the protein, and mouse aS is reported to aggregate more rapidly than human aS in vitro. Nuclear magnetic resonance experiments of mouse and human aS at supercooled temperatures (263 K) are used to understand the effect of sequence on conformational fluctuations in the disordered ensembles and to relate these to differences in propensities to aggregate. We show that both aS are natively unfolded at low temperature with different propensities to secondary structure, backbone dynamics and long-range contacts across the protein. Mouse aS exhibits a higher propensity to helical conformation around the C-terminal substitutions as well as the loss of transient long-range contacts from the C- to the N-terminal end and hydrophobic central regions of the protein relative to human aS. Lack of back-folding from the C-terminal end of mouse aS exposes the N-terminal region, which is shown, by ¹⁵N relaxation experiments, to be very restricted in mobility relative to human aS. We propose that the restricted mobility in the N-terminal region may arise from transient interchain interactions, suggesting that the N-terminal KTK(E/Q)GV repeats may serve as initiation sites for aggregation in mouse aS. These transient interchain interactions coupled with a non-Aβ amyloid component (NAC) region that is both more exposed and has a higher propensity to β structure may accelerate the rate of fibril formation of aS.     

Structure and dynamics of the force-generating domain of myosin probed by multifrequency electron paramagnetic resonance

Spin-labeling and multifrequency EPR spectroscopy were used to probe the dynamic local structure of skeletal myosin in the region of force generation. Subfragment 1 (S1) of rabbit skeletal myosin was labeled with an iodoacetamide spin label at C707 (SH1). X-and W-band EPR spectra were recorded for the apo state and in the presence of ADP and nucleotide analogs. EPR spectra were analyzed in terms of spin-label rotational motion within myosin by fitting them with simulated spectra. Two models were considered: rapid-limit oscillation (spectrum-dependent on the orientational distribution only) and slow restricted motion (spectrum-dependent on the rotational correlation time and the orientational distribution). The global analysis of spectra obtained at two microwave frequencies (9.4 GHz and 94 GHz) produced clear support for the second model and enabled detailed determination of rates and amplitudes of rotational motion and resolution of multiple conformational states. The apo biochemical state is well-described by a single structural state of myosin (M) with very restricted slow motion of the spin label. The ADP-bound biochemical state of myosin also reveals a single structural state (M*, shown previously to be the same as the post-powerstroke ATP-bound state), with less restricted slow motion of the spin label. In contrast, the extra resolution available at 94 GHz reveals that the EPR spectrum of the S1.ADP.V_i-bound biochemical state of myosin, which presumably mimics the S1.ADP.P_i state, is resolved clearly into three spectral components (structural states). One state is indistinguishable from that of the ADP-bound state (M*) and is characterized by moderate restriction and slow motion, with a mole fraction of 16%. The remaining 84% (M**) contains two additional components and is characterized by fast rotation about the x axis of the spin label. After analyzing EPR spectra, myosin ATPase activity, and available structural information for myosin II, we conclude that post-powerstroke and pre-powerstroke structural states (M* and M**) coexist in the S1.ADP.V_i biochemical state. We propose that the pre-powerstroke state M** is characterized by two structural states that could reflect flexibility between the converter and N-terminal domains of myosin.     

Heme-heme and heme-ligand interactions in the di-heme oxygen-reducing site of cytochrome bd from Escherichia coli revealed by nanosecond absorption spectroscopy

Cytochrome bd is a terminal quinol:O₂ oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b₅₅₈, b₅₉₅, and d. The role of heme b₅₉₅ remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b₅₉₅ causing loss of an absorption band centered at 435 nm, thus supporting interactions between heme b₅₉₅ and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with τ ∼ 12 μs, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with τ ∼ 14 ns, 14 μs, and 280 μs. The spectra of the absorption changes associated with these components are different in line shape. The 14 ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-μs component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in ∼ 4% of the enzyme population. The final, 280-μs component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b₅₉₅, and not that of heme b₅₅₈, controls the pathway(s) by which CO migrates between heme d and the medium.     

The denatured state of N-PGK is compact and predominantly disordered

The organisation of the structure present in the chemically denatured N-terminal domain of phosphoglycerate kinase (N-PGK) has been determined by paramagnetic relaxation enhancements (PREs) to define the conformational landscape accessible to the domain. Below 2.0 M guanidine hydrochloride (GuHCl), a species of N-PGK (denoted I_b) is detected, distinct from those previously characterised by kinetic experiments [folded (F), kinetic intermediate (I_k) and denatured (D)]. The transition to I_b is never completed at equilibrium, because F predominates below 1.0 M GuHCl. Therefore, the ability of PREs to report on transient or low population species has been exploited to characterise I_b. Five single cysteine variants of N-PGK were labelled with the nitroxide electron spin-label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate] and the denaturant dependences of the relaxation properties of the amide NMR signals between 1.2 and 3.6 M GuHCl were determined. Significant PREs for I_b were obtained, but these were distributed almost uniformly throughout the sequence. Furthermore, the PREs indicate that no specific short tertiary contacts persist. The data indicate a collapsed state with no coherent three-dimensional structure, but with a restricted radius beyond which the protein chain rarely reaches. The NMR characteristics of I_b indicate that it forms from the fully denatured state within 100 μs, and therefore a rapid collapse is the initial stage of folding of N-PGK from its chemically denatured state. By extrapolation, I_b is the predominant form of the denatured state under native conditions, and the non-specifically collapsed structure implies that many non-native contacts and chain reversals form early in protein folding and must be broken prior to attaining the native state topology.     

A divalent-ion binding site on the 16-kDa proton channel from Nephrops norvegicus—revealed by EPR spectroscopy

As purified from the hepatopancreas of Nephrops norvegicus, the 16-kDa proton channel proteolipid is found to contain an endogenous divalent ion binding site that is occupied by Cu²⁺. The EPR spectrum has g-values and hyperfine splittings that are characteristic of type 2 Cu²⁺. The copper may be removed by extensive washing with EDTA. Titration with Ni²⁺ then induces spin-spin interactions with nitroxyl spin labels that are attached either to the unique Cys⁵⁴, or to fatty acids intercalated in the membrane. Paramagnetic relaxation enhancement by the fast-relaxing Ni²⁺ is used to characterise the binding and to estimate distances from the dipolar interactions. The Ni²⁺-binding site on the protein is situated around 14-18 Å from the spin label on Cys⁵⁴, and is at a similar distance from a lipid chain spin-labelled on the 5 C-atom, but is more remote from the C-9 and C-14 positions of the lipid chains.     

Liquid domains in vesicles investigated by NMR and fluorescence microscopy

We use (2)H-NMR, (1)H-MAS NMR, and fluorescence microscopy to detect immiscibility in three particular phospholipid ratios mixed with 30% cholesterol: 2:1 DOPC/DPPC, 1:1 DOPC/DPPC, and 1:2 DOPC/DPPC. Large-scale (>160 nm) phase separation into liquid-ordered (L(o)) and liquid-crystalline (L(alpha)) phases is observed by both NMR and fluorescence microscopy. By fitting superimposed (2)H-NMR spectra, we quantitatively determine that the L(o) phase is strongly enriched in DPPC and moderately enriched in cholesterol. Tie-lines estimated at different temperatures and membrane compositions are based on both (2)H-NMR observations and a previously published ternary phase diagram. (2)H- and (1)H-MAS NMR techniques probe significantly smaller length scales than microscopy experiments (submicron versus micron-scalp), and complex behavior is observed near the miscibility transition. Fluorescence microscopy of giant unilamellar vesicles shows micrometer-scale domains below the miscibility transition. In contrast, NMR of multilamellar vesicles gives evidence for smaller ( approximately 80 nm) domains just below the miscibility transition, whereas large-scale demixing occurs at a lower temperature, T(low). A transition at T(low) is also evident in fluorescence microscopy measurements of the surface area fraction of ordered phase in giant unilamellar vesicles. Our results reemphasize the complex phase behavior of cholesterol-containing membranes and provide a framework for interpreting (2)H-NMR experiments in similar membranes.     

Probing the structure of the Ff bacteriophage major coat protein transmembrane helix dimer by solution NMR

The transmembrane (TM) segment of the major coat protein from Ff bacteriophage has been extensively studied as an example of dimerization in detergent and lipid bilayer systems. However, almost all the information regarding this interaction has been gained through mutagenesis studies, with little direct structural information being available. To this end solution NMR has the potential to provide new insights into structure of the dimer. In order to evaluate the utility of this approach we have studied a selectively ¹⁵N-labeled peptide containing the TM segment of MCP (MCP_TM) by solution NMR. This peptide was found to give rise to detergent concentration-dependent spectra that were assigned to monomeric and dimeric forms. The standard free energy of this interaction in SDS was estimated from these spectra and found to be consistent with weak but specific dimerization. In addition, similar spectra could be obtained in β-octyl glucoside with intermolecular paramagnetic relaxation experiments demonstrating a parallel arrangement of TM helices in the dimer. In both detergents backbone chemical shift differences between monomeric and dimeric forms of MCP_TM showed that the largest changes occur around its GXXXG motif. The resulting structural model is consistent with observations made for MCP mutants previously characterized in biological membranes, opening the door to detailed structural characterization of this form of MCP. These results also have general implications for the study of weakly interacting TM segments by solution NMR since the use of similar sample conditions should allow structural data to be accessed for oligomeric states from a wide range systems that undergo biologically relevant but weak associations in the membrane.     

Analysis of internal motions of interleukin-13 variant associated with severe bronchial asthma using (15)N NMR relaxation measurements

The single nucleotide polymorphism interleukin-13 (IL-13) R110Q is associated with severe bronchial asthma because its lower affinity leads to the augmentation of local IL-13 concentration, resulting in an increase in the signal transduction via IL-13R. Since the mutation site does not directly bind to IL-13Ralpha2, we carried out NMR relaxation analyses of the wild-type IL-13 and IL-13-R110Q in order to examine whether the R110Q mutation affects the internal motions in IL-13 molecules. The results showed that the internal motion in the micro- to millisecond time scale on helix D, which is suggested to be important for the interaction between IL-13 and IL-13Ralpha2, is increased in IL-13-R110Q compared with that in the wild-type IL-13. It therefore appears that the difference in the internal motions on helix D between the wild-type IL-13 and IL-13-R110Q may be involved in their affinity differences with IL-13Ralpha2.     

IF1 limits the apoptotic-signalling cascade by preventing mitochondrial remodelling

Mitochondrial structure has a central role both in energy conversion and in the regulation of cell death. We have previously shown that IF₁ protects cells from necrotic cell death and supports cristae structure by promoting the oligomerisation of the F₁F_o-ATPsynthase. As IF₁ is upregulated in a large proportion of human cancers, we have here explored its contribution to the progression of apoptosis and report that an increased expression of IF₁, relative to the F₁F_o-ATPsynthase, protects cells from apoptotic death. We show that IF₁ expression serves as a checkpoint for the release of Cytochrome c (Cyt c) and hence the completion of the apoptotic program. We show that the progression of apoptosis engages an amplification pathway mediated by: (i) Cyt c-dependent release of ER Ca²⁺, (ii) Ca²⁺-dependent recruitment of the GTPase Dynamin-related protein 1 (Drp1), (iii) Bax insertion into the outer mitochondrial membrane and (iv) further release of Cyt c. This pathway is accelerated by suppression of IF₁ and delayed by its overexpression. IF₁ overexpression is associated with the preservation of mitochondrial morphology and ultrastructure, consistent with a central role for IF₁ as a determinant of the inner membrane architecture and with the role of mitochondrial ultrastructure in the regulation of Cyt c release. These data suggest that IF₁ is an antiapoptotic and potentially tumorigenic factor and may be a valuable predictor of responsiveness to chemotherapy.     

Structural dynamics of the actomyosin complex probed by a bifunctional spin label that cross-links SH1 and SH2

We have used a bifunctional spin label (BSL) to cross-link Cys⁷⁰⁷ (SH1) and Cys⁶⁹⁷ (SH2) in the catalytic domain of myosin subfragment 1 (S1). BSL induces the same weakened ATPase activity and actin-binding affinity that is observed when SH1 and SH2 are cross-linked with pPDM, which traps an analog of the post-hydrolysis state A·M·ADP·P. Electron paramagnetic resonance showed that BSL reports the global orientation and dynamics of S1. When bound to actin in oriented muscle fibers in the absence of ATP, BSL-S1 showed almost complete orientational disorder, as reported previously for the weakly bound A·M·ADP. In contrast, helical order is observed for the strongly bound state A·M. Saturation transfer electron paramagnetic resonance showed that the disorder of cross-linked S1 on actin is nearly static on the microsecond timescale, at least 30 times slower than that of A·M·ADP. We conclude that cross-linked S1 exhibits rotational disorder comparable to that of A·M·ADP, slow rotational mobility comparable to that of A·M, and intermediate actin affinity. These results support the hypothesis that the catalytic domain of myosin is orientationally disordered on actin in a post-hydrolysis state in the early stages of force generation.     

Solution NMR structure of putidaredoxin-cytochrome P450cam complex via a combined residual dipolar coupling-spin labeling approach suggests a role for Trp106 of putidaredoxin in complex formation

The 58-kDa complex formed between the [2Fe-2S] ferredoxin, putidaredoxin (Pdx), and cytochrome P450cam (CYP101) from the bacterium Pseudomonas putida has been investigated by high-resolution solution NMR spectroscopy. Pdx serves as both the physiological reductant and effector for CYP101 in the enzymatic reaction involving conversion of substrate camphor to 5-exo-hydroxycamphor. In order to obtain an experimental structure for the oxidized Pdx-CYP101 complex, a combined approach using orientational data on the two proteins derived from residual dipolar couplings and distance restraints from site-specific spin labeling of Pdx has been applied. Spectral changes for residues in and near the paramagnetic metal cluster region of Pdx in complex with CYP101 have also been mapped for the first time using ¹⁵N and ¹³C NMR spectroscopy, leading to direct identification of the residues strongly affected by CYP101 binding. The new NMR structure of the Pdx-CYP101 complex agrees well with results from previous mutagenesis and biophysical studies involving residues at the binding interface such as formation of a salt bridge between Asp38 of Pdx and Arg112 of CYP101, while at the same time identifying key features different from those of earlier modeling studies. Analysis of the binding interface of the complex reveals that the side chain of Trp106, the C-terminal residue of Pdx and critical for binding to CYP101, is located across from the heme-binding loop of CYP101 and forms non-polar contacts with several residues in the vicinity of the heme group on CYP101, pointing to a potentially important role in complex formation.     

Backbone dynamics of the regulatory domain of calcium vector protein, studied by 15N relaxation at four fields, reveals unique mobility characteristics of the intermotif linker

CaVP is a calcium-binding protein from amphioxus. It has a modular composition with two domains, but only the two EF-hand motifs localized in the C-terminal domain are functional. We recently determined the solution structure of this regulatory half (C-CaVP) in the Ca(2+)-saturated form and characterized the stepwise ion binding. This paper reports the (15)N nuclear relaxation rates of the Ca(2+)-saturated C-CaVP, measured at four different NMR fields (9.39, 11.74, 14.1, and 18.7 T), which were used to map the spectral density function for the majority of the amide H(N)-N vectors. Fitting the spectral density values at eight frequencies by a model-free approach, we obtained the microdynamic parameters characterizing the global and internal movements of the polypeptide backbone. The two EF-hand motifs, including the ion binding loops, behave like compact structural units with restricted mobility as reflected in the quite uniform order parameter and short internal correlation time (< 20 nsec). Comparative analysis of the two Ca(2+) binding sites shows that site III, having a larger affinity for the metal ion, is generally more rigid, and the amide vector in the second residue of each loop is significantly less restricted. The linker fragment is animated simultaneously by a larger amplitude fast motion and a slow conformational exchange on a microsecond to millisecond time scale. The backbone dynamics of C-CaVP characterized here is discussed in relation with other well-characterized Ca(2+)-binding proteins. Supplemental material: See www.proteinscience.org     

Methionine acts as a "magnet" in photoaffinity crosslinking experiments

Photoaffinity crosslinking has been utilized to probe the nature of the ligand-receptor interface for a number of G protein-coupled receptor systems. Often the photoreactive benzophenone moiety incorporated in the ligand is found to react with a methionine in the receptor. We introduced methionines one-at-a-time into the region 163-176 of the parathyroid hormone receptor, and find that crosslinking occurs to the side-chain of methionine over a range of 11 amino acids. We call this the "Magnet Effect" of methionine. Hence, crosslinking contact points can be significantly shifted by the presence of methionine in a receptor domain.     

Enhancement of YD spin relaxation by the CaMn4 cluster in photosystem II detected at room temperature: A new probe for the S-cycle

The long-lived, light-induced radical Y_D^• of the Tyr161 residue in the D2 protein of Photosystem II (PSII) is known to magnetically interact with the CaMn₄ cluster, situated ∼ 30 Å away. In this study we report a transient step-change increase in Y_D^• EPR intensity upon the application of a single laser flash to S₁ state-synchronised PSII-enriched membranes from spinach. This transient effect was observed at room temperature and high applied microwave power (100 mW) in samples containing PpBQ, as well as those containing DCMU. The subsequent decay lifetimes were found to differ depending on the additive used. We propose that this flash-induced signal increase was caused by enhanced spin relaxation of Y_D^• by the OEC in the S₂ state, as a consequence of the single laser flash turnover. The post-flash decay reflected S₂ → S₁ back-turnover, as confirmed by their correlations with independent measurements of S₂ multiline EPR signal and flash-induced variable fluorescence decay kinetics under corresponding experimental conditions. This flash-induced effect opens up the possibility to study the kinetic behaviour of S-state transitions at room temperature using Y_D^• as a probe.     

Class III compensation,represented by KRP2 overexpression,depends on V-ATPase activity in proliferative cells

Compensation refers to an increase in cell size when the cell number is significantly decreased due to the mutation or gain of function of a gene that negatively affects the cell cycle. Given the importance of coordinated growth during organogenesis in both animal and plant systems, compensation is important to understand the mechanism of size regulation. In leaves, cell division precedes cell differentiation (which involves cell expansion); therefore, a decrease in cell number triggers enhanced cell expansion (compensated cell expansion; hereafter, CCE). Functional analyses of genes for which a loss or gain of function triggers compensation have increased our understanding of the molecular mechanisms underlying the decrease in cell number. Nevertheless, the mechanisms that induce enhanced cell expansion (the link between cell cycling and expansion), as well as the cellular machinery mediating CCE, have not been characterized. We recently characterized an important pathway involved in cell enlargement in KRP2-overexpressing plants. Here, we discuss the potential role of plant KRPs in triggering enlargement in cells with meristematic features.