Structures of Staphylococcus aureus cell-wall complexes with vancomycin, eremomycin, and chloroeremomycin derivatives by 13C{19F} and 15N{19F} rotational-echo double resonance

Solid-state NMR has been used to examine isolated cell walls and intact whole cells of Staphylococcus aureus complexed to five different vancomycin, eremomycin, and chloroeremomycin derivatives. The cell walls and whole cells were specifically labeled with d-[1-(13)C]alanine, or a combination of [1-(13)C]glycine and [epsilon-(15)N]lysine. Each of the bound glycopeptides had a (19)F-labeled substituent at either its C-terminus or its disaccharide position. The (13)C{(19)F} rotational-echo double-resonance (REDOR) dephasing for the cell-wall (13)C-labeled bridging pentaglycyl segment connecting a glycopeptide-complexed peptidoglycan stem with its neighboring stem indicates that the fluorine labels for all bound glycopeptides are positioned at one or the other end of the bridge. An exception is N'-(p-trifluoromethoxybenzyl)chloroeremomycin, whose hydrophobic substituent differs in length by one phenyl group compared to that of oritavancin, N'-4-[(4-chlorophenyl)benzyl)]chloroeremomycin. For this drug, the fluorine label is near the middle of the pentaglycyl segment. (15)N{(19)F} REDOR dephasing shows the proximity of the fluorine to the bridge-link site of the pentaglycyl bridge for C-terminus-substituted moieties and the cross-link site for disaccharide-substituted moieties. Full-echo REDOR spectra of cell-wall complexes from cells labeled by d-[1-(13)C]alanine (in the presence of an alanine racemase inhibitor) reveal three different carbonyl carbon chemical-shift environments, arising from the d-Ala-d-Ala binding site and the d-Ala-Gly-1 cross-link site. The REDOR results indicate a single fluorine dephasing center in each peptidoglycan complex. Molecular models of the mature cell-wall complexes that are consistent with internuclear distances obtained from (13)C{(19)F} and (15)N{(19)F} REDOR dephasing allow a correlation of structure and antimicrobial activity of the glycopeptides.     

Spectral editing of two-dimensional magic-angle-spinning solid-state NMR spectra for protein resonance assignment and structure determination

Several techniques for spectral editing of 2D ¹³C?C¹³C correlation NMR of proteins are introduced. They greatly reduce the spectral overlap for five common amino acid types, thus simplifying spectral assignment and conformational analysis. The carboxyl (COO) signals of glutamate and aspartate are selected by suppressing the overlapping amide N?CCO peaks through ¹³C?C¹⁵N dipolar dephasing. The sidechain methine (CH) signals of valine, lecuine, and isoleucine are separated from the overlapping methylene (CH₂) signals of long-chain amino acids using a multiple-quantum dipolar transfer technique. Both the COO and CH selection methods take advantage of improved dipolar dephasing by asymmetric rotational-echo double resonance (REDOR), where every other ??-pulse is shifted from the center of a rotor period t_r by about 0.15 t_r. This asymmetry produces a deeper minimum in the REDOR dephasing curve and enables complete suppression of the undesired signals of immobile segments. Residual signals of mobile sidechains are positively identified by dynamics editing using recoupled ¹³C?C¹H dipolar dephasing. In all three experiments, the signals of carbons within a three-bond distance from the selected carbons are detected in the second spectral dimension via ¹³C spin exchange. The efficiencies of these spectral editing techniques range from 60?% for the COO and dynamic selection experiments to 25?% for the CH selection experiment, and are demonstrated on well-characterized model proteins GB1 and ubiquitin.     

Rotational-echo double-resonance NMR-restrained model of the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase

The 46-kD enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form EPSP. The reaction is inhibited by N-(phosphonomethyl)-glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. We have used solid-state NMR and molecular modeling to characterize the EPSP synthase-S3P-Glp ternary complex. Modeling began with the crystal coordinates of the unliganded protein, published distance restraints, and information from the chemical modification and mutagenesis literature on EPSP synthase. New inter-ligand and ligand-protein distances were obtained. These measurements utilized the native (31)P in S3P and Glp, biosynthetically (13)C-labeled S3P, specifically (13)C and (15)N labeled Glp, and a variety of protein-(15)N labels. Several models were investigated and tested for accuracy using the results of both new and previously published rotational-echo double resonance (REDOR) NMR experiments. The REDOR model is compared with the recently published X-ray crystal structure of the ternary complex, PDB code 1G6S. There is general agreement between the REDOR model and the crystal structure with respect to the global folding of the two domains of EPSP synthase and the relative positioning of S3P and Glp in the binding pocket. However, some of the REDOR data are in disagreement with predictions based on the coordinates of 1G6S, particularly those of the five arginines lining the binding site. We attribute these discrepancies to substantive differences in sample preparation for REDOR and X-ray crystallography. We applied the REDOR restraints to the 1G6S coordinates and created a REDOR-refined xray structure that agrees with the NMR results.     

REDOR: an assessment of the efficacy of dipolar recoupling with adiabatic inversion pulses

A numerical assessment of the efficacy of REDOR recoupling of heteronuclear dipolar interactions employing adiabatic dephasing pulses has been carried out by considering an isolated dipolar coupled spin 1/2 I-S system. At moderate magic angle spinning frequencies in the range of 3–6 kHz and when the CSA of the dephased spins is large, it is shown that efficient broadband heteronuclear dipolar recoupling and reliable distance estimates can be achieved even under conditions where a significant fraction of the rotor period is occupied by the adiabatic pulse. The efficacy of REDOR with adiabatic inversion pulses has been demonstrated experimentally in two model ¹⁵N-¹³C spin systems, (¹³C,¹⁵N) Aib-(¹⁵N) Aib-NH₂ (Aib = -aminoisobutyric acid) and (1-¹³C,¹⁵N) glycine.     

Location of fluorotryptophan sequestered in an amphiphilic nanoparticle by rotational-echo double-resonance NMR.

Rotational-echo double-resonance (REDOR) 13C NMR spectra (with 19F dephasing) have been obtained of 6-fluorotryptophan complexed by a polymeric amphiphilic nanosphere consisting of a polystyrene core covalently attached to a poly(acrylic acid)-polyacrylamide shell. The REDOR spectra show that aromatic carbons from the polystyrene core and oxygenated carbons in the poly(acrylic acid)-polyacrylamide shell are both proximate to the 19F of 6-fluorotryptophan. Molecular modeling restrained by distances inferred from the REDOR spectra suggests that all of the 6-fluorotryptophans are in the shell but within 10 A of the core-shell interface.     

Rapid measurement of long-range distances in proteins by multidimensional <Superscript>13</Superscript>C–<Superscript>19</Superscript>F REDOR NMR under fast magic-angle spinning

The ability to simultaneously measure many long-range distances is critical to efficient and accurate determination of protein structures by solid-state NMR (SSNMR). So far, the most common distance constraints for proteins are ¹³C–¹⁵N distances, which are usually measured using the rotational-echo double-resonance (REDOR) technique. However, these measurements are restricted to distances of up to ~?5 Å due to the low gyromagnetic ratios of ¹⁵N and ¹³C. Here we present a robust 2D ¹³C–¹⁹F REDOR experiment to measure multiple distances to ~?10 Å. The technique targets proteins that contain a small number of recombinantly or synthetically incorporated fluorines. The ¹³C–¹⁹F REDOR sequence is combined with 2D ¹³C–¹³C correlation to resolve multiple distances in highly ¹³C-labeled proteins. We show that, at the high magnetic fields which are important for obtaining well resolved ¹³C spectra, the deleterious effect of the large ¹⁹F chemical shift anisotropy for REDOR is ameliorated by fast magic-angle spinning and is further taken into account in numerical simulations. We demonstrate this 2D ¹³C–¹³C resolved ¹³C–¹⁹F REDOR technique on ¹³C, ¹⁵N-labeled GB1. A 5-¹⁹F-Trp tagged GB1 sample shows the extraction of distances to a single fluorine atom, while a 3-¹⁹F-Tyr labeled GB1 sample allows us to evaluate the effects of multi-spin coupling and statistical ¹⁹F labeling on distance measurement. Finally, we apply this 2D REDOR experiment to membrane-bound influenza B M2 transmembrane peptide, and show that the distance between the proton-selective histidine residue and the gating tryptophan residue differs from the distances in the solution NMR structure of detergent-bound BM2. This 2D ¹³C–¹⁹F REDOR technique should facilitate SSNMR-based protein structure determination by increasing the measurable distances to the ~?10 Å range.     

Possible conformation of amphotericin B dimer in membrane-bound assembly as deduced from solid-state NMR

Aiming for structural analysis of amphotericin B (AmB) ion-channel assemblies in membrane, a covalent dimer was synthesized between ¹³C-labled AmB methyl ester and ¹⁹F-labled AmB. The dimer showed slightly weaker but significant biological activities against fungi and red blood cells compared with those of monomeric AmB. Then the dimer was subjected to ¹³C{¹⁹F}REDOR (Rotational-Echo Double Resonance) experiments in hydrated lipid bilayers. The obtained REDOR dephasing effects were explained by two components; a short ¹³C/¹⁹F distance (6.9 Å) accounting for 23% of the REDOR dephasing, and a longer one (14 Å) comprising the rest of the dephasing. The shorter distance is likely to reflect the formation of barrel-stave ion channel.     

Dominant formation of a single-length channel by amphotericin B in dimyristoylphosphatidylcholine membrane evidenced by 13C-31P rotational echo double resonance

(13)C-Labeled amphotericin B (AmB) was prepared by feeding the producing organism Streptomyces nodosus with [3-(13)C]propionate. The REDOR experiments for dimyristoylphosphatidylcholine (DMPC) membrane using the (13)C-labeled AmB showed the prominent dephasing effects between the phosphate group in PC and C41 carboxyl carbon in the polar head. In addition, C39/C40 methyl carbons also gave rise to the significant reduction of their (13)C NMR signals, implying that both terminal parts of AmB reside close to the surface of the DMPC membrane. Conversely, the same REDOR experiments with use of distearoylphosphatidylcholine (DSPC) showed no dephasing for the C39/C40 methyl signals while a marked reduction of the C41 carbonyl signal was again observed. These findings should be most reasonably accounted for by the notion that AmB can span across the DMPC membrane with a single-length interaction but cannot span the DSPC membrane due to its greater thickness. To our knowledge, the results provide the first direct spectroscopic evidence for the formation of a single-length channel across a biomembrane, which was previously suggested by channel current recording experiments.     

Off-resonance 13C–2H REDOR NMR for site-resolved studies of molecular motion

We introduce a ¹³C–²H Rotational Echo DOuble Resonance (REDOR) technique that uses the difference between on-resonance and off-resonance ²H irradiation to detect dynamic segments in deuterated molecules. By selectively inverting specific regions of the ²H magic-angle spinning (MAS) sideband manifold to recouple some of the deuterons to nearby carbons, we distinguish dynamic and rigid residues in 1D and 2D ¹³C spectra. We demonstrate this approach on deuterated GB1, H/D exchanged GB1, and perdeuterated bacterial cellulose. Numerical simulations reproduce the measured mixing-time and ²H carrier-frequency dependence of the REDOR dephasing of bacterial cellulose. Combining numerical simulations with experiments thus allow the extraction of motionally averaged quadrupolar couplings from REDOR dephasing values.

     

Membrane interaction of amphotericin B as single-length assembly examined by solid state NMR for uniformly 13C-enriched agent

The membrane interaction of amphotericin B (AmB), one of the most important anti-fungal drugs, was investigated by solid state NMR measurements of uniformly 13C-enriched AmB, which was prepared by the culture of the drug-producing microorganism in the presence of [u-13C6]glucose. All the 13C NMR signals of AmB upon binding to DLPC membrane were successfully assigned on the basis of the 13C-13C correlation spectrum. 13C-31P RDX (Rotational-Echo Double Resonance for X-clusters) experiments clearly revealed the REDOR dephasing effects for carbon atoms residing in the both terminal parts, whereas no dephasing was observed for the middle parts including polyolefinic C20-C33 and hydroxyl-bearing C8/C9 parts. These observations suggest that AmB binds to DLPC membrane with a high affinity to the phospholipid and spans the membrane with a single molecular length.     

Head-to-tail interaction between amphotericin B and ergosterol occurs in hydrated phospholipid membrane

Amphotericin B (AmB) is thought to exert its antifungal activity by forming an ion-channel assembly in the presence of ergosterol. In the present study we aimed to elucidate the mode of molecular interactions between AmB and ergosterol in hydrated phospholipid bilayers using the rotational echo double resonance (REDOR) spectra. We first performed (13)C{(19)F}REDOR experiments with C14-(19)F-labeled AmB and biosynthetically (13)C-labeled ergosterol and implied that both "head-to-head" and "head-to-tail" orientations occur for AmB-ergosterol interaction in the bilayers. To further confirm the "head-to-tail" pairing, (13)C-labeled ergosterol at the dimethyl terminus (C26/C27) was synthesized and subjected to the REDOR measurements. The spectra unambiguously demonstrated the presence of a "head-to-tail" orientation for AmB-ergosterol pairing. In order to obtain information on the position of the dimethyl terminus of ergosterol in membrane, (13)C{(31)P}REDOR were carried out using the labeled ergosterol and the phosphorus atom of a POPC headgroup. Significant REDOR dephasing was observed at the C26/C27 signal of ergosterol in the presence of AmB, but not in the absence of AmB, clearly indicating that the side-chain terminus of ergosterol in the AmB complex comes close to the bilayer surface.     

Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR

Local dynamics of interhelical loops in bacteriorhodopsin (bR), the extracellular BC, DE and FG, and cytoplasmic AB and CD loops, and helix B were determined on the basis of a variety of relaxation parameters for the resolved 13C and 15N signals of [1-13C]Tyr-, [15N]Pro- and [1-13C]Val-, [15N]Pro-labeled bR. Rotational echo double resonance (REDOR) filter experiments were used to assign [1-13C]Val-, [15N]Pro signals to the specific residues in bR. The previous assignments of [1-13C]Val-labeled peaks, 172.9 or 171.1 ppm, to Val69 were revised: the assignment of peak, 172.1 ppm, to Val69 was made in view of the additional information of conformation-dependent 15N chemical shifts of Pro bonded to Val in the presence of 13C-15N correlation, although no assignment of peak is feasible for 13C nuclei not bonded to Pro. 13C or 15N spin-lattice relaxation times (T1), spin-spin relaxation times under the condition of CP-MAS (T2), and cross relaxation times (TCH and TNH) for 13C and 15N nuclei and carbon or nitrogen-resolved, 1H spin-lattice relaxation times in the rotating flame (1H T1 rho) for the assigned signals were measured in [1-13C]Val-, [15N]Pro-bR. It turned out that V69-P70 in the BC loop in the extracellular side has a rigid beta-sheet in spite of longer loop and possesses large amplitude motions as revealed from 13C and 15N conformation-dependent chemical shifts and T1, T2, 1H T1 rho and cross relaxation times. In addition, breakage of the beta-sheet structure in the BC loop was seen in bacterio-opsin (bO) in the absence of retinal.     

Residue-specific membrane location of peptides and proteins using specifically and extensively deuterated lipids and 13C–2H rotational-echo double-resonance solid-state NMR

Residue-specific location of peptides in the hydrophobic core of membranes was examined using ¹³C–²H REDOR and samples in which the lipids were selectively deuterated. The transmembrane topology of the KALP peptide was validated with this approach with substantial dephasing observed for deuteration in the bilayer center and reduced or no dephasing for deuteration closer to the headgroups. Insertion of β sheet HIV and helical and β sheet influenza virus fusion peptides into the hydrophobic core of the membrane was validated in samples with extensively deuterated lipids.     

Detection of closed influenza virus hemagglutinin fusion peptide structures in membranes by backbone 13CO-15N rotational-echo double-resonance solid-state NMR

The influenza virus fusion peptide is the N-terminal ~20 residues of the HA2 subunit of the hemagglutinin protein and this peptide plays a key role in the fusion of the viral and endosomal membranes during initial infection of a cell. The fusion peptide adopts N-helix/turn/C-helix structure in both detergent and membranes with reports of both open and closed interhelical topologies. In the present study, backbone ¹³CO-¹⁵N REDOR solid-state NMR was applied to the membrane-associated fusion peptide to detect the distribution of interhelical distances. The data clearly showed a large fraction of closed and semi-closed topologies and were best-fitted to a mixture of two structures that do not exchange. One of the earlier open structural models may have incorrect G13 dihedral angles derived from TALOS analysis of experimentally correct ¹³C shifts.     

Vancomycin derivative with damaged D-Ala-D-Ala binding cleft binds to cross-linked peptidoglycan in the cell wall of Staphylococcus aureus

Des-N-methylleucyl-4-(4-fluorophenyl)benzyl-vancomycin (DFPBV) retains activity against vancomycin-resistant pathogens despite its damaged d-Ala-d-Ala binding cleft. Using solid-state nuclear magnetic resonance (NMR), a DFPBV binding site in the cell walls of whole cells of Staphylococcus aureus has been identified. The cell walls were labeled with d-[1-(13)C]alanine, [1-(13)C]glycine, and l-[epsilon-(15)N]lysine. Internuclear distances from (19)F of the DFPBV to the (13)C and (15)N labels of the cell-wall peptidoglycan were determined by rotational-echo double-resonance (REDOR) NMR. The (13)C{(19)F} and (15)N{(19)F} REDOR spectra show that, in situ, DFPBV binds to the peptidoglycan as a monomer with its vancosamine hydrophobic side chain positioned near a pentaglycyl bridge. This result suggests that the antimicrobial activity of other vancosamine-modified glycopeptides depends upon both d-Ala-d-Ala stem-terminus recognition (primary binding site) and stem-bridge recognition (secondary binding site).     

Secondary structure and lipid contact of a peptide antibiotic in phospholipid bilayers by REDOR

The chemical shifts of specific (13)C and (15)N labels distributed throughout KIAGKIA-KIAGKIA-KIAGKIA (K3), an amphiphilic 21-residue antimicrobial peptide, prove that the peptide is in an all alpha-helical conformation in the bilayers of multilamellar vesicles (MLVs) containing dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (1:1). Rotational-echo double-resonance (REDOR) (13)C[(31)P] and (15)N[(31)P] experiments on the same labeled MLVs show that on partitioning into the bilayer, the peptide chains remain in contact with lipid headgroups. The amphipathic lysine side chains of K3 in particular appear to play a key role in the electrostatic interactions with the acidic lipid headgroups. In addition to the extensive peptide-headgroup contact, (13)C[(19)F] REDOR experiments on MLVs containing specifically (19)F-labeled lipid tails suggest that a portion of the peptide is surrounded by a large number of lipid acyl chains. Complementary (31)P[(19)F] REDOR experiments on these MLVs show an enhanced headgroup-lipid tail contact resulting from the presence of K3. Despite these distortions, static (31)P NMR lineshapes indicate that the lamellar structure of the membrane is preserved.     

Structure of tightly membrane-bound mastoparan-X, a G-protein-activating peptide, determined by solid-state NMR

The structure of mastoparan-X (MP-X), a G-protein activating peptide from wasp venom, in the state tightly bound to anionic phospholipid bilayers was determined by solid-state NMR spectroscopy. Carbon-13 and nitrogen-15 NMR signals of uniformly labeled MP-X were completely assigned by multidimensional intraresidue C-C, N-CalphaCbeta, and N-Calpha-C', and interresidue Calpha-CalphaCbeta, N-CalphaCbeta, and N-C'-Calpha correlation experiments. The backbone torsion angles were predicted from the chemical shifts of 13C', 13Calpha, 13Cbeta, and 15N signals with the aid of protein NMR database programs. In addition, two 13C-13C and three 13C-15N distances between backbone nuclei were precisely measured by rotational resonance and REDOR experiments, respectively. The backbone structure of MP-X was determined from the 26 dihedral angle restraints and five distances with an average root-mean-square deviation of 0.6 A. Peptide MP-X in the bilayer-bound state formed an amphiphilic alpha-helix for residues Trp3-Leu14 and adopted an extended conformation for Asn2. This membrane-bound conformation is discussed in relation to the peptide's activities to form pores in membranes and to activate G-proteins. This study demonstrates the power of multidimensional solid-state NMR of uniformly isotope-labeled molecules and distance measurements for determining the structures of peptides bound to lipid membranes.     

Characterization of slow conformational dynamics in solids: dipolar CODEX

A solid state NMR experiment is introduced for probing relatively slow conformational exchange, based on dephasing and refocusing dipolar couplings. The method is closely related to the previously described Centerband-Only Detection of Exchange or CODEX experiment. The use of dipolar couplings for this application is advantageous because their values are known a priori from molecular structures, and their orientations and reorientations relate in a simple way to molecular geometry and motion. Furthermore the use of dipolar couplings in conjunction with selective isotopic enrichment schemes is consistent with selection for unique sites in complex biopolymers. We used this experiment to probe the correlation time for the motion of ¹³C, ¹⁵N enriched urea molecules within their crystalline lattice.     

Structure and Orientation of Bovine Lactoferrampin in the Mimetic Bacterial Membrane as Revealed by Solid-State NMR and Molecular Dynamics Simulation

Bovine lactoferrampin (LFampinB) is a newly discovered antimicrobial peptide found in the N1-domain of bovine lactoferrin (268–284), and consists of 17 amino-acid residues. It is important to determine the orientation and structure of LFampinB in bacterial membranes to reveal the antimicrobial mechanism. We therefore performed ¹³C and ³¹P NMR, ¹³C-³¹P rotational echo double resonance (REDOR), potassium ion-selective electrode, and quartz-crystal microbalance measurements for LFampinB with mimetic bacterial membrane and molecular-dynamics simulation in acidic membrane. ³¹P NMR results indicated that LFampinB caused a defect in mimetic bacterial membranes. Ion-selective electrode measurements showed that ion leakage occurred for the mimetic bacterial membrane containing cardiolipin. Quartz-crystal microbalance measurements revealed that LFampinB had greater affinity to acidic phospholipids than that to neutral phospholipids. ¹³C DD-MAS and static NMR spectra showed that LFampinB formed an α-helix in the N-terminus region and tilted 45° to the bilayer normal. REDOR dephasing patterns between carbonyl carbon nucleus in LFampinB and phosphorus nuclei in lipid phosphate groups were measured by ¹³C-³¹P REDOR and the results revealed that LFampinB is located in the interfacial region of the membrane. Molecular-dynamics simulation showed the tilt angle to be 42° and the rotation angle to be 92.5° for Leu³, which are in excellent agreement with the experimental values.     

Structure of (KIAGKIA)3 aggregates in phospholipid bilayers by solid-state NMR

The interchain (13)C-(19)F dipolar coupling measured in a rotational-echo double-resonance (REDOR) experiment performed on mixtures of differently labeled KIAGKIA-KIAGKIA-KIAGKIA (K3) peptides (one specifically (13)C labeled, and the other specifically (19)F labeled) in multilamellar vesicles of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (1:1) shows that K3 forms close-packed clusters, primarily dimers, in bilayers at a lipid/peptide molar ratio (L/P) of 20. Dipolar coupling to additional peptides is weaker than that within the dimers, consistent with aggregates of monomers and dimers. Analysis of the sideband dephasing rates indicates a preferred orientation between the peptide chains of the dimers. The combination of the distance and orientation information from REDOR is consistent with a parallel (N-N) dimer structure in which two K3 helices intersect at a cross-angle of approximately 20 degrees. Static (19)F NMR experiments performed on K3 in oriented lipid bilayers show that between L/P = 200 and L/P = 20, K3 chains change their absolute orientation with respect to the membrane normal. This result suggests that the K3 dimers detected by REDOR at L/P = 20 are not on the surface of the bilayer but are in a membrane pore.