Molecular Dynamics Simulation of Atomic Interactions in the Vancomycin Binding Site

Abstract

Vancomycin is a glycopeptide antibiotic produced by Amycolaptopsis orientalis used to treat serious infections by Gram-positive pathogens including methicillin-resistant Staphylococcus aureus. Vancomycin inhibits cell wall biosynthesis by targeting lipid II, which is the membrane-bound peptidoglycan precursor. The heptapeptide aglycon structure of vancomycin binds to the d-Ala-d-Ala of the pentapeptide stem structure in lipid II. The third residue of vancomycin aglycon is asparagine, which is not directly involved in the dipeptide binding. Nonetheless, asparagine plays a crucial role in substrate recognition, as the vancomycin analogue with asparagine substituted by aspartic acid (V_D) shows a reduction in antibacterial activities. To characterize the function of asparagine, binding of vancomycin and its aspartic-acid-substituted analogue V_D to l-Lys-d-Ala-d-Ala and l-Lys-d-Ala-d-Lac was investigated using molecular dynamic simulations. Binding interactions were analyzed using root-mean-square deviation (RMSD), two-dimensional (2D) contour plots, hydrogen bond analysis, and free energy calculations of the complexes. The analysis shows that the aspartate substitution introduced a negative charge to the binding cleft of V_D, which altered the aglycon conformation that minimized the repulsive lone pair interaction in the binding of a depsipeptide. Our findings provide new insight for the development of novel glycopeptide antibiotics against the emerging vancomycin-resistant pathogens by chemical modification at the third residue in vancomycin to improve its binding affinity to the d-Ala-d-Lac-terminated peptidoglycan in lipid II found in vancomycin-resistant enterococci and vancomycin-resistant S. aureus.

Figure 1

Chemical structures of S. aureus peptidoglycan (PG) and vancomycin. (a) The PG-repeat unit in S. aureus consists of a GlcNAc-MurNAc disaccharide, a pentapeptide stem with a sequence l-Ala-d-iso-Glu-l-Lys-d-Ala-d-Ala, and a pentaglycine bridge structure that is attached to the ε-nitrogen of the side chain L-Lys. Vancomycin binds to the d-Ala-d-Ala dipeptide of the PG-stem structure (green circle). In vancomycin-resistant pathogens, including vancomycin-resistant enterococci (VRE) and vancomycin-resistant S. aureus (VRSA), the dipeptide is replaced by a depsipeptide d-Ala-d-Lac. (b) Chemical structure of vancomycin and its interactions with the bound PG-stem structure. The vancomycin binding affinity to depsipeptide (X = O) is 1000-fold less than that to dipeptide (X = NH). The minimal inhibitory concentration of vancomycin against VRE and VRSA increases by 1000-fold. The lost efficacy is attributed to the depsipeptide substitution, which replaces a hydrogen bond with an electrostatic repulsion (red dotted line). (c) A model structure of vancomycin bound to an acyl-d-Ala-d-Ala. Vancomycin is shown as a space-filling model with an electrostatic surface and the dipeptide as a stick-and-ball model.

Figure 2

RMSD plots of three independent 30 ns MD simulations for the complexes (a) V_N-Ala, (b) V_N-Lac, (c) V_D-Lac, and (d) V_D-Ala. The RMSD values of three independent simulations are plotted using red, blue, and black colors. The average RMSD values for each complex is shown as a figure inset.

Figure 3

Initial structure of the V_D-Ala complex at t = 0 ns and the final structure at 30 ns MD simulation. (a) The initial structure of the V_D-Ala complex showing the acyl-d-Ala-d-Ala bound to the aglycon structure of the glycopeptide. (b) The final structure of the V_D-Ala complex after 30 ns of MD simulation. The C-terminus of the d-Ala is displaced from the binding cleft, resulting in the loss of three hydrogen bonds between the C-terminus of d-Ala and the amide protons of residues 1, 2, and 3 of the aglycon.

Figure 4

Comparative 2D RMSD (Å) contour plots of the V_N-Ala complex relative to V_D-Lac, V_N-Lac, and V_D-Ala complexes. Comparative 2D RMSD contour plots of (a) V_N-Ala vs V_D-Lac, (b) V_N-Ala vs V_N-Lac, and (c) V_N-Ala vs V_D-Ala complexes. The contour lines represent the density of trajectories that are located within the area. The red color denotes the region of highest density and the blue color denotes the region of lowest density.

Figure 5

Number of intermolecular H-bonds formed per time frame between the C-terminus of the ligand and the glycopeptide. The number of stable H-bonds formed between the (a) C-terminus of d-Ala-d-Ala and vancomycin in the V_N-Ala complex, (b) C-terminus of d-Ala-d-Lac and vancomycin in the V_N-Lac complex, (c) C-terminus of d-Ala-d-Lac and the modified vancomycin in the V_D-Lac complex, and (d) C-terminus of d-Ala-d-Ala and the modified vancomycin in the V_D-Ala complex.

Figure 6

Monitoring intermolecular distance between the bound ligand and the side chain at the 3rd amino acid position in vancomycin per time frame during the 30 ns MD simulation. Intermolecular distances between the amide nitrogen of the asparagine side chain of residue 3 in vancomycin and (a) the C-terminus amide nitrogen of d-Ala-d-Ala for the V_N-Ala complex and (b) the ester oxygen of d-Ala-d-Lac for V_N-Lac complex. The fluctuating intermolecular distances in the V_N-Lac complex, compared to the V_N-Ala complex, indicate conformational changes associated with the depsipeptide binding. Intermolecular distances between the carbonyl oxygen atom of the aspartate side chain of residue 3 in the glycopeptide and (c) the C-terminus ester oxygen of d-Ala-d-Lac for the V_D-Lac complex and (d) the C-terminus amide nitrogen of d-Ala-d-Ala for the V_D-Ala complex. In the V_D-Ala complex, the aspartate substitution in vancomycin introduces a negative charge that destabilizes d-Ala-d-Ala binding through electrostatic repulsion with the C-terminus of the bound ligand. After 3 ns, the formation of a new H-bond between the aspartate side chain and the amide proton of the terminal d-Ala is associated with the removal of the terminal d-Ala out of the binding pocket (Figures 5d and 3b).

Figure 7

Average atomic distance per time frame during the 30 ns MD simulation of oxygen–oxygen atoms involved in repulsive interactions between the peptidoglycan precursor and the glycopeptide in the different complexes. Average atomic distance between (a) d-Ala-d-Lac ester oxygen of the ligand and the carbonyl oxygen of residue 4 in vancomycin of the V_N-Lac complex and (b) d-Ala-d-Lac ester oxygen of the ligand and the carbonyl oxygen of residue 4 in the modified vancomycin of the V_D-Lac complex.