Vancomycin: ligand recognition, dimerization ... - Wiley Online Library

Vancomycin: ligand recognition, dimerization and super-complex formation ZhiGuang Jia1, Megan L. O’Mara2, Johannes Zuegg2, Matthew A. Cooper2 and Alan E. Mark1,2 1 School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Qld, Australia 2 Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld, Australia

Keywords antibiotic resistance; computer simulation; glycopeptide antibiotics; lipid II; molecular dynamics Correspondence A. E. Mark, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Qld 4072, Australia Fax: +61 7 3365 3872 Tel: +61 7 3365 4180 E-mail: [email protected] (Received 1 November 2012, revised 19 December 2012, accepted 21 December 2012) doi:10.1111/febs.12121

The antibiotic vancomycin targets lipid II, blocking cell wall synthesis in Gram-positive bacteria. Despite extensive study, questions remain regarding how it recognizes its primary ligand and what is the most biologically relevant form of vancomycin. In this study, molecular dynamics simulation techniques have been used to examine the process of ligand binding and dimerization of vancomycin. Starting from one or more vancomycin monomers in solution, together with different peptide ligands derived from lipid II, the simulations predict the structures of the ligated monomeric and dimeric complexes to within 0.1 nm rmsd of the structures determined experimentally. The simulations reproduce the conformation transitions observed by NMR and suggest that proposed differences between the crystal structure and the solution structure are an artifact of the way the NMR data has been interpreted in terms of a structural model. The spontaneous formation of both back-to-back and face-to-face dimers was observed in the simulations. This has allowed a detailed analysis of the origin of the cooperatively between ligand binding and dimerization and suggests that the formation of face-to-face dimers could be functionally significant. The work also highlights the possible role of structural water in stabilizing the vancomycin ligand complex and its role in the manifestation of vancomycin resistance.

Introduction Vancomycin was once the antibiotic of last resort against Gram-positive bacteria. However, in recent years, the utility of vancomycin has decreased because of the increasing prevalence of resistance [1,2]. To overcome this, a detailed understanding of the interaction between vancomycin and its various derivatives with both antibiotic-sensitive and -resistant variants of lipid II is needed. Vancomycin is a glycosylated heptapeptide: N-methyl-D-leucine (NLEU)–m-chloro-bhydroxy-D-tyrosine (HDPY)–asparagine (ASN)–p-(2-[a4-L-epi-vancosaminyl]-b-1-D-glucosyl)-D-phenylglycine (DPYG)–p-hydroxy-D-phenylglycine (DHPG)–HDPY–

m,m-dihydroxy-L-phenylglycine (LPGH). A disaccharide composed of vancosamine and glucose is linked to the para-position of the phenyl group of DPYG4 (Fig. 1). Covalent cross-links between the side chains of HDPY2 and DPYG4, DPYG4 and HDPY6, and DPHG5 and LPGH7 results in a tricyclic structure [3] with limited flexibility [3–5]. Vancomycin has six titratable groups, but only the protonation state of the amine group of vancosamine (pKa = 7.75 at 298 K) is uncertain under physiological conditions [6]. Vancomycin targets lipid II, blocking a key step in the synthesis of the bacterial cell wall. Lipid II (Fig. 2)

Abbreviations ASN, asparagine; DHPG, p-hydroxy-D-phenylglycine; DMSO, dimethylsulfoxide; DPYG, p-(2-[a-4-L-epi-vancosaminyl]-b-1-D-glucosyl)-Dpheny1glycine; HDPY, m-chloro-b-hydroxy-D-tyrosine; LPGH, m,m-dihydroxy-L-phenylglycine; NLEU, N-methyl-D-leucine; NOE, nuclear Overhauser effect.

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Simulation study of vancomycin

HO pKa = 7.8 +H N 3

HO

O

D-NAc-Glu

OH

HO HO

O O

O

O

O

NH

O

Cl

O O P O P O O O OO

OH

(NAG)

OH

O O

A

OH

HN

O

O

O

NAc-Mur (NAM)

NH

OH Cl O H O H H 2 4 3 N 6 N N NH N O 5 H 1 NH2+ O O pKa = 2.2 NH O pKa = 8.9 7 O O NH2 pKa = 12.0 O OH HO OH pKa = 10.4 pK = 9.6 O Oa H N N O H O HO

O

-

NH O

O

B

O

NH O

C

HN +

H3N

NH

O

O O-

Fig. 1. The chemical structure of vancomycin showing pKa values for titratable groups determined experimentally. The seven amino acid residues in vancomycin are numbered 1–7 from the N-terminal to C-terminal. Titratable groups are colored blue. The dashed lines indicate hydrogen bonds between vancomycin and the ligand acetyl-D-Ala-D-Ala which is also shown. The thick solid arrow indicates a charge–charge interaction between the N-terminus of vancomycin and the C-terminus of the ligand.

D O

Lipid II

NH O HN

O N H

O

NH O -

O

is comprised of the glycopeptide N-acetyl-muramyl-Nacetyl-glucosaminyl-L-Ala-c-D-Glu-L-Lys-D-Ala-D-Ala attached to a C55 hydrophobic tail via a pyrophosphate linker [7]. After synthesis, lipid II is translocated to the periplasmic side of the plasma membrane where the peptidoglycan group is cleaved from the lipid tail and incorporated into the nascent cell wall [8,9]. Vancomycin primarily interacts with the peptidic region of lipid II and the interaction between vancomycin and a range of lipid II peptidoglycan analogs has been studied using a variety of techniques. Lipid II is insoluble in water and thus most investigations of the recognition of lipid II by vancomycin have involved either chemically modified or truncated fragments of lipid II [10]. NMR and X-ray crystallographic studies [11–17] showed that the ligand bound to the concave (front face) of vancomycin, the C-terminal carboxylic acid group of D-Ala-D-Ala interacting with the backbone amide groups of resides NLEU1, HDPY2 and ASN3. In addition, the amide group of Ala1 forms a stable hydrogen bond with the carbonyl oxygen of DPYG4. This in part explained why resistance to vancomycin is primarily associated with changes within the terminal D-Ala-D-Ala motif of lipid II. In most resistant strains, the terminal Ala is mutated to D-Lac (the amide group of the Ala replaced by oxygen) [2,18]. This results in the loss of

Fig. 2. The chemical structures of alternative vancomycin ligands. (A) Lipid II (undecaprenyl-pyrophosphoryl-N-acetyl-muramyl(N-acetylglucosaminyl)-L-alanyl-c-D-glutamyl-L-lysyl-D-alanyl-D-alanine), (B) acetylLys-D-Ala-D-Ala, (C) acetyl-D-Ala-D-Ala and (D) Ac2-Lys-D-Ala-D-Ala.

the hydrogen bond involving the Ala residue and a 1000-fold drop in binding affinity [19]. In addition to binding lipid II, vancomycin is known to readily self-associate in solution, it has been proposed that the ability to self-associate plays a critical role in biological activity. X-Ray crystallographic [11,12,15–17] and NMR studies [20] suggest that vancomycin preferentially forms a back-to-back dimer. As illustrated in Fig. 3, the back-to-back dimer is stabilized by four backbone–backbone hydrogen bonds. The dimer has only pseudo-C2 symmetry because orientation of the sugar group on DPYG4 varies between the monomers. At 298 K and physiological pH, the dimerization constant of unligated vancomycin is in the low millimolar range [21–23]. In the presence of ligand, the extent of the association increases by one to two orders of magnitude [23]. In addition to back-to-back dimers, crystallographic studies suggest that vancomycin also forms stable face-to-face and


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Fig. 3. Structure of the vancomycin backbone residues in a backto-back dimer (PDB entry 1FVM). The vancomycin backbone is displayed in a licorice representation. Hydrogen bonds are presented as red dotted lines. The four hydrogen bonds are indicated by the numbers 1–4. There is a twofold symmetry between the backbones of the two vancomycin molecules. This symmetry is broken by the disaccharide in the side chain of DPYG (residue 4) of vancomycin, which, are not shown.

side-to-side interactions [12,17]. In the face-to-face dimer, the N- and C-termini of vancomycin are in close proximity, with potentially two ligand molecules sandwiched within the interface. Side-to-side interactions between back-to-back and face-to-face dimers can potentially lead to the formation of a hexameric cluster as shown in Fig. 4. Although there is little direct experimental evidence to support the formation of a stable face-to-face dimer in solution, size-exclusion chromatography does indicate the presence of species with molecular masses of between 7000 and 10 000 a.m.u., consistent with the presence of hexamers [17]. The size of vancomycin, the fact that it rapidly and reversibly self-associates, and the extent of association being ligand dependent make it difficult to characterize in detail experimentally either how vancomycin binds ligand in aqueous solution, or how the binding of ligand influences self-association. For example, NMR studies [4,5,24] suggest that there are small but significant differences between the structure found in the crystal and that in solution; in particular in the orientation of HDPY2 and ASN3, potentially affecting both ligand binding and dimerization. Furthermore, although it is clear that vancomycin primarily forms back-to-back dimers in solutions, evidence suggests 1296

Fig. 4. A higher order assembly of vancomycin cluster (PDB entry 1FVM) containing three pairs of back-to-back dimers. Vancomycin molecules are represented as licorice backbones with a transparent surface and colored by dimer (green, pink or yellow). The ligand molecules (Ac2-Lys-D–Ala-D-Ala) are shown in CPK representation. There are ligand-mediated face-to-face interactions between the pink and yellow dimers, and side-to-side interactions at the contact interface between the green and pink dimers and the green and yellow dimers.

that face-to-face dimer and higher order oligomers may be functionally important. Because the ability to probe the system experimentally is limited one must turn to theoretical approaches, in particular molecular dynamics simulation techniques, in order to characterize the process of ligand binding in detail. For example, Jusuf et al. [25] used simulation techniques to estimate the change in the configurational entropy of vancomycin on binding ligand. Lee et al. [26] used density functional theory and an implicit solvation model to estimate the energy of interaction between vancomycin and various ligands and Yang et al. [27] used a combination of mass spectrometry, quantum mechanics and molecular dynamics simulations in a vacuum to study the stability of vancomycin in complex with Ac2-L-Lys-D-Ala-D-Ala. The utility of these previous studies is limited, however, as they have either involved relatively short simulations (~ 1 ns) or been performed under conditions where critical contributions to the binding free energy are potentially ignored such as in a vacuum or using an implicit solvent model. Recently, Jia et al. [28] have used molecular dynamics simulations and free energy calculations to examine the interaction of vancomycin with lipid II in a membrane environment. The work showed that the recognition of lipid II by vancomycin occurred via the N-terminal amine group of vancomycin and the C-terminal carboxyl group of lipid II. The interaction


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of vancomycin with lipid II at the membrane–water interface was found to be essentially identical to that of soluble tripeptide analogs of lipid II. The work also suggested that the relative binding affinity of vancomycin for native, resistant and synthetic forms of membrane-bound lipid II was unaffected by the membrane environment. In this study, we build on our earlier work and examine in detail the mechanism by which two specific lipid II analogs, Ac-D-Ala-D-Ala and Ac2-Lys-D-Ala-DAla, are recognized by vancomycin. In particular how the binding of ligand promotes dimerization and potentially stabilizes higher order complexes is investigated. The simulations reproduce the structures of both the monomeric and dimeric complexes accurately and suggest that differences between the crystal- and NMR-derived structures primarily reflect the effects of motional averaging. We show that changes in the N-terminal region on binding to ligand can account for the fact that ligand binding and dimerization are cooperative and how the presence of certain ligands can promote the formation of face-to-face dimers and stabilize higher order complexes.

Results Parameter validation As an initial validation of the parameters used to describe vancomycin, vancomycin–acetate was simulated in a crystalline environment with anisotropic pressure coupling (Tables 1–3). The starting configuration was taken from PDB entry 1AA5. 1AA5 was solved at 98 K at a resolution of 0.089 nm. A complete unit cell consisting of eight vancomycin dimers arranged in P43212 symmetry, eight acetate molecules, 32 chloride ions, 24 sodium ions and 833 water molecules was simulated for 10 ns. Because the pH at which vancomycin was crystalized was 4.6, the vancosamine group was assumed to be protonated. In general the agreement between the crystal structure and the simulations was excellent. The average RMSD of the eight copies of vancomycin dimer with respect to the initial structure was 0.12 0.03 nm. The average box volume in the simulation was 52.79 0.22 nm3, which was within 0.1% of the unit cell in the crystal (53.30 nm3). There was a close correspondence between the average of the root mean square fluctuations observed in the simulations and the crystallographic B-factors (see Fig. S1). The average root mean square fluctuations considering all heavy atoms in each of the two vancomycin molecules in the asymmetric unit were 0.05 0.03 and 0.04 0.02 nm, respectively.


These can be compared with values of 0.04 0.01 and 0.05 0.01 nm estimated from the corresponding Bfactors. There was, however, a very slight distortion of the overall box. The crystal has P43212 symmetry, implying that each of the cell angles (a, b and c) was 90°. When averaged over the full 10 ns simulated, these angles were a = 90.3 0.6°, b = 90.9 1.0° and c = 88.6 1.2°. In addition, although the overall volume of the box was essentially unchanged there were small differences in the box dimensions associated with the change in the box shape. The final dimensions of the box after 10 ns were a = 2.78, b = 2.77 and c = 6.96 nm compared with a = b = 2.84 and c = 6.58 nm in the crystal. Note that a slight expansion in the box is expected because the simulations were performed at 298 K. To validate the parameters further, a series of simulations of an unligated vancomycin monomer and dimer in aqueous solution were performed. The starting configurations for these simulations were taken from the PDB entry 1AA5. The average distances observed during the simulations were calculated using 1/3 or 1/6 averaging between pairs of hydrogens in the peptide core of vancomycin (without the disaccharide) observed experimentally in NOESYNMR experiments of monomeric vancomycin in dimethylsulfoxide (DMSO) at 298 K [5]. The distances obtained from the simulations are shown in Table S1 together with the upper bound distances inferred from the nuclear Overhauser effect (NOE) intensities and the distances between the same pairs of hydrogens (Fig. S2) in the 1AA5 crystal structure constructed assuming ideal geometries. Note, the NOE data presented in Table S1 correspond to measurements in DMSO rather than in water. To our knowledge, no comparable NOE data for either monomeric or dimeric vancomycin is available in water. In general, there is excellent agreement between the simulations and the available NOE data. The structure of vancomycin in solution is almost identical to that in the crystal. Table 4 shows the number of distances within a given range (violations) between those distances obtained during the simulations, those inferred from the experimental NOE data and those obtained from the crystal structure. As indicated in Table 4, of the 61 pairs of hydrogens for which NOEs could be observed experimentally in DMSO, the distance observed in the crystal is within 0.05 nm of the distance inferred from the NOE intensities in 46 cases. In only five cases did the distance differ by > 0.1 nm. In all cases the distance inferred from the NMR spectra is lower than that observed in the crystal. Although this might be due to the molecule becoming more compact in


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Table 1. A summary of the simulations performed to validate the vancomycin parameters.

Description Series ID Vancosamine protonation state Ligand No. of simulations Initial configuration Time (ns) Initial box size (nm) Temperature (K)

Simulation of vancomycin crystal

Simulation of vancomycin monomer without ligand

Simulation of vancomycin dimer without ligand

Simulation of vancomycin dimer with ligand

Series 1 Protonated

Series 2 Deprotonated



Acetate 2 (a, b) Back-to-back dimer 10 2.881 9 2.881 9 6.613 298

No ligand 1 (a) Monomer

No ligand 2 (a, b) Back-to-back dimer

Ac2-Lys-D-Ala-D-Ala 2 (a, b) Back-to-back dimer

100 4.5 9 4.5 9 4.5

40 4.5 9 4.5 9 4.5

40 4.5 9 4.5 9 4.5

310

310

310

Table 2. A summary of the simulations performed to investigate the spontaneous binding of vancomycin to ligand and the formation of vancomycin dimers. Description

Ligand reorganization

Series ID Vancosamine protonation state Ligand

Series 6 Protonated

Series 7 Protonated



Series 10 Protonated



Ac-D-Ala D-Ala 2 (a, b) Separated 100 59595 310

Ac2-Lys-DAla-D-Ala 2 (a, b) Separated 100 59595 310

Ac-D-Ala -D-Ala

Ac2-LysD-Ala-D-Ala 2 (a, b) Separated 100 59595 310

No ligand

Ac-D-Ala-D-Ala

4 (a, b, c, d ) Separated 200 59595 310

4 (a, b, c, d ) Separated 200 59595 310

Ac-Lys-D-AlaD-Ala 4 (a, b, c, d ) Separated 200 59595 310

No. of simulations Initial configuration Time (ns) Initial box size (nm) Temperature (K)

Dimer formation

2 (a, b) Separated 100 59595 310

Table 3. A summary of the simulations performed to investigate the ligand-mediated face-to-face dimer and stability of larger vancomycin clusters.

Description

Simulation of ligand-mediated face-to-face dimer

Simulation of vancomycin cluster

Series ID Vancosamine protonation state Ligand No. of simulations Initial configuration Time (ns) Initial box size (nm) Temperature (K)

Series 13 Deprotonated Ac2-Lys-D-Ala-D-Ala 4 (a, b, c, d ) Face-to-face dimer 200 59595 310

Series 15 Deprotonated Ac2-Lys-D-Ala-D-Ala 3 (a, b, c ) Cluster 200 79797 310

Series 14 Deprotonated Ac-D-Ala-D-Ala 4 (a, b, c, d ) Face-to-face dimer 200 59595 310

DMSO or the effect of dimerization, it is most likely simply an artifact owing to structural fluctuations in solution, in combination with the fact that the NOE intensity is nonlinearly dependent on the distance. In the simulation of the monomer in solution, the average distance between 51 of the 61 pairs considered was within 0.05 nm of the distance observed in the crystal. 1298

Series 16 Deprotonated Ac-D-Ala-D-Ala 3 (a, b, c ) Cluster 200 79797 310

In only one case did the distance differ by > 0.1 nm. In the simulations of the dimer, the average distance in 54 of the 61 cases is within 0.05 nm of the distance in the crystal. None deviate by > 0.1 nm. Using 1/6 averaging during the simulations led to relatively poor agreement with the distances extracted from the crystal. Likewise simply averaging the dis-


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Table 4. A summary of the differences between the distances, r, extracted from the simulations, inferred from the experimental NOE measurements or obtained from the crystal structures. Number of violations (nm) Data set

Reference

0.05

> 0.05 0.1

> 0.1

NOEa monomerb dimer 1/6 monomerb 1/6 dimer monomer dimer 1/6 monomer 1/6 dimer

Crystalc Crystal Crystal Crystal Crystal NOE NOE NOE NOE

46 51 54 41 41 47 44 50 50

10 9 7 8 8 7 10 6 5

5 1 0 2 2 7 7 5 6

NOE data of monomeric vancomycin in DMSO at 298 K [5]. b Formula used to average the distances from the simulations of either the monomer or dimer. c Crystal structure PDB entry 1AA5 [11].

DPYG4. That, in solution, ASN3 can rotate into the binding pocket is supported by the fact that NOEs between the amide hydrogen of ASN3 and the a hydrogen of HDPY2 as well as between the hydrogen atoms on the side chain of ASN3 and the aromatic hydrogen of HDPY2 and DPYG4 are observed experimentally [4,20]. The rotation of ASN2 into the binding pocket was also observed to occur in the simulation of the dimer. Although this conformation appeared to be less stable in the case of the dimer, it was not possible to draw a definitive conclusion given the limited sampling. Ligand binding

a

tances during the simulations led to poor agreement between the simulations and the available NOE data for both the monomer and the dimer. However, using 1/6 averaging for the simulation of the monomer in solution, the distance is within 0.05 nm of the NOE distances for 50 of the 61 pairs, whereas five deviate by > 0.1 nm. For simulation of the dimer in solution, the distance between 50 of the 61 pairs obtained using 1/6 averaging is within 0.05 nm of the NOE distance, whereas six deviate by > 0.1 nm. Thus, using 1/6 averaging, the simulations are in better agreement with the distances inferred from the NOE experiments in DMSO than the distances observed in the crystal structure. The results obtained using 1/3 averaging were intermediate between linear and 1/6 averaging. This suggests that the very short distances inferred from the NMR experiments result from a combination of thermal motion and the nonlinear dependence of the NOE on distance. They do not necessarily reflect a difference between the dominant structure in solution and the crystal. For the monomer in solution, the largest deviations in the interatomic distances with respect to that in the crystal structure occur for atoms pairs in the vicinity of the side chain of ASN3, between HDPY2 and the terminal methyl and pairs involving the ring of DPHG5. For the dimer, the largest deviations with respect to the crystal structure occur for pairs involving atoms from HDPY2 and for pairs involving atoms on the ring of DPHG5. The largest structural deviations in the monomer are associated with the rotation of ASN3 into the binding pocket and the formation of a hydrogen bond between the carboxyl oxygen in the side chain of ASN3 and the amide hydrogen of

To examine the mechanism by which vancomycin recognizes a ligand, a series of eight simulations were performed. Each 100 ns simulation consisted of a single vancomycin molecule in a box of water containing either Ac-D-Ala-D-Ala or Ac2-Lys-D-Ala-DAla. Four simulations were performed for each ligand, two in which the vancosamine group was protonated, and two in which the vancosamine group was deprotonated. In seven of the eight simulations, a complex containing all five hydrogen bonds between the ligand and vancomycin formed after 11, 12, 22, 35, 47, 48 and 90 ns respectively. In each case, this complex remained stable for the remainder of the simulation. The complexes that formed were essentially identical to those observed crystallographically. The heavy atom rmsd with respect to the corresponding crystal structure averaged over all frames after the last hydrogen bond had formed was 0.11 nm in the case of Ac-D-Ala-D-Ala and 0.15 nm in the case of Ac2-Lys-D-Ala-D-Ala. The higher average rmsd in the case of Ac2-Lys-D-Ala-D-Ala compared with Ac-D-Ala-D-Ala was due to the conformation of the side chain of Lys. In the crystal 1FVM the side chain of lysine adopts two different conformations, one projecting away from the C-terminus of vancomycin and one in which it lies in the region between LPGH7 and the disaccharide group of vancomycin. Likewise in the simulations, the side chain of Lys either projected out of the binding pocket and was fully exposed to solvent or folded back toward the front face of vancomycin in the region between the LPGH7 and the disaccharide group of vancomycin. The average rmsd was calculated, however, only with respect to the first configuration in 1FVM in which the side chain of lysine projects away from the binding pocket. The binding of ligand was a two-step process. First, an initial complex formed in which the -NH2MeLeu+


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group of vancomycin interacted directly with the carboxyl group of the terminal D-Ala of the ligand, indicated by the thick solid arrow in Fig. 1. Although other encounter complexes did occur, the formation of the -NH2MeLeu+–carboxyl interaction was required for the ligand to enter the binding pocket. Note that experimentally the neutralization of the terminal amine group (e.g. by mutation to NCO) results in a significant loss of affinity [29,30]. Second, the ligand rotated toward the front face of the vancomycin forming the five intermolecular hydrogen bonds. The time taken for the ligand to rotate towards the binding pocket varied between 3 and 30 ns. Once the ligand rotated into an appropriate position, all five intermolecular hydrogen bonds (indicated by the thin dashed lines in Fig. 1) formed within 2 ns. In five of the seven simulations, the first three hydrogen bonds involving the C-termini of the ligand and the backbone amide of residues 2, 3 and 4 formed in rapid succession, whereas the final two hydrogen bonds involving the carboxyl group of residue 4 and the backbone amide of residue 7 of vancomyicin formed 0.2–1.6 ns later. In the remaining two simulations, however, the hydrogen bond involving residue 7 formed first. The results of this study are consistent with the results from our previous study of a vancomycin dimer interacting with membrane-bound lipid II [28]. The arrangement of hydrogen bonds between vancomycin and the lipid II analogs examined in this work is identical to that between vancomycin and the D-Ala-D-Ala terminus of membrane-bound lipid II found earlier. [28]. In addition, a similar initial complex was observed in simula-

tions of the binding of vancomycin to membranebound lipid II. Namely, the NH2MeLeu+ group of vancomycin interacted directly with the carboxyl group of the terminal D-Ala in the initial complex [28]. Structural waters The role of water in stabilizing the dimeric vancomycin–ligand complex was also examined. Figure 5 shows a stereoview of the spatial distribution of water surrounding the vancomycin–Ac-D-Ala-D-Ala complex. The spatial distribution of water was calculated from the last 40 ns of simulation b, series 8 (Table 2) in which Ac-D-Ala-D-Ala bound spontaneously to monomeric vancomycin. The mesh in Fig. 5 is contoured at a level of 0.036 nm3, enclosing a volume in which the probability of finding water is at least three times greater than the bulk. Also superimposed on Fig. 5 are the positions of structural waters observed in the crystal structure of the vancomycin–Ac-D-Ala-D-Ala complex (CCDC code 704975). All except one of the waters lying within 0.3 nm of the ligand in each of the six vancomycin–ligand complexes in the asymmetric unit are shown. In one of the vancomycin–ligand complexes in the asymmetric unit, the hydrogen bond between the ligand and the backbone amide of LPGH7 of vancomyicin does not form. Instead a water molecule (O_H2O_41) interacts with LPGH7. Because this configuration is incompatible with the predominant structure, this water is not shown. Two distinct regions of high water density are found in close proximity to the ligand (pink mesh Fig. 5). Two

Fig. 5. A stereoview of the spatial distribution of water molecules surrounding the vancomycin–Ac-D-Ala-D-Ala complex. The spatial distribution of water was averaged over the last 40 ns of simulation b, series 8 (Table 2) and is represented by both the pink and green mesh surfaces contoured at 0.036 atomsnm3. The mesh surfaces enclose regions where the probability of finding a water molecule is three times that of bulk water. The distribution is superimposed onto the final configuration of the simulation with vancomycin shown in licorice representation and the ligand in CPK. For comparison, the positions of the crystallographic water molecules in the vancomycin–Ac-DAla-D-Ala complex (CCDC code 704975) are shown. Here the crystallographic water molecules lying within 0.3 nm of the ligand in any of the six vancomycin–Ac-D-Ala-D-Ala complexes in the asymmetric unit are represented as red spheres.

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small regions of well-defined density lie near the C-terminus of the ligand. One in a cavity formed between the vancosamine–glucose disaccharide, NLEU1 and the aromatic side chain of HDPY2. A second region of high water density lies between NLEU1, ASN3 and the side chains of DPHG5 and LPGH7. The other regions of high water density (green mesh Fig. 5) are located in regions associated with the dimerization interface of vancomycin. In four of the six complexes in the asymmetric unit, a structural water has been placed in or close to the region of high density near the C-termini of the ligands. A number of structural waters have been found in a range of positions to the side of the ligand. Although individual water molecules reside in these two regions of high density only transiently, collectively these water molecules form stable hydrogen bonding networks with both vancomycin and the ligand that persists throughout the simulation. Animations of the trajectories in which these networks of water molecules are clearly visible are provided in the Supporting information. The average residence time for waters interacting with the C-terminus of the ligand that lie in the region of high density between the vancosamine–glucose disaccharide, NLEU1 and the aromatic side chain of HDPY2 was ~ 40 ps. The longest residence time was ~550 ps. These values are approximately twice that observed for waters interacting with the C-termini of the ligand free in solution (~ 20 and ~ 170 ps, respectively) suggesting that the water molecules contribute to the stability of the complex. Dimer formation Experimentally, vancomycin is known to dimerize with an association constant of 2.0 9 102 M1 at pH 5 and 298 K [22]. Dimerization is also believed to play a critical role in the antibiotic activity of vancomycin. To investigate the process of dimer formation, 12 simulations were initiated in which two copies of vancomycin or a vancomycin–ligand complex were placed randomly within the simulation box and allowed to associate spontaneously. Of the 12 simulations, four involved just vancomycin, four involved vancomycin/ Ac-D-Ala-D-Ala and four involved vancomycin/ Ac-Lys-D-Ala-D-Ala. In two cases, both involving the presence of ligand, a stable a back-to-back dimer formed within 200 ns. In one the ligand was Ac-D-Ala-D-Ala, in the other Ac-Lys-D-Ala-D-Ala. Figure 6A shows a plot of the rmsd with respect to the X-ray structure of the vancomycin back-to-back dimer (PDB entry 1FVM) as a function of time for the two cases. The large initial rmsd (~ 1.5 nm) reflects the fact


that the two vancomycin molecules were well separated. The sharp decrease in rmsd at ~ 10 ns corresponds to the formation of an initial encounter complex. After 1 and 20 ns respectively the rmsd decreased to ~ 0.12 nm. At this point the dimeric complex formed spontaneously in solution was essentially identical to that observed crystallographically in both cases. In one of the simulations involving Ac-D-Ala-D-Ala (simulation c, series 11), the two vancomycin molecules formed a ligand-mediated face-to-face dimer. As shown in Fig. 7, the face-to-face dimer had C2-symmetry and was stabilized by four hydrogens. Two involved interactions between the phenyl hydroxyl hydrogen of LPGH7 of one vancomycin with the C-terminal carboxyl oxygen of the ligand bound to the other vancomycin, and two were interactions between the backbone of the two ligands [12,17]. Although NMR data suggest that the back-to-back dimer is the predominate form in solution, the crystal structures suggest that other packing arrangements are also possible. Indeed, although the formation of the faceto-face dimer appeared less cooperative than the formation of the back-to-back dimer (Fig. 6B) the average rmsd between the face-to-face dimer, which formed spontaneously during the simulations, and that observed crystallographically was only 0.12 nm. This suggests the crystallographic face-to-face dimer is at least transiently stable. To examine whether the faceto-face dimer was stable for an extended period in solution a series of eight simulations, four involving vancomycin–Ac-D-Ala-D-Ala and four involving vancomycin–Ac2-Lys-D-Ala-D-Ala were initiated starting from the appropriate crystal structures. In seven of the eight simulations the face-to-face interaction was lost within 200 ns. In one case, the two vancomycin molecules, not only fully separated but, spontaneously reassociated to form a back-to-back dimer within 200 ns. Clearly both the simulations and a range of NMR data suggest that the back-to-back dimer is more stable than the face-to-face dimer in solution. Nevertheless, there has been much debate as to the possible role of face-to-face dimers in the formation of higher order oligomers. For example, there is evidence from size-exclusion chromatography that a hexameric complex may form in solution. To test this possibility a series of six simulations of the asymmetric unit from PDB entry 1FVM and CCDC structure 704975 were performed. In five of the six cases, two involving Ac-D-Ala-D-Ala and three involving Ac2-Lys-D-Ala-DAla, the hexameric cluster was stable throughout the 200 ns simulated with the rmsd with respect to the


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Time (ns) Fig. 6. Root mean-squared positional deviation of all heavy atoms of the vancomycin dimer with respect to the crystallographic dimer (PDB entry 1FVM). (A) The back-to-back dimer formed in series 11 (black) and 12 (red) (Table 2). (B) The face-to-face dimer formed series 11 (Table 2).

initial configuration remaining below 0.3 nm. The remaining cluster disassociated into three back-to-back dimers after 50 ns.

Discussion In this work, the binding of different ligands to vancomycin and the spontaneous formation of back-to-back and face-to-face dimers has been examined. The binding of ligand and the dimerization of vancomycin are known to be cooperative processes [20]. Based on sedimentation equilibrium data, Linsdell et al. [22], reported the dimerization constant for vancomycin to be 3.9 9 103 M1 in the absence of ligand and 8.5 9 103 M1 in the presence of Ac2-Lys-D-Ala-D-Ala. Using isothermal titration calorimetry, McPhail and Cooper [23] found that the dimerization constant was 4.7 9 102 M1 in the absence of ligand and 5.0 9 103 M1 in the presence of Ac2-Lys-D-Ala-D-Ala. This corresponds to a change in the free energy of dimerization of between 1.9 and 5.8 kJmol1 at pH 7 and 298 K. However, there is some uncertainty in the value of the dimerization constant of vancomycin itself. 1302

Fig. 7. Snapshots of the face-to-face dimer of vancomycin that formed spontaneously during the simulation (200 ns, simulation c, series 11). (A) Vancomycin molecules are represented as licorice backbones with a transparent surface. The ligand (Ac2-Lys-D-Ala-DAla) molecule is shown in a CPK representation with a mesh surface. Vancomycin is colored cyan. (B) Vancomycin is shown in stick representation. The four hydrogen bonds involved in the faceto-face interactions are shown as back dashed lines. The ligand molecules are colored according to atom type (C, green; N, blue; O, red; H, white).

Depending on the technique used and the experimental conditions, published values at approximately pH 7 without ligand range from 5.1 9 10 to 3.9 9 103 M1 [21,22]. Although free in solution vancomycin is only dimeric at millimolar concentrations, dimerization is believed to be clinically important. Several groups have noted that there is a general correlation between the propensity of glycopeptides that target lipid II to dimerize and their ability to bind both to model bacterial membranes and to bacterial cell walls [31,32]. Moreover, the activity of covalently linked vancomycin dimers is normally higher than that of the monomer [33,34]. For example, linking the vancosamine groups


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of two vancomycin monomers reduced the minimum inhibitory concentration to vancomycin-resistant Enterococcus faecium from > 16 to 1 lgmL1 [34] Likewise, the introduction of a cross-link between the C-terminal carboxyl groups of two vancomycin molecules increased the binding constant to vancomycinresistant form of ligand Ac2-Lys-D-Ala-D-Lac from 5.5 9 102 to 5.6 9 103 M1 [33]. The question of how ligand binding is correlated with dimerization has yet to be fully addressed. NMR chemical shift data suggest that atoms in the dimer interface such as the Ha of DPYG4 are less solvent exposed in the presence of ligand. This is consistent with a strengthening of the interaction between two vancomycin molecules after ligand binding [20]. The simulations suggest that the binding of ligand stabilizes both the N-terminal region of vancomycin and the dimer interface. In the simulation of the ligand-free monomer, the N-terminal region of vancomycin, including NLEU1, HDPY2 and ASN3, is more flexible than the rest of the molecule. Experimentally, NOE data suggest that in the absence of ligand, HDPY2 and ASN3 can adopt two alternative conformations [4,5,35]. In the available crystal structures (dimeric vancomycin), the amide hydrogen of ASN3 points towards the front face of vancomycin forming part of the binding pocket. However, NMR data in DMSO are consistent with the molecule also adopting a conformation in which the amide hydrogen pointed away from the binding pocket toward the back face of vancomycin [3]. During the simulations in water, NLEU1 and ASN3 were observed to adopt multiple configurations associated with the rotation of the backbone dihedrals between HDPY2 and ASN3. In fact, the largest deviations between the structure of the monomer in solution and the crystal structure involved the side chain of ASN3, HDPY2 and the terminal methyl group. However, once ligand entered the binding pocket, the conformational flexibility of both NLEU1 and ASN3 was severely restricted. This is due to the formation of a hydrogen bond between the amide group of ASN3 and the carboxyl oxygen of the terminal D-Ala, as well as the interaction between the positively charged -NH2MeLeu+ group of NLEU1 and the negative charged carboxyl group of the ligand. Together these interactions lock the molecule into the conformation observed in the crystal, thus promoting dimer formation. Steric effects are also expected to lead to some degree of cooperativity between ligand binding and dimerization. In the simulations, ligand binding is a two-step process. There is an initial formation of an encounter complex, in which there is an interaction between the positively charged N-terminal group of vancomycin and the negatively charged C-terminal


group of the ligand. To form the final complex, the ligand must rotate into the binding pocket on the front face of vancomycin. In the monomeric encounter complex, the ligand can interact with both faces of vancomycin. However, in the dimer, the interaction with the back face is hindered, thus promoting the formation of the final complex. In addition to the formation of back-to-back dimers, the formation of face-to-face dimers, and to a lesser extent side-to-side dimers, may lead to the formation of large-scale vancomycin and lipid II aggregates on the surface of target cells. The formation of large-scale aggregates has been proposed to be important for antimicrobial activity [12]. Specifically, a decrease in the propensity to form face-to-face dimers, and thus the ability to form stable large-scale aggregates, has been proposed to explain (at least in part) why the D-Ala-DLac variant of lipid II leads to vancomycin resistance in vivo [12]. A possible role for face-to-face dimer formation in glycopeptide activity is also suggested if one compares the minimum inhibitory concentration values of vancomycin, THRX-689909 and telavancin. THRX689909, a derivative of vancomycin, has decyl-aminoethyl attached to the amine group of vancosamine to act as a membrane anchor. In addition, telavancin has a phosphonomethyl–aminomethyl substitute group in the para-position of the aromatic ring of LPGH7. In the face-to-face dimer, the aromatic ring of LPGH7 lies close to the N-terminal amine group of the opposing monomer. Thus the phosphonomethyl–aminomethyl substitution is expected to strengthen the face-to-face interaction. Indeed, although the affinity of vancomycin for ligand is five times greater than that of telavancin [36], the minimum inhibitory concentration values of vancomycin, THRX-689909 and telavancin to methicillin-resistant S. aureus are 1, 0.4 and 0.25 lgmL1, respectively [37,38]. This suggests that stabilizing the face-to-face dimer enhances activity. The simulations suggest that although not as stable as the back-to-back dimer, face-to-face dimers can form spontaneously in solution. In addition, the face-to-face dimer and larger hexameric clusters of vancomycin appeared to be more stable in the presence of Ac2-LysD-Ala-D-Ala as compared with Ac-D-Ala-D-Ala. This was due to interactions between the side chain of Lys with the vancosamine and glucose groups of the opposing vancomycin and suggests that the tendency to form face-to-face dimers, and as a consequence larger clusters, would be enhanced in the presence of lipid II, as opposed to short peptides analogs. It would also lead to the binding of vancomycin being nonlinearly dependent on the concentration of lipid II. Indeed, in vitro studies using fluorescently labeled


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vancomycin show a strong accumulation of signal just along the septum of the bacteria in regions of active peptidoglycan synthesis where the concentration of lipid II is believed to be the highest [39]. In addition to shedding light on the origins of the cooperativity between binding and dimerization, the simulations also highlighted the role structural waters play in stabilizing ligand within the binding pocket and the roles these interactions might play in resistance. Two distinct regions of high water density were found in simulations, both located in vicinity of the C-terminal D-Ala of the ligand. The first lay between NLEU1, ASN3 and the side chains of DPHG5 and LPGH7. This network of structural water lies within 0.3–0.5 nm of the amide group of the terminal D-Ala and would likely be disrupted on the mutation of this amide group to oxygen (D-Lac). The second was in a cavity formed by the vancosamine-glucose disaccharide, NLEU1 and the aromatic side chain of HDPY2. This cavity lies in the vicinity the side chain methyl atom of the terminal D-Ala. The replacement of D-Ala by D-Ser, the second most common resistant form of lipid II, would result in a –CH2OH projecting into this pocket. The close correlation between the positions of the structural waters observed in the simulations and the two most common resistance mutations suggests that the role of water should also be considered when designing novel glycopeptides targeted at specific variants of lipid II.

Methods Simulations All simulations were performed under periodic conditions using the GROMACS package v. 4.0.5 [40] in conjunction with the GROMOS 54A7 force field [41]. Vancomyicin (NLEU–HDPY–ASN–DPYG–DPGH–HDPY–LPGH) contains nonstandard amino acids. In order to build a molecular topology for vancomycin, the Automated Topology Builder was used to identify all bonds, bond angles and dihedral angles [42]. The interaction parameters were then assigned by identifying similar atom, bond, angle and dihedral angle types from amino acids within the GROMOS 54A7 force field [41]. The parameters for vancosamine were based on those of glucose. The parameters for the amino group were taken from the side chain of lysine. Topology files in GROMOS and GROMACS format are available for download via the Automated Topology Builder. Note two topologies for vancomycin were generated, one in which the vancosamine was protonated and one in which vancosamine was deprotonated in order to investigate the effect of pH on bonding and self-association. The protonation state of vancosamine together with the conditions under which the sim-

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ulations were performed are listed in Tables 1–3. The solvent water was described using the simple point charge water model [43]. The solvent and solute (Tables 1–3) were coupled independently to an external temperature bath with a coupling constant of 0.1 ps using a Berendsen thermostat [44]. The pressure was maintained at 1 bar using a Berendsen barostat with a time constant of 4 ps. The compressibility was 4.0 9 105 bar1. The LINCS algorithm [45] was used to constrain the length of covalent bonds within the peptide and the SETTLE algorithm [46] was used to constrain the geometry of the water molecules. In order to further extend the timescale that could be simulated, the mass of hydrogen atoms was increased to 4 a.m.u. by transferring mass from the atom to which it was attached. This allowed a time step of 4 fs to be used to integrate the equations of motion without affecting the thermodynamic properties of the system significantly [47].

Crystal simulations To simulate the vancomycin crystal, a complete unit cell containing eight vancomycin dimers was constructed based on the asymmetric unit given in the PDB entry 1AA5 using the program MERCURY [48] by applying P43212 symmetry. The dimensions of the unit cell were 2.845, 2.845 and 6.584 nm [11]. In PDB entry 1AA5, the asymmetric unit contains two vancomycin molecules, one acetate molecule, four chloride ions and 48 water molecules. The acetate lies in the binding pocket of one of the two vancomycin molecules. Note in 1AA5 the side chain of ASN3, and the disaccharide group on DPYG4 of both vancomycin molecules as well as the side chain of NLEU1 in the vancomycin to which acetate was bound have been modeled in two alternative conformations with populations of ~ 2/3 and 1/3 respectively. In all cases, the A conformation was selected. The crystallographic waters were removed and the system was solvated using an equilibrated box of simple point charge water. To maintain the overall neutrality of the system, two water molecules selected randomly were replaced by sodium ions. The simulations of the crystal were performed using anisotropic pressure coupling. The long-range electrostatic interactions were calculated using the particlemesh Ewald [49] method with a Fourier grid spacing of 0.12 nm, a fourth order cubic interpolation, a cutoff of 0.9 nm for real-space interactions, and a relative accuracy of 1.0 9 105.

Solution simulations Simulations of vancomycin in solution were performed in a cubic periodic box using isotropic pressure coupling. The nonbonded interactions were evaluated using a twin-range method. Interactions within the short-range cut-off of 0.8 nm were updated every step. Interactions within the


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long-range cut-off of 1.4 nm were recalculated every three steps, together with the pair list. To correct for the truncation of electrostatic interactions beyond the long-range cutoff, a reaction-field correction was applied using a value of 78 for the relative dielectric permittivity [50]. The initial structures were taken from the corresponding crystal structure. In order to simulate the spontaneous association of vancomycin with a given ligand, or the self-association of two vancomycin molecules, the two molecules of interest were placed randomly within the simulation box, separated by a distance of at least 1 nm. The combined system was then resolvated. The size of the box was chosen such that the vancomycin and the ligand were at least 0.9 nm from the nearest box wall. Each system was then minimized for 500 steps using a steepest descent algorithm. To further relax the solvent, 50 ps of dynamics at 310 K was then performed in which the positions of the heavy atoms of both vancomycin and the ligand were harmonically restrained using a force constant of 1000 kJmol1nm1. A series of unrestrained simulations were then performed. The details of these simulations are given in Tables 1–3.

Analysis During the simulations, configurations were saved for analysis every 0.1 ns, unless stated otherwise. To compare the configurations obtained from the simulation to the available experimental structures, the rmsd was calculated using the following equation: vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u N u1 X rmsd ¼ t ðri ðtÞ ri ðRÞÞ2 N i¼1

(1)

after performing a least squares fit of each frame of the trajectory to the reference structure, R. Here ri(t) and ri(R) are the coordinates of the particle i in the structure at time, t, and in the reference state, respectively. The B-factors (B) were calculated from root mean square fluctuation (rmsf) using the following equation: B ¼ 8ðp rmsfÞ2 =3

(2)

atoms of vancomycin. A cubic grid with a spacing of 0.05 nm, the origin of which was aligned with the reference box, was then superimposed onto the aligned system. The average water density during simulation was then determined by counting the frequency with which any atom in a given water molecule was located within each 1.25 9 104 nm3 voxel. The spacing of the grid was such that two atoms could not simultaneously occupy the same voxel. The average residence time for water near the C-terminus of the ligand was also calculated using the configurations saved every 0.004 ns. The residence time was counted from the point at which the distance between any atom of a specific water molecule and any atom (carbon or oxygen) of the carboxylate group was < 0.3 nm. The residence time was counted until the point a water molecule no longer interacted with the carboxylate group. A water molecule was considered to no longer interact with the solute if no atom lay within 0.3 nm of the solute for more than 0.008 ns (two time frames). The distances between specific pairs of hydrogens as inferred from NOE NMR intensities were compared with the average distances observed during the simulations. The average distance during the simulation between a specific pair of hydrogen atoms was calculated as either rðtÞ ¼ < r3 >1=3

(4)

rðtÞ ¼ < r6 >1=6

(5)

or

where the angular brackets denote an average over the ensemble of configurations in the stored trajectory. Note in cases where the individual hydrogens could not be resolved during the experiment and the measured NOE corresponds to an average value (such as in the case of methyl hydrogens), the values from the simulations were obtained by averaging over the distances for each possible equivalent pair using Eqns (4) or (5). In this way, the results from the simulations can be compared directly with the results from the experiment and thus it is not necessary to incorporate pseudo-atom corrections.

The rmsf was calculated using the equation: vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u T u1 X rmsf ¼ t ðri ðtÞ ri Þ2 T i¼1

Acknowledgements (3)

where ri(t) is the position of particle i at time t. ri is the average position of particle i. T is the time over which the average fluctuation is determined. The spatial distribution of water was calculated based on configurations saved every 0.004 ns using the program G_SPATIAL [40]. First, each frame of the trajectory was aligned to the starting configuration by performing a least-square rotational and translational fit using all heavy

We thank Dr Alpeshkumar K. Malde for assistance in the generation of the topology for vancomycin. This work was supported by the Australian Grants Commission and the Australian Health and Medical Research Council grants DP110100327 and APP1026922 MAC is the recipient of an NHMRC Australia Fellowship AF51105. The authors would also like to thank Dr Mark Blaskovich, Dr Patrick Groves and Prof Joel Mackay for helpful discussions and provision of NMR data.


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Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site: Fig. S1. A plot comparing the root mean squared fluctuations extracted from the simulations together with those inferred from the crystallographic B-factors. Fig. S2. The hydrogen atom identifiers taken from Grdadolnik et al. 1998. An animation of a trajectory showing the positions of structural water molecules in the vicinity of the C-terminus of vancomycin is also provided. Table S1. The distances between pairs of hydrogens inferred from NOESY-NMR experiments of monomeric vancomycin, obtained from the simulations, and from the crystal structure 1AA5. Movie S1. An animation showing the formation of stable networks of structural waters.


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