Solid-state NMR structure determination of melittin in a lipid environment

Solid-state (13)C NMR spectroscopy was used to investigate the three-dimensional structure of melittin as lyophilized powder and in ditetradecylphosphatidylcholine (DTPC) membranes. The distance between specifically labeled carbons in analogs [1-(13)C]Gly3-[2-(13)C]Ala4, [1-(13)C]Gly3-[2-(13)C]Leu6, [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 was measured by rotational resonance. As expected, the internuclear distances measured in [1-(13)C]Gly3-[2-(13)C]Ala4 and [1-(13)C]Gly3-[2-(13)C]Leu6 were consistent with alpha-helical structure in the N-terminus irrespective of environment. The internuclear distances measured in [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 revealed, via molecular modeling, some dependence upon environment for conformation in the region of the bend in helical structure induced by Pro14. A slightly larger interhelical angle between the N- and C-terminal helices was indicated for peptide in dry or hydrated gel state DTPC (139 degrees -145 degrees ) than in lyophilized powder (121 degrees -139 degrees ) or crystals (129 degrees ). The angle, however, is not as great as deduced for melittin in aligned bilayers of DTPC in the liquid-crystalline state (approximately 160 degrees ). The study illustrates the utility of rotational resonance in determining local structure within peptide-lipid complexes.     

Carbon-13 nuclear magnetic resonance studies of the selectively isotope-labeled reactive site peptide bond of the basic pancreatic trypsin inhibitor in the complexes with trypsin, trypsinogen, and anhydrotrypsin

A previously characterized modification of the basic pancreatic trypsin inhibitor (BPTI), with the carbonyl carbon atom of Lys-15 selectively enriched in 13C, the peptide bond Arg-39--Ala-40 cleaved, and Arg-39 removed, was used for 13C NMR studies of the reactive site peptide bond Lys-15--Ala-16 in the complexes with trypsin, trypsinogen, and anhydrotrypsin. The chemical shift of [1-13C]Lys-15 was 175.7 ppm in the free inhibitor, 176.4 ppm in the complexes with trypsin and anhydrotrypsin and the ternary complex with trypsinogen and H-Ile-Val-OH, and 175.7 ppm in a neutral solution containing the inhibitor and trypsinogen. These data show that the trypsin--BPTI complex does not contain a covalent tetrahedral carbon atom in the position of the reactive site peptide carbonyl of the inhibitor. They would be consistent with the formation of a noncovalent complex but cannot at present be used to further characterize the degree of a possible pyramidalization of the carbonyl carbon of Lys-15 in such a complex. The identical chemical shifts in the complexes with trypsin and anhydrotrypsin indicate that the gamma-hydroxyl group of Ser-195 of trypsin does not have an important role in the binding of the inhibitor. The previously described [Perkins, S. J. & Wüthrich, K. (1980) J. Mol. Biol. 138, 43--64] stepwise transition from the trypsinogen conformation to an intermediate conformational state in the trypsinogen--BPTI complex and a trypsin-like conformation in the ternary complex trypsinogen--BPTI--H-Ile-Val-OH appears to be manifested also in the chemical shift of [1-13C]Lys-15 of labeled BPTI.     

Structure and solvation of melittin in hexafluoroacetone/water

Intermolecular (1)H[(19)F] and (1)H[(1)H] nuclear Overhauser effects have been used to explore interaction of solvent components with melittin dissolved in 50% hexafluoroacetone trihydrate (HFA)/water. Standard nuclear Overhauser effect experiments and an analysis of C(alpha)H proton chemical shifts confirm that the conformation of the peptide in this solvent is alpha-helical from residues Ala4 to Thr11 and from Leu13 to Arg24. The two helical regions are not collinear; the interhelix angle (144 +/- 20 degrees ) found in this work is near that observed in the solid state and previous NMR studies. Intermolecular NOEs arising from interactions between spins of the solvent and the solute indicate that both fluoroalcohol and water molecules are strongly enough bound to the peptide that solvent-solute complexes persist for > or =2 ns. Preferential interactions of HFA with many hydrophobic side chains of the peptide are apparent while water molecules appear to be localized near hydrophilic side chains. These results indicate that interactions of both HFA and water are qualitatively different from those present when the peptide is dissolved in 35% hexafluoro-2-propanol/water, a chemically similar helix-supporting solvent system.     

Characterization of selectively 13C-labeled synthetic melittin and melittin analogues in isotropic solvents by circular dichroism, fluorescence, and NMR spectroscopy

The spectroscopic and functional characterization of 13C-labeled synthetic melittin and three analogues is described. Selectively 13C-enriched tryptophan ( [13C delta 1]-L-Trp) and glycine ( [13C alpha]Gly) were incorporated into melittin and three analogues by de novo peptide synthesis. 13C-Labeled tryptophan was incorporated into melittin at position 19 and into single-tryptophan analogues of melittin at positions 17, 11, and 9, respectively. Each of the synthetic peptides contained 13C-labeled glycine at position 12 only. The peptides were characterized functionally in a cytolytic assay, and spectroscopically by CD, fluorescence, and NMR. The behavior of 13C-labeled synthetic melittin was, in all respects, indistinguishable from that of the naturally occurring peptide. All of the analogues were found to be efficient lytic agents and thus were functionally similar to the native peptide, yet no evidence was found for formation of a melittin-like tetramer by any of the analogues in aqueous media, although there was a propensity for apparently nonspecific peptide aggregation, especially for MLT-W9. Since the analogues did exhibit fractional helicities by CD comparable to or even greater than melittin itself in the presence of methanol, we infer that tetramer assembly requires not only the ability to form alpha-helix but also a very precise packing of amino acid side chains of the constituent monomers. The 13C chemical shift of the Gly-12 C alpha was found to be a sensitive marker for helix formation in all of the peptides. For melittin itself, 13C NMR spectra revealed a downfield shift of approximately 1.8 ppm for the Gly-12 13C alpha resonance of the tetramer relative to that observed for the free monomer in D2O. In mixed samples containing melittin monomer and tetramer, two discrete Gly-12 13C alpha peaks were observed simultaneously, suggestive of slow exchange between the two species. We conclude that melittin's ability to form a soluble tetramer is not a prerequisite for cytolytic activity, nor is cytolytic potential precisely correlated with the ability to form an amphiphilic helix.     

Assignment of the 13C-NMR spectra of virgin and reactive-site modified turkey ovomucoid third domain

The virgin (reactive-site Leu18-Glu19 peptide bond intact) and modified (reactive-site Leu18-Glu19 peptide bond hydrolyzed) forms of turkey ovomucoid third domain (OMTKY3 and OMTKY3*, respectively) have been analyzed by proton-detected 1H(13C) two-dimensional single-bond correlation (1H[13C]SBC) spectroscopy. Previous 1H-nmr assignments of these proteins [A.D. Robertson, W.M. Westler, and J.L Markley (1988) Biochemistry, 27, 2519-2529; G. I. Rhyu and J. L. Markley (1988) Biochemistry, 27, 2529-2539] have been extended to directly bonded 13C atoms. Assignments have been made to 52 of the 56 backbone 13C alpha-1H units and numerous side-chain 13C-1H groups in both OMTKY3 and OMTKY3*. The largest changes in the 13C chemical shift upon conversion of OMTKY3 to OMTKY3* occur at or near the reactive site, and tend toward values observed in small peptides. Moreover, the side-chain prochiral methylene protons attached to the C gamma of Glu19 and C delta of Arg21 show nonequivalent chemical shifts in OMTKY3 but more equivalent chemical shifts in OMTKY3*. These results suggest that the reactive site region becomes less ordered upon hydrolysis of the Leu18-Glu19 peptide bond. Comparison of 13C alpha chemical shifts of OMTKY3 and bovine pancreatic trypsin inhibitor [D. Brühuiler and G. Wagner (1986) Biochemistry 25, 5839-5843; N. R. Nirmala and G. Wagner (1988) Journal of the American Chemical Society, 110, 7557-7558] with small peptide values [R. Richarz and K. Wüthrich (1978) Biopolymers, 17, 2133-2141] suggests that 13C alpha chemical shifts of residues residing in helices are generally about 2 ppm downfield of resonances from nonhelical residues.     

Evidence for a tetrahedral intermediate complex during serpin-proteinase interactions

Proteinase inhibitors in the serpin family form complexes with serine proteinases by interactions between the gamma-OH group at serine 195 of the enzyme and a specific peptide bond within the reactive site loop of the inhibitor. However, the type of complex formed (i.e. Michaelis, acyl, or tetrahedral) is unknown. Until now, 13C NMR spectroscopy studies have only been useful in examining complexes formed with either peptide-related or small protein inhibitors, where 13C-labeled amino acids can be inserted semi-synthetically. Recombinant DNA technology has, however, made it possible to specifically enrich larger proteins with 13C. In the case of serpins we have examined the structure of the complex formed between human alpha 1-proteinase inhibitor uniformally labeled with [13C]methionine and porcine pancreatic elastase. 13C NMR spectroscopic studies revealed a large upfield chemical shift of the carbonyl signal of Met-358 upon complex formation suggesting for the first time that a tetrahedral adduct is formed between a serpin inhibitor and a serine proteinase.     

Structural characterization by nuclear magnetic resonance of a reactive-site 13carbon-labelled basic pancreatic trypsin inhibitor with the peptide bond Arg-39--Ala-40 cleaved and Arg-39 removed

With the use of an enzymatic replacement method, 90%-enriched [1-13C]lysine was introduced into the reactive site of the basic pancreatic trypsin inhibitor. Characterization of the labelled inhibitor with 13C nuclear magnetic resonance (NMR), 1H NMR and chemical methods showed that while the reactive-site peptide bond Lys-15--Ala-16 was properly resynthesized, the polypeptide chain was cleaved at the peptide bond Arg-39--Ala-40 and Arg-39 was removed. Detailed 1H NMR studies showed further that, with the exception of the immediate environment of the modification site, the average spatial structure of the native inhibitor was preserved in the modified protein. Compared to the native inhibitor, the thermal stability of the globular conformation was found to be reduced, interior amide protons exchanged at a faster rate and the internal mobility of aromatic rings located outside the immediate environment of the cleaved peptide bond was essentially unchanged. These observations coincide closely with previous reports on different modifications of the inhibitor and can be explained by a recently proposed dynamic multi-state model for globular proteins. Since the fundamental structural properties of the native inhibitor and full inhibitory activity are preserved after resynthesis, the [1-13C]lys-15-labelled inhibitor with the peptide bond Arg-39--Ala-40 cleaved and Arg-39 removed should be suitable for 13C NMR studies of mechanistic aspects of proteinase-inhibitor interactions.     

Calix[8]arene-mediated self-assembly of tetrapeptide H-Leu-Leu-Ile-Leu-OMe

Conformational analysis of peptide 1, H-Leu-Leu-Ile-Leu-OMe on complexing with macro cycle calix[8]arene has been carried out using (1)H-NMR and FTIR spectroscopic techniques. Stoichiometry of the complex formed in the 1:8 ratio was evidenced by a Job plot. NMR studies of the above peptide show a marked downfield shift and an increase in (3)J values for NH resonances on complexing with calix[8]arene. The characteristic NOE connectivity between N(i+1)H and C(ialpha)H confirm beta-sheet conformation in the complexed state. Both (1)H-NMR and FTIR results indicate that the alpha-amino group of Leu I is proximal to the macrocycle and is involved in hydrogen bond formation with phenolic hydrogen atom of the calix[8]arene. This suggests that calix[8]arene provides a suitable platform for peptide 1 to self-assemble in a parallel beta-sheet conformation. The nature of calix[8]arene interaction with peptide 1 has been studied using dynamic NMR studies, which concludes that a bifurcated hydrogen bonding interaction exists in the molecular interfaces of the assembly.     

Conformational Studies of a Melittin—Inhibitor Complex

The conformation of a melittin—inhibitor complex was studied by solution NMR, solid-state NMR, and circular dichroism. In solution, binding was studied by titrating inhibitor against melittin in dimethyl sulfoxide, methanol, aqueous buffer, and dodecylphosphocholine micelles. The change in chemical shift of Trp19 resonances and the formation of a precipitate at 1:1 molar ratio indicated that the inhibitor was bound to melittin. Solid-state NMR also showed a change in chemical shift of two labeled carbons of melittin near Pro14 and a change in ¹H T ₁ relaxation times when complexed with inhibitor. Rotational resonance experiments of melittin labeled in the proline region indicated a change in conformation for melittin complexed with inhibitor. This observation was also supported by circular dichroism measurements, indicating a reduction in α-helical structure for increasing ratios of inhibitor bound to melittin.     

Hydrophobic residues of melittin mediate its binding to αA−crystallin

The molecular chaperone αA‐crystallin, mainly localized in the human ocular lens, is believed to protect the lens from opacification and cataract, by suppressing the aggregation of the other lens proteins. The present study provides structural and thermodynamic insights into the ability of human αA‐crystallin (HAA) to bind to its partially unfolded clients in the lens, using a small peptide, melittin from bee venom, as a model client. We characterized the thermodynamic parameters of the binding process between melittin and HAA through isothermal titration calorimetry (ITC), and found the binding to be endothermic and entropy‐driven. We identified the amino acids in melittin important for binding to HAA by saturation‐transfer difference (STD) nuclear magnetic resonance (NMR) experiments, and analysis of NMR line broadening upon titration of melittin with HAA. Our results suggest that hydrophobic residues Ile17 and Ile20 on the C‐terminal region of melittin are in close contact with HAA in the melittin‐HAA complex. Information obtained from NMR experiments was used to generate structural models of the melittin‐HAA complex by molecular docking with high‐ambiguity driven docking (HADDOCK). Structural models of the melittin‐HAA complex reveal important principles underlying the interaction of HAA with its clients.     

Structural and thermodynamic analyses of the interaction between melittin and lipopolysaccharide

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is the very first site of interactions with the antimicrobial peptides. In this work, we have determined a solution conformation of melittin, a well-known membrane active amphiphilic peptide from honey bee venom, by transferred nuclear Overhauser effect (Tr-NOE) spectroscopy in its bound state with lipopolysaccharide. The LPS bound conformation of melittin is characterized by a helical structure restricted only to the C-terminus region (residues A15-R24) of the molecule. Saturation transfer difference (STD) NMR studies reveal that several C-terminal residues of melittin including Trp19 are in close proximity with LPS. Isothermal titration calorimetry (ITC) data demonstrates that melittin binding to LPS or lipid A is an endothermic process. The interaction between melittin and lipid A is further characterized by an equilibrium association constant (Ka) of 2.85 x 10(6) M(-1) and a stoichiometry of 0.80, melittin/lipid A. The estimated free energy of binding (delta G0), -8.8 kcal mol(-1), obtained from ITC experiments correlates well with a partial helical structure of melittin in complex with LPS. Moreover, a synthetic peptide fragment, residues L13-Q26 or mel-C, derived from the C-terminus of melittin has been found to contain comparable outer membrane permeabilizing activity against Escherichia coli cells. Intrinsic tryptophan fluorescence experiments of melittin and mel-C demonstrate very similar emission maxima and quenching in presence of LPS micelles. The Red Edge Excitation Shift (REES) studies of tryptophan residue indicate that both peptides are located in very similar environment in complex with LPS. Collectively, these results suggest that a helical conformation of melittin, at its C-terminus, could be an important element in recognition of LPS in the outer membrane.     

The binding site of sodium in the gramicidin A channel: comparison of molecular dynamics with solid-state NMR data.

The location of the main binding site for sodium in the gramicidin A (GA) channel was investigated with molecular dynamics simulations, using an atomic model of the channel embedded in a fully hydrated dimyristoyl phosphatidycholine (DMPC) bilayer. Twenty-four separate simulations in which a sodium was restrained at different locations along the channel axis were generated. The results are compared with carbonyl 13C chemical shift anisotropy solid-state NMR experimental data previously obtained with oriented GA:DMPC samples. Predictions are made for other solid-state NMR properties that could be observed experimentally. The combined information from experiment and simulation strongly suggests that the main binding sites for sodium are near the channel's mouth, approximately 9.2 A from the center of the dimer channel. The 13C chemical shift anisotropy of Leu10 is the most affected by the presence of a sodium ion in the binding site. In the binding site, the sodium ion is lying off-axis, making contact with two carbonyl oxygens and two single-file water molecules. The main channel ligand is provided by the carbonyl group of the Leu10-Trp11 peptide linkage, which exhibits the largest deviation from the ion-free channel structure. Transient contacts with the carbonyl group of Val8 and Trp15 are also present. The influence of the tryptophan side chains on the channel conductance is examined based on the current information about the binding site.     

Conformational studies of a melittin-inhibitor complex

The conformation of a melittin—inhibitor complex was studied by solution NMR, solid-state NMR, and circular dichroism. In solution, binding was studied by titrating inhibitor against melittin in dimethyl sulfoxide, methanol, aqueous buffer, and dodecylphosphocholine micelles. The change in chemical shift of Trp19 resonances and the formation of a precipitate at 1:1 molar ratio indicated that the inhibitor was bound to melittin. Solid-state NMR also showed a change in chemical shift of two labeled carbons of melittin near Pro14 and a change in ¹H T ₁ relaxation times when complexed with inhibitor. Rotational resonance experiments of melittin labeled in the proline region indicated a change in conformation for melittin complexed with inhibitor. This observation was also supported by circular dichroism measurements, indicating a reduction in -helical structure for increasing ratios of inhibitor bound to melittin.     

Allosteric inhibition of rat neuronal nitric-oxide synthase caused by interference with the binding of calmodulin to the enzyme

A sigmoid-type dependence on the inhibitor concentration was observed in the cytochrome c reductase activity for peptide inhibitors (mastoparan and melittin), calmodulin antagonists (W-7 and tamoxifen) and monobutyltin in a reconstituted system comprised of recombinant rat neuronal nitric-oxide synthase (nNOS) and calmodulin (CaM). The increase in the concentration of CaM in the system induced a decrease in the inhibitory effect, indicating that the inhibitors might interfere with the interaction between nNOS and CaM. The changes in the fluorescence spectra of dansylated CaM caused by the addition of mastoparan, melittin and monobutyltin indicated complex formation between CaM and those compounds, which led to the decrease in the effective concentration of CaM available to nNOS. The sigmoid-type inhibition of mastoparan and melittin fit the theoretical equations quite well, assuming that two CaM molecules bind cooperatively to one nNOS homodimer. Monobutyltin, tamoxifen and W-7 were found to inhibit nNOS activity by binding to the CaM binding site of the nNOS homodimer, in addition to the binding of the inhibitors to calmodulin. These compounds inhibited the L-citrulline formation of nNOS from L-arginine, and the inhibitory effects were abrogated by raising the concentration of calmodulin. It became clear that the binding of calmodulin to nNOS can be interfered with in two ways: (1) via a decrease in the effective concentration of calmodulin caused by complex formation between the inhibitor and calmodulin, and (2) via the inhibition of the binding of calmodulin to nNOS caused by the occupation of the binding site by the inhibitor.     

Conformational and quantitative characterization of oritavancin-peptidoglycan complexes in whole cells of Staphylococcus aureus by in vivo 13C and 15N labeling

Solid-state NMR has been used to examine the cell walls of intact whole cells of Staphyloccus aureus grown on media containing D-[1-(13)C]alanine, [(15)N]glycine, and the alanine racemase inhibitor, alaphosphin. The results of in situ site-selective, four-frequency NMR experiments show directly for the first time that (i) 54% of the cell-wall peptidoglycan stems have D-alanine termini and 46%, D-alanine-D-alanine termini; (ii) the molar ratio of stems ending in D-alanine to esterified alditol repeats of cell-wall teichoic and lipoteichoic acids is 3:2; and (iii) 50% of the mature cell-wall binding sites for a fluorinated oritavancin analogue consist of two nearest-neighbor peptide stems of different glycan strands. The drug is bound to the D-Ala-D-Ala terminus of one stem and is proximate to the bridging pentaglycyl segment that cross-links the two stems. Structural details of the binding site are revealed in a model of the glycopeptide-peptidoglycan interaction produced by molecular dynamics simulations with internuclear distance restraints determined by NMR.     

Identification of oxygen nucleophiles in tetrahedral intermediates: 2H and 18O induced isotope shifts in 13C NMR spectra of pepsin-bound peptide ketone pseudosubstrates

The synthetic ketone peptide analogue of pepstatin, isovaleryl-L-valyl-[3-13C]-(3-oxo-4S)-amino-6-methylheptanoyl-L-al anyl-isoamylamide is a strong inhibitor of aspartyl proteases. When the peptide is added to porcine pepsin in H2O at pH 5.1, the 13C NMR chemical shift of the ketone carbon moves from 208 ppm for the inhibitor in solution to 99.07 ppm when bound to the enzyme active site. In 2H2O the bound shift is 98.71 ppm, 0.36 ppm upfield. For the analogous experiment contrasting H216O and H218O, the 13C chemical shift was 0.05 ppm to higher field for the heavier isotope. These data show that water, and not an enzyme nucleophile, adds to the peptide carbonyl to yield a tetrahedral diol adduct in the enzyme-catalyzed reaction, and provide a method for differentiating between covalent and non-covalent mechanisms.     

Comparative structure-function studies with analogs of dynorphin-(1-13) and [Leu5]enkephalin

Analogs of dynorphin-(1-13) with modifications in the enkephalin segment were compared with correspondingly modified analogs of [Leu5]enkephalin in the guinea pig ileum (GPI) and mouse vas deferens (MVD) assay as well as in mu- and delta-receptor selective binding assays. The obtained results indicate that a) the enkephalin binding domain of the dynorphin (kappa) receptor has structural requirements which are distinct from those of the enkephalin binding site at the mu-receptor and b) the introduction of an identical conformational constraint in [Leu5]enkephalin and in the enkephalin segment of dynorphin-(1-13) produces a superpotent agonist in both cases. Fluorescence energy transfer measurements with the active [4-tryptophan]analogs of dynorphin-(1-13) and [Leu5]enkephalin and with dynorphin-(1-17) demonstrated a more extended conformation of the N-terminal tetrapeptide segment in [Trp4]dynorphin-(1-13) than in [Trp4, Leu5]enkephalin as well as the absence of an interaction between the N- and C-terminal segments of dynorphin-(1-17).     

Nuclear magnetic resonance determination of metal-proton distances in a synthetic calcium binding site of rabbit skeletal troponin C

The binding of gadolinium to a synthetic peptide of 13 amino acid residues representing the calcium binding loop of site 3 of rabbit skeletal troponin C [AcSTnC(103-115)amide] has been studied by using proton nuclear magnetic resonance (1H NMR) spectroscopy. In particular, the proton line broadening and enhanced spin-lattice relaxation have been used to determine proton-metal ion distances for several assigned nuclei in the peptide-metal ion complex. These distances have been used in conjunction with other constraints and a distance algorithm procedure to demonstrate that the structure of the peptide-metal complex as shown by 1H NMR is consistent with the structure of the EF calcium binding loop in the X-ray structure of parvalbumin but that the available 1H NMR distances do not uniquely define the solution structure.     

Helical structure of phospholamban in membrane bilayers.

The regulation of calcium levels across the membrane of the sarcoplasmic reticulum involves the complex interplay of several membrane proteins. Phospholamban is a 52 residue integral membrane protein that is involved in reversibly inhibiting the Ca(2+) pump and regulating the flow of Ca ions across the sarcoplasmic reticulum membrane during muscle contraction and relaxation. The structure of phospholamban is central to its regulatory role. Using homonuclear rotational resonance NMR methods, we show that the internuclear distances between [1-(13)C]Leu7 and [3-(13)C]Ala11 in the cytoplasmic region, between [1-(13)C]Pro21 and [3-(13)C]Ala24 in the juxtamembrane region and between [1-(13)C]Leu42 and [3-(13)C]Cys46 in the transmembrane domain of phospholamban are consistent with alpha-helical secondary structure. Additional heteronuclear rotational-echo double-resonance NMR measurements confirm that the secondary structure is helical in the region of Pro21 and that there are no large conformational changes upon phosphorylation. These results support the model of the phospholamban pentamer as a bundle of five long alpha-helices. The long extended helices provide a mechanism by which the cytoplasmic region of phospholamban interacts with residues in the cytoplasmic domain of the Ca(2+) pump.     

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).