Interactions of {alpha}- and ß-avoparcin with bacterial cell-wall receptor-mimicking peptides studied by electrospray ionization mass spectrometry

Anca van de Kerk-van Hoof and Albert J. R. Heck*

Department of Biomolecular Mass Spectrometry Bijvoet, Center for Biomolecular Research, Department of Chemistry and Department of Pharmacy, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Discussion
 References
 
Solution phase affinity constants of the glycopeptide antibiotic {alpha}- and ß-avoparcin, with a range of bacterial cell-wall receptor-mimicking model peptides, were determined by a relatively new method: affinity electrospray ionization mass spectrometry (ESI-MS). This method is relatively efficient and allows the parallel determination of several affinity constants in mixtures of antibiotics and receptors. The determined binding constants for {alpha}- and ß-avoparcin were compared with those of the related glycopeptide antibiotic vancomycin. The solution phase binding affinities of {alpha}- and ß-avoparcin on one hand, and vancomycin on the other, were found to be in the same order, at least for the range of receptor-mimicking peptides studied. However, ß-avoparcin displayed slightly higher binding affinities than {alpha}-avoparcin, particularly for strong binding receptor-mimicking peptides. The evidence that {alpha}- and ß-avoparcin and vancomycin are structurally similar, combined with the present data revealing their similar affinity for bacterial cell-wall receptor-mimicking peptides, supports the hypothesis that the appearance of vancomycin-resistant enterococci (VRE) might be linked to the widespread use of avoparcin.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Discussion
 References
 
Since many bacteria have become resistant to penicillin and a range of other antibiotics, glycopeptide antibiotics now play an important role against Gram-positive bacterial infections. This clinically important group of antibiotics act by binding to bacterial cell-wall precursors, terminating in the sequence -Lys-D-Ala-D-Ala. This interaction inhibits cross-linking of the growing cell wall, leading to bacterial cell death. Avoparcin is a glycopeptide antibiotic of the vancomycin group and is structurally related to vancomycin (Figure 1).1,2 The major application of avoparcin is as an antibacterial, growth-promoting food additive used primarily in animal feed.3 In Europe it was brought on to the market under the tradename avotan. The recent appearance of vancomycin-resistant enterococci (VRE) has been linked to the widespread use of avoparcin by farmers.4,56 In contrast to Europe, environmental VRE isolates have not been observed in the USA, where these antibiotics have never been licensed.7 As a consequence, the use of avoparcin as an animal food additive has recently been banned by the European Union. VRE are, however, emerging rapidly worldwide, not only due to the use of animal food additives, but also due to the increased clinical use of vancomycin.8,9 Microbes may be able to adapt quite quickly when vancomycin or other glycopeptide antibiotics are introduced into their environment, by modifying their genes and becoming resistant.9 In addition, there is the possibility that the conjugative transposons carrying resistance in one strain of bacteria may be transferred to other bacteria, possibly even human enterococci.9,10 Consequently, there may be a connection between human VRE, animal VRE and the use of farmyard antibiotics. A link between avoparcin and human VRE is supported by the strong similarities between the chemical structures of vancomycin and avoparcin (Figure 1).9,11 Prompted by the recent commotion concerning VRE and the possible role of animal-food antibiotic additives, the present study was performed to evaluate the thermodynamic aspects of the molecular interactions of avoparcin with bacterial cell-wall precursor, compared with vancomycin.



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Figure 1. Chemical structures of (a) vancomycin and (b) {alpha}- and ß-avoparcin.

 
The commercial product used in our studies consists of a mixture of two antibiotics, {alpha}- and ß-avoparcin, which differ only in the substitution of a Cl by an H on one of the aromatic rings (Figure 1b). The MICs of {alpha}-avoparcin and vancomycin for several strains of staphylococci, streptococci and enterococci have been found to be mostly comparable, although {alpha}-avoparcin was generally less potent against staphylococci.12 In addition, an earlier report showed that the MIC values of {alpha}- and ß-avoparcin differ by a factor of approximately two, ß-avoparcin being the more potent antibiotic.2 The recent appearance of VRE infections is in most cases caused by strains of bacteria in which peptidoglycan precursors terminate in D-Ala-D-Lac rather than D-Ala-D-Ala. 13 A different form of natural resistance has been reported, particularly in the case of Enterococcus gallinarum and Enterococcus casseliflavus, where peptidoglycan precursors terminating in D-Ala-D-Ser were found.9,14 Experimental studies have shown that vancomycin binds approximately 10 times more weakly to receptor-mimicking peptides such as N, N'-Ac2-L-Lys-D-Ala-D-Ser, and approximately 1000 times more weakly to N, N'-Ac2-L-Lys-D-Ala-DLac, although the mechanism of binding seems to be similar as for N, N'-Ac2-L-Lys-D-Ala-DAla.15,16 Binding constants of vancomycin and to a lesser extent {alpha}- and ß-avoparcin with several bacterial cell-wall receptor-mimicking precursor peptides have been measured by various methods, such as 1H nuclear magnetic resonance (NMR), capillary electrophoresis, UV difference spectroscopy and microcalorimetry.17,18,19,20 Some of these previously reported data are summarized in the last columns of Tables I and II.


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Table I. Affiny constants for {alpha}-avoparcin and ß-avoparcin determined by ESI-MS
 

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Table II. Affinity constants for vancomycin determined by ESI-MS
 
In recent years, relatively ‘soft’ ionization methods have been developed in mass spectrometry, which allow the detection of even weakly bound, intact, noncovalent complexes.21,22,23,2425 In particular, nanoflow electrospray ionization mass spectrometry (ESI-MS)26 has great potential in this important biochemical area. With some success, nanospray ionization has even been used to assess quantitatively the binding of protein–protein and protein–ligand interactions.27,28,29,30,31 In this study, nanoflow ESI-MS has been used to measure quantitatively the binding of the antibacterial food additives {alpha}- and ß-avoparcin to peptidoglycan receptor-mimicking peptides.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Discussion
 References
 
Electrospray ionization mass spectra were recorded on a Finnigan/Thermoquest LC-Q (San Jose, CA, USA) ion trap mass spectrometer, operating the mass spectrometer in positive ion mode. All samples were introduced using a nanoflow electrospray source (Protana, Odense, Denmark). Solutions of antibiotic and receptor peptides at concentrations of typically 15–150 µM (depending on the strength of noncovalent binding) were made up in aqueous 5 mM ammonium acetate, pH 5.1 (acidified using acetic acid), 298 K. Approximately 2 µL of these solutions were introduced into the electrospray needles. The applied electrospray voltage between the needle and the capillary was typically 500 V. The capillary temperature was 383 K. The offset between the capillary voltage and the tube–lens voltage was usually <5 V. For quantitative measurements, ion intensities were measured in the ‘selected-ion-monitoring’ mode, whereby only small mass (m/z) windows are monitored. Peak intensities were obtained by integrating over the whole isotope envelope of the ions. The general procedures for obtaining binding constants from the nanoflow electrospray mass spectra have been reported previously.30 In short, the equilibrium concentrations of the antibiotics, [{alpha}_Avop]eq. and [ß_Avop]eq. can be derived from the peak intensities in the mass spectra using the following equations (square brackets denote concentrations):

where {alpha}_Avop and {alpha}_AvopL are the integrated peak intensities of the antibiotic and its complex with the ligand, respectively. [{alpha}_Avop]0 is the initial concentration of the antibiotic. The concentration of the complex between the antibiotic and the ligand is

The concentration of the other antibiotic ( [ß_Avop]eq.) and its complex with the ligand ( [ß_AvopL]eq.) can be derived in an analogous manner. For the concentration of unbound ligand, the relation

holds, where [Ligand]0 is the total initial concentration of the ligand. The binding constant of the antibiotic to the ligand can now simply be calculated from the equation

Similar equations can be used and derived for mixtures of two antibiotics and several peptides. This mass spectrometric method is based on the assumption that the ionization probability of the free antibiotics is identical to the ionization probability of the antibiotic–ligand complexes. This assumption was validated by performing quantitative measurements at different concentrations of antibiotic(s) and ligand(s).30

The commercially available antibiotic avoparcin (Wyeth-Ayerst Research, Lederle Laboratories, NY, USA) used in the present study consists of a 1:2 mixture of two products, {alpha}- and ß-avoparcin, which differ by the substitution of a Cl for an H on one of the aromatic ring side chains (Figure 1b). The 1:2 ratio was confirmed by capillary electrophoresis analysis using UV detection.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Discussion
 References
 
The ionization probablities, using ESI, for both {alpha}- and ß-avoparcin were found to be identical. {alpha}- and ß-avoparcin were detected, in a 1:2 ratio, almost exclusively as doubly protonated ions. Figure 2 shows a nanoflow ESI mass spectrum of a 15 µM 1:2 mixture of {alpha}- and ß-avoparcin, to which 30 µM of the receptor-mimicking peptide N-Ac-D-Ala-D-Ala-D-Ala was added. The signals at m/z values of approximately 955 and 973 Th originate from the doubly protonated {alpha}- and ß-avoparcin, respectively. The ion signals observed at 1093 and 1111 Th originate from the doubly protonated noncovalent complexes of N-Ac-D-Ala-D-Ala-D-Ala with protonated {alpha}- and ß>-avoparcin, respectively. Besides these signals the spectrum is quite clean. At the start of the experiment the concentrations of {alpha}-avoparcin, ß-avoparcin and N-Ac-D-Ala-D-Ala-D-Ala were 5, 10 and 30 µM, respectively. Using the procedures outlined in the Materials and methods, it may be calculated that according to the measured mass spectrum in Figure 2 the equilibrium concentrations in solution were 2.35, 4.12 and 21.47 µM for {alpha}-avoparcin, ß-avoparcin and N-Ac-D-Ala-D-Ala-D-Ala, respectively. The noncovalent complexes of the antibiotic with the peptides had concentrations of 2.65 and 5.88 µM for the {alpha}- and ß-forms. Binding constants of 53 000/M and 66 000/M for {alpha}- and ß-avoparcin, respectively, were determined from this particular spectrum. Table I summarizes the binding constants obtained for a wide range of precursor peptides. The binding constants are averaged over several measurements using typically different mixtures of receptor-mimicking peptides. The absolute error in the binding constants is approximately 30%. The error in relative binding constants is expected to be much smaller, i.e. within 10%.



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Figure 2. Nanoflow ESI mass spectrum of a 5 mM aqueous ammonium acetate solution containing a 15 µM 1:2 mixture of {alpha}- and ß-avoparcin and 30 µM of the receptor-mimicking peptide N-Ac-D-Ala-D-Ala-DAla.

 
Figure 3 shows selected ion monitoring mode ESI mass spectra obtained from such a solution containing a ‘complex’ mixture of {alpha}- and ß-avoparcin (5 and 10 µM, respectively) and two equimolar (50 µM) receptor-mimicking peptides, i.e. N, N'-Ac2-L-Lys-D-Ala-D-Ser and N-Ac-D-Ala-D-Ala. -L-Lys-D-Ala-D-Ser termini have been observed in the peptidoglycan structure of several VRE. The relative ion signal intensities observed in this spectrum indicate immediately that the affinities of {alpha}- and ß-avoparcin for N-Ac-D-Ala-D-Ala are similar, whereas ß-avoparcin reveals a relatively higher affinity for N, N'-Ac2-L-Lys-D-Ala-D-Ser than {alpha}-avoparcin. Additionally, the binding affinity of {alpha}-avoparcin for N-Ac-D-Ala-D-Ala is approximately equal to the affinity for N, N'-Ac2-L-Lys-D-Ala-D-Ser. Therefore, from this single measurement, which takes, experimentally, only seconds, one can derive several (in this case, four) binding constants simultaneously, revealing one of the strengths of this mass spectrometrical approach. Binding constants for {alpha}-avoparcin, ß-avoparcin and vancomycin, all determined by ESI-MS, are summarized in Tables I and II. The values determined by ESI-MS for vancomycin were reported previously,30 except for N, N'-Ac2-L-Lys-D-Ala-D-Ser. For comparison, several previously reported binding constants, obtained by alternative, direct solution-phase methods are also given.



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Figure 3. Selected ion monitoring mode nanoflow ESI mass spectra of a 5 mM aqueous ammonium acetate solution containing a 15 µM 1:2 mixture of {alpha}- and ß-avoparcin and two equimolar (50µM ) receptor-mimicking peptides, N,N'-Ac2-L-Lys-D-Ala-D-Ser and N-Ac-D-Ala-D-Ala.

 
Most antibiotics of the vancomycin group self-associate and these dimers may be important in promoting antibiotic activity.32,33 For a range of antibiotics, a large variation in the dimerization constants has been observed. A mixture of two glycopeptide antibiotics might form hetero-dimers. In this study, the self-association constants of {alpha}- and ß-avoparcin have been assessed in a similar manner to that described previously for a range of related antibiotics.30 The determined dimerization constants were all rather low, around 2000/M, for the {alpha}{alpha}-, ßß-homo-dimers and the {alpha}ß-avoparcin hetero-dimer. As the dimer ion signals were quite small in the mass spectra measured, the error in these self-association constants is approximately 50%. Reported dimerization constants of vancomycin range from 50 to 4000/M.34,35,36 The tendency for self-association of avoparcin was found to be rather poor, but comparable to that of vancomycin.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Discussion
 References
 
The main aim of this study was to investigate whether the structurally related antibiotics of {alpha}-avoparcin, ß-avoparcin and vancomycin have (dis)similar affinities for peptides that resemble the receptor sites of bacterial peptidoglycans. While an extensive amount of affinity data was already available for vancomycin, few figures had been reported for avoparcin. The data presented in this study reveal that {alpha}- and ß-avoparcin display similar affinities to vancomycin for the whole range of investigated receptor-mimicking peptides. In common with vancomycin, {alpha}- and ß-avoparcin exhibit strongly reduced affinities for N, N'-Ac2-L-Lys-D-Ala-D-Lac, and to a lesser extent for N, N'-Ac2-L-Lys-D-Ala-D-Ser, two peptides that mimick the peptidoglycan receptors of VRE.

Subtle differences in molecular recognition could, however, be observed. For instance, vancomycin displays a higher affinity for Ac-D-Ala-D-Ala than for Ac-Gly-D-Ala by a factor of two, which is reflected by their binding constants of 19,000 and 11,000/M (see Table II). This difference in affinity is markedly enhanced in the case of {alpha}- and ß-avoparcin. For example, the binding constants for Ac-D-Ala-D-Ala and Ac-Gly-D-Ala differ by a factor of 10, i.e. 15,000 and 2000/M, respectively (see Table I). The effect that the methyl (Ala) to hydrogen (Gly) substitution has on the noncovalent association is remarkable, maybe even more so than the fact that this effect of <2 kJ/mol in binding energy can be monitored quantitatively by ESI-MS. Differences in affinity were also observed when {alpha}- and ß-avoparcin were compared. ß-avoparcin exhibited affinities up to twice as high as those of {alpha}-avoparcin, especially for strong binding, receptor-mimicking peptides. The tendency to self-associate is poor for {alpha}-avoparcin, ß-avoparcin and vancomycin.

In general, ESI-MS was observed to be a reliable method for screening the binding affinities of several glycopeptide antibiotics with a range of receptor-mimicking peptides.


    Acknowledgments
 
We would like to acknowledge the following people for generous gifts of antibiotics and receptor-mimicking peptides: Dr Marshall Siegel (Wyett-Aherst, Pearl River, USA) for {alpha}-avoparcin, ß-avoparcin and vancomycin, Dr Thomas Staroske and Professor Dudley H. Williams (Cambridge University, UK) for Ac2-L-Lys-D-Ala-D-Ser, and Dr Thomas J. D. Jørgensen and Professor Peter Roepstorff (Odense University, Denmark) for Ac-Gly-D-Ala, Ac2-L-Lys-L-Ala-L-Ala and Ac-D-Ala-D-Ala-D-Ala.


    Notes
 
* Corresponding author. Fax: +31-30-251-8219; E-mail: a.j.r.heck{at}chem.uu.ul Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Discussion
 References
 
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Received 22 February 1999; returned 17 May 1999; revised 2 June 1999; accepted 11 June 1999





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