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Structural basis of triclosan resistance
Journal of Structural Biology 174 (2011) 173–179
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Structural basis of triclosan resistance N. Jiten Singh a,1, Dongkyu Shin b,1, Han Myoung Lee a,1, Hyun Tae Kim b, Ho-Jin Chang b, Joong Myung Cho b, Kwang S. Kim a,⇑, Seonggu Ro b,⇑ a b
Center for Superfunctional Materials, Department of Chemistry, Pohang University of Science and Technology, Pohang 780-790, Republic of Korea CrystalGenomics, Inc., Asan Institute for Life Sciences, 388-1 Pungnap-2dong, Songpa-gu, Seoul 138-736, Republic of Korea
a r t i c l e
i n f o
Article history: Received 16 September 2010 Received in revised form 22 October 2010 Accepted 4 November 2010 Available online 19 November 2010 Keywords: Enoyl–acyl carrier protein reductase ENR inhibitor Triclosan Protein–ligand interaction p-interaction Molecular dynamics
a b s t r a c t Triclosan (5-chloro-2-(2,4-dichloro-phenoxy)-phenol, TCL) is a well known inhibitor against enoyl–acyl carrier protein reductase (ENR), an enzyme critical for cell-wall synthesis of bacteria. The inhibitory concentration at 50% inhibition (IC50) of TCL against the Escherichia coli ENR is 150 nM for wild type (WT), 380, 470 and 68,500 nM for Ala, Ser and Val mutants, respectively. To understand this high TCL resistance in the G93V mutant, we obtained the crystal structures of mutated ENRs complexed with TCL and NAD+. The X-ray structural analysis along with the ab initio calculations and molecular dynamics simulations explains the serious consequence in the G93V mutant complex. The major interactions around TCL due to the aromatic(cation)–aromatic and hydrogen bonding interactions are found to be conserved both in WT and mutant complexes. Thus, the overall structural change of protein is minimal except that a flexible a-helical turn around TCL is slightly pushed away due to the presence of the bulky valine group. However, TCL shows substantial edge-to-face aromatic (p)-interactions with both the flexible R192-F203 region and the residues in the close vicinity of G93. The weakening of some edge-to-face aromatic interactions around TCL in the G93V mutant results in serious resistance to TCL. This understanding is beneficial to design new generation of antibiotics which will effectively act on the mutant ENRs. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Triclosan (5-chloro-2-(2,4-dichloro-phenoxy)-phenol, TCL) is a broad-spectrum biocide (Levy, 2001; Schweizer, 2001). Currently, its usage is expanded in consumer products such as toothpaste, mouthwashes, deodorants, hand soaps, and lotions. It is also incorporated in children’s toys, cutting boards, and plastic films to wrap meat products. Although several mechanisms of resistance to TCL were reported including activating efflux activities, capturing the TCL and increasing the expression of important regulators or metabolic enzymes (Yu et al., 2010), mutation of ENR gene showed dramatic change in MIC value. When triclosan-resistant strains were isolated from laboratories and hospitals, the mutations included G93S, G93V, M159T and F203L (McMurry et al., 1998; Chen et al., 2009). The ENR encoded by the fabI gene is an enzyme critical for cell-wall synthesis of bacteria. Subsequent studies show that TCL is an inhibitor of ENR (Heath et al., 1998, 1999; Levy et al., 1999; Qiu et al., 1999). Because TCL is broadly used in our daily life, the resistance may cause serious global health problems (Levy, 2001; Aiello and Larson, 2003; Russell, 2004). ENR is now one of the most important drug targets (Fidock et al., 2004; Kuo
⇑ Corresponding authors. 1
E-mail addresses: [email protected] (K.S. Kim), [email protected] (S. Ro). These authors contributed equally to this work.
1047-8477/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2010.11.008
et al., 2003; Wang et al., 2006) for methicillin-resistant staphylococcus aureus (MRSA) (Priyadarshi et al., 2010), Tuberculosis (TB) (Gagneux et al., 2006) and Malaria (Hall et al., 2005), three of major infectious problems of the world. Thus, the structural origin of TCL resistance should be understood to solve the resistance problems and discover new generation antibiotics for global infectious problems. Hence, it is vital to explore the structural changes of ENRs caused by mutation. Here we report the crystal structures of mutated ENRs complexed with TCL and NAD+ using X-ray crystallography along with the energetic analysis by molecular/quantum mechanical computation. We elucidate why the single G93V mutation of ENR raises serious consequences on the triclosan resistance. Among the identified single amino-acid mutations in ENR of Escherichia coli K12 i.e., G93V, M159T and F203L, the highest level of TCL resistance occurs in G93V mutants (McMurry et al., 1998). The G93A/S/C/V substitution greatly reduces diazaborine (an anti-TB drug) binding to E. coli ENR (de Boer et al., 1999). The X-ray data for structures of ENR of E. coli bound with TCL and cofactor NAD+ are reported (Levy et al., 1999; Qiu et al., 1999). Despite various mutational analysis of ENR of E. coli (Levy, 2001; McMurry et al., 1998; Qiu et al., 1999), P. falciparum (Kapoor et al., 2004), and MRSA (Takahata et al., 2007) as well as crystal structural studies of mutant Mycobacterium tuberculosis ENRNAD+ complexes (Oliveira et al., 2006) are already been reported, the structural data for mutant ENRs complexed with TCL and cofactor NAD+ are still lacking.
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2. Material and methods
the hexagonal space group P6122 with two molecules per asymmetric unit.
2.1. Protein expression and purification 2.4. Structure determination and refinement The full length of E. coli ENR (residue 1-262) was cloned into the Nde I/Xho I sites of pET21b expression vector (Novagen) with a C-terminal 6 His-tag. The sequence information is (1-262a.a + LE (restriction site) + His-6) MGFLSGKRILVTGVASKLSIAYGIAQAMH REGAELAFTYQNDKLKGRVEEFAAQLGSDIVLQCDVAEDASIDTMFAEL GKVWPKFDGFVHSIGFAPGDQLDGDYVNAVTREGFKIAHDISSYSFVA MAKACRSMLNPGSALLTLSYLGAERAIPNYNVMGLAKASLEANVRYM ANAMGPEGVRVNAISAGPIRMGFLSGKRILVTGVASKLSIAYGIAQAMH REGAELAFTYQNDKLKGRVEEFAAQLGSDIVLQCDVAEDASIDTMF. The His tag was not cleaved. The E. coli ENR mutants of Gly 93 to Ala (G93A), Ser (G93S) and Val (G93V) were generated by the QuickChange method (Stratagene) using the E. coli ENR as a template. The entire coding sequences of all the constructs were confirmed by DNA sequencing. The E. coli BL21 (DE3) transformed with the ENR expression constructs was grown and induced with IPTG for 15 h at 18 °C. Cells were resuspended in lysis buffer (50 mM Tris–HCl, pH 7.5, 200 mM NaCl, 5 mM b-mercaptoethanol, 5% (w/v) glycerol, 1 mM PMSF and complete EDTA-free protease inhibitor cocktail mixture) and disrupted by sonication. Cell lysate was clarified by centrifugation at 12,000 rpm for 1 h and the supernatant was loaded onto a Ni column (GE Healthcare). The column was washed with buffer A (50 mM Tris–HCl (pH 7.5), 200 mM NaCl, 5 mM BME, 5% (w/v) Glycerol) containing 50 mM imidazole and the protein was eluted with buffer A containing 250 mM imidazole. The ENR protein was further purified using a Superdex 200 gel filtration column (GE Healthcare) with buffer B (50 mM Tris (pH7.5), 5 mM b-mercaptoethanol, 5% (w/v) Glycerol). Peak fractions were pooled, concentrated to 25 mg/ml and stored at 80 °C before use. 2.2. Enzyme assay An enzyme activity of purified ENR proteins was determined in 50 mM sodium phosphate buffer (pH 7.5) containing 400 lM crotonyl-CoA and 100 lM NADH. Enzyme reaction was initiated with the addition of 500 nM of ENR followed by incubation for 1 h at RT and the oxidation of NADH–NAD was detected at 340 nM. IC50 values of TCL against the wild type ENR and two mutants (G93A and G93S) were determined using various concentrations of TCL when 50% of ENR activity is inhibited. 2.3. Crystallization and data collection The purified protein was incubated for 1 h at 4 °C with 5-fold molar excess of triclosan and NAD+. Cocrystallization experiments were set up using the hanging drop vapor diffusion technique. The crystals of G93A and G93S mutants were grown in 0.1 M HEPES pH 7.5, 2 M ammonium sulfate, and 5% (w/v) PEG400. The crystals of G93V mutant were grown in 0.5 M ammonium citrate/ammonium hydroxide, pH8.5, 15% (w/v) PEG 8 K. Diffraction data of G93A and G93S mutants were collected at room temperature to a resolution of 2.5 Å using RAXIS IV imaging plates coupled to a Rigaku X-ray generator with a copper rotating anode (k = 1.5418). The data were processed and scaled using CrystalClear (Pflugrath, 1999)20. The crystals of G93V mutants were cryoprotected in reservoir solution containing 20% (v/v) ethylene glycol for 3 min and flashfrozen in liquid nitrogen. Diffraction data were collected at 4A beamline in PAL (Pohang Accelerator Laboratory). The data were processed and scaled using HKL2000 (Otwinowski and Minor, 1997)21. The crystals of G93A, G93S and G93V mutants belong to
The structures of G93A, G93S and G93V were solved by molecular replacement with the E. coli ENR (PDB code: 1c14) as an initial model. Refinement of the structure was carried out with CNS. Several refinement cycles consisting of alternating cycles of simulated annealing, positional and B-factor refinement in CNS (Brünger et al., 1998) and model building with Quanta. Accelrys, Inc. (San Dego, USA) are performed. All three structures were refined in the same manner. The relevant data collection and refinement statistics are summarized in Table 1. 2.5. Data deposition The atomic coordinates structure factors are deposited in the Protein Data Bank (accession codes 3PJD for ENR G93A, 3PJE for ENR G93S, and 3PJF for ENR G93V). 2.6. Computational analysis Each of the X-ray structures of the E. coli ENR-NAD+-TCL (PDB ID: 1c14, 1d8a and new G93A, G93S and G93V mutant complexes) consists of two configurations A and B. Water molecules which are not hydrogen bonded to any of the protein residues are removed. The force field parameters and partial charges of NAD+ were taken from the work of Ryde and co-workers (Holmberg et al., 1999). The partial charges of triclosan were derived using the RED (RESP ESP charge Derive) program (Pigache et al., 2004). Amber atom types and force field parameters were assigned using the antechamber in Amber8 (Case et al., 2004). First of all, hydrogen atoms were added. The geometry was solvated with the truncated octahedron solvation method which gave a more uniform distribution of solvent around the solute, and the final system contained 9600 water molecules. The geometry was minimized with molecular mechanics. Residual decomposed molecular mechanics energies in the gas phase and free energies in water were obtained for the minimized geometries of WT/G93A/G93V mutant complexes using the Molecular Mechanics Generalized Born Surface Area (MM-GBSA) method (Massova and Kollman, 2000). Based on these results, important interactions were selected. Then, ab initio MP2/ aug-cc-pVDZ interaction energies were calculated by using the Gaussian suite of programs (Frisch et al., 2004). We performed the molecular dynamics (MD) simulations of WT (PDB ID 1c14 and 1d8a) and G93V for 2.5 ns MD run 25 ps constant volume molecular dynamics with the ENR-NAD+-TCL complex fixed, followed by constant pressure MD with cartesian restraint applied to NAD+ and TCL which was subsequently removed step by step. Then it is followed by 2.3 ns equilibration (800 ps) plus production (1.5 ns). We have also calculated the B-factor of the a-carbon atoms of the protein of each of the MD trajectories. Using a few selected geometries of G93V (with and without the TCL) from the production run of molecular dynamics (MD) simulations, we calculated the free energy change for the 93 V ! 93G mutation using thermodynamic integration method of free energy perturbation (Kollman, 1993). Calculations were done for both configurations A and B, and their average values are reported. 3. Results and discussion We have prepared the wild type (WT) and glycine 93 mutated (G93A, G93S and G93V mutants) E. coli ENRs. Inhibitory activity of TCL was assayed against the purified proteins. Compared with
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N. Jiten Singh et al. / Journal of Structural Biology 174 (2011) 173–179 Table 1 Data collection and refinement statistics. ENR G93A
ENR G93S
ENR G93V
Data collection Space group
P6122
P6122
P6122
Cell dimensions a, b, c (Å) a, b, c (°) Resolution (Å) Rsym or Rmerge I/r I Completeness (%) Redundancy
80.83, 80.83,328.40 90.0, 90.0, 120.0 30–2.50(2.59–2.50) 0.242(0.383) 2.5(1.4) 97.9(97.9) 9.2(8.9)
80.85, 80.85,328.40 90.0, 90.0, 120.0 30–2.50(2.59–2.50) 0.285(0.404) 2.1(1.6) 95.5(95.5) 7.5(10.9)
79.48, 79.48, 323.27 90.0, 90.0, 120.0 50–1.90(1.97–1.90) 0.083(0.276) 35.7(4.4) 94.7(90.1) 11.4(6.9)
Refinement Resolution (Å) No. reflections Rwork/Rfree No. atoms Protein Triclosan/NAD+ Water
30–2.5 22,600 0.210/0.234 4030 3885 34/88 84
30–2.5 21,986 0.220/0.248 4031 3887 34/88 83
35–1.9 46,530 0.219/0.247 4091 3814 34/88 216
B-factors Protein Triclosan/NAD+ Water
36.6 45.5/35.9 34.1
34.8 32.6/31.6 33.5
28.3 14.6/26.8 30.3
r.m.s. deviations Bond lengths (Å) Bond angles (°)
0.009 1.27
0.009 1.29
0.006 1.22
Diffraction data from one crystal were used to determine each structure. Values in parentheses are for the highest-resolution shell.
the activity against WT (IC50 = 150 nM), those against the Ala and Ser mutants are IC50 = 380 and 470 nM, respectively, while that against the G93V mutant is 68,500 nM. This result is well correlated with minimum inhibition concentration (MIC) of TCL against resistant strains containing the mutant ENRs (G93S: 4 times, and G93V: 60–500 times higher than WT) (Levy, 2001; McMurry et al., 1998; Qiu et al., 1999). Therefore, strains containing G93V mutant are highly resistant to TCL, while G93A and G93S show low resistance.
Additionally, we have collected the X-ray data for the corresponding G93A/S/V mutants (Table 1). The overall folding structures of the four proteins and NAD+ are almost fully overlapped (Fig. 1A). The major interactions around TCL which are found to be conserved in both WT and mutant complexes (Fig. 1B) are (i) the aromatic(cation)-aromatic [pcaion–p] interaction (Kim et al., 2000, 1994; Singh et al., 2009) between positively charged nicotinamide (NIC) and neutral TCL, (ii) the H-bond interactions (Pak et al., 2005) of the phenoxyl group of 5-(mono)Chloro-phenol
Fig. 1. (A) Overlapped crystal structures of the WT (the average geometry of PDB IDs (4,5) 1c14 and 1d8a) and mutant ENRs (G93A/S/V) complexed with NAD+-TCL. Protein backbones are superimposed (complexes for WT: blue, G93A: yellow, G93S: cyan, and G93V: red color). (X93: G/A/S/V93); (B) well conserved important interactions around TCL and NAD+ among the WT and mutant complexes. Only the G93A complex is shown for clarity. (C, D and E) Stereo views of the omit map contoured at 2.0r level of NAD and TCL respectively for G93A, G93S, and G93V mutant (left figure), electron density map contoured at 1.0r near the site of X93 (A93, S93 and V93) (middle figure), and electron density map contoured at 1.0r level in the flexible region (R192-F203) respectively for G93A, G93S and G93V mutant (right figure).
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Fig. 2. Overlapped crystal structures of the WT (PDB ID: 1C14) and mutant ENRs (G93A/S/V) complexed with NAD+-TCL (complexes for WT: blue, G93A: yellow, G93S: cyan, and G93V: red color).
(MCP) with both the ribose of NAD+ and the phenolic OH group of Y156 (2.6 ± 0.1 Å). The electron density maps of all three mutant complexes (G93A, G93S and G93V) reveal the mode of binding of TCL adjacent to the NIC ring of the nucleotide cofactor (Fig. 1C–E left figure), near the mutation site (Fig. 1C–E middle figure) and also with the flexible regions (R192-F203) (Fig. 1C–E right figure). Fig. 2 shows the overlapped crystal structures of the WT (PDB ID: 1C14) and mutant ENRs (G93A/S/V) complexed with NAD+-TCL in particular showing the difference in the loop area of the a-helix region. For p-stacked structure between aromatic rings, the interaction is maximal when two p-rings are displaced by forming a half overlapped sandwich with the plane-to-plane separation 3.5 ± 0.1 Å (Hunter and Sanders, 1990; Kim et al., 2002). The complexation of positively charged NIC with TCL in the WT and mutant complexes show similar distances and overlap patterns (Fig. 3). The face-to-face p+–p interaction (Singh et al., 2009) between the phenol ring of NIC and TCL ring is 3.5 Å. In addition, as shown
Fig. 3. Structural differences for the TCL and X93 (X = G/A/S/V) for WT, G93A, and G93V in crystal complexes – Side and Top views. G93V is distinctively different from others. The DCP ring in G93V is rotated by h = 45° with respect to WT to avoid the bump due to the bulky arms of V93. Complexes for WT: blue, G93A: yellow, G93S: cyan, and G93V: red color. (X93: G/A/S/V93).
in Fig. 1B the H-bonds between T194-OG1 and pyrophosphate (PP)-O1 N (2.9 ± 0.1 Å), the H-bond between I192-O and NIC-N7 N (3.4 ± 0.1 Å) and the H-bond between I192-N and NIC-O7 N (2.7 ± 0.1 Å) are well kept (Fig. 1B). An interesting difference is noted in the structure of TCL which is the ether linkage by two independent moieties: 5-(mono)chlorophenol (MCP) ring and 2,4-dichloro-phenyl (DCP) ring. The DCP rings of the G93A/S mutants are almost overlapped with that of WT (the DCP ring is rotated by only 10°/15°), whereas in the G93V mutant the DCP ring is rotated by 45° (Fig. 3). It seems to be well correlated to the inhibitory activities of TCL against the G93V mutant (Table 2). However, the differences in the inhibitory activity are not directly related to the configurational energy of TCL in the WT and mutants complexes, because the energy differences in the rotational conformers of the DCP ring are calculated to be negligible (<0.2 kcal/mol). The DCP ring rotates to avoid the bump due to the bulky arms [–CH(CH3)2] in the G93V mutant complex. These rotations are attributed from the configurational changes of residues in the active site around the DCP ring by virtue of the mutation. Therefore, these should reflect the interaction energies of TCL with residues including the loop part (R192-F203) of an a-helical turn (Fig. 4). Indeed, this is reflected in the differences in the hydrogen-aromatic ring (H–p) interactions (Burley and Petsko, 1985, 1986; Tarakeshwar et al., 2001; Lee et al., 2005) between alkyl hydrogen atoms of amino acids and aromatic rings of TCL among the WT and mutant complexes. For the edge-to-face conformation involving p rings, the optimal carbon-to-ring-center distance for the maximal H–p interaction is 3.5 Å when perpendicular to the facial ring center (or 4 Å when slightly slanted) (Lee et al., 2007). In the WT/G93A/ G93V complex, the distance between the methyl carbon of A196 and the DCP ring center is 4.32/4.37/5.94 Å and the average distance between the terminal carbon atom of I200 and that of DCP ring is 4.5/5.1/5.7 Å (Fig. 4). Thus, the H–p interactions of TCL with A196, A197 and I200 are strong in G93A/S mutants but weak in the G93V mutant. G93V also shows the longer H-bond distance
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N. Jiten Singh et al. / Journal of Structural Biology 174 (2011) 173–179 Table 2 Experimental inhibitory activity of TCL against ENRs and X-ray geometrical distances (Å) of important residues from TCL (MCP/DCP). Type
IC50 (nM)a
DDGb
hc
WT G93A G93V
150 380 68,500
0.0 0.5 3.6
0 10 45
MCPd
DCPe
NIC
Rib
Y156
I200
A197
X93
F94
M159
A196
3.47;1.39 3.47;1.37 3.47;1.40
2.65 2.60 2.56
2.52 2.78 2.65
4.75 5.10 5.72
5.05 5.06 5.47
5.51 4.73 3.87
5.27;7.84 5.13;7.57 5.45;7.94
4.58 4.03 5.94
4.03 4.32 5.94
a
Inhibitory activity of TCL against ENRs is expressed in terms of the concentration (nM) for the 50% inhibition of ENRs [IC50]. Relative free energy change [DDG in kcal/mol] was obtained based on the relative selectivity of IC50. Rotated angle [in degree] as shown in Fig. 1B. d The distances around MCP are described as follows. NIC: distance (left) between the NIC ring center to the MCP ring plane and distance (right) for the NIC ring displacement from the MCP ring center. Rib: distance between ribose O2 N and TCL-O17. Y156: H-bond distance. I200/A197: distance from the MCP ring center to the terminal methyl carbon of I200/A197. X93: distance from the MCP ring center to the C a (for G93)/terminal methyl carbon (for A93)/nearest terminal methyl carbon (for V93). e The distances around DCP (F94, M159, and A196) are from the DCP ring center to the C a/phenyl ring center of F94, the terminal methyl carbon atom of M159, and the terminal methyl carbon of A196, respectively. Reported distances are the average distance of A and B conformers in the crystal structures. For the WT, PDB ID 1c14 and 1d8a are the average geometrical distances. b
c
Fig. 4. pcation–p and H–p interactions around TCL (MCP + DCP) for (A) G93A and (B) G93V mutant complexes. Because of the bulky group in 93 V, M159 is pushed out from its original position and the DCP ring is significantly rotated, resulting in the a-helices pushed away from the original position in WT. This, in turn, reduces the H–p interaction of DCP with A196 and the H–p interactions of MCP with A197 and I200.
between A196-N and PP-O1A (3.27/3.40/4.45 Å for G93A/S/V), reflecting the weaker H-bonding (between the ENR and the cofactor NAD+). This weaker H-bond is the consequence of the weaker H–p interactions between TCL and A196/A197/I200, causing significant changes in the binding of TCL to the active site of ENR (Fig. 4B). The intriguing observation is that the R192-F203 region in the G93V mutant includes yet an a-helical turn, but is slightly pushed away. It is quite plausible that this region has more flexible backbones, due to the weakened H–p interaction with DCP. Therefore, the high TCL resistance of the G93V mutation of ENR is due to the weakening of p-involving interactions around TCL which are associated with both the flexible R192-F203 region and the residues in the close vicinity of V93 in the G93V complex. Thus, we note that while the strong pcation–p interaction and H bonds play the role in keeping the skeleton of the WT ENR, a few weakened H–p interactions are responsible for the high TCL resistance. As the overall structure does not change significantly (with all the binding sites still kept), it is the H–p interactions that play the key role in this unusual drug resistance. In order to obtain the structure-energy relationship, we analyze the residual energy decomposition of the total interaction energy using the molecular mechanics minimized geometry of the X-ray structures of WT and G93V complexes. It shows that NIC and ribose moieties of NAD+, Y146, Y156, P191, I192, T194, A197, I200, and F203 are the interaction sites around MCP. It is also weakly bound by Y146, P191, I192, T194, A197 and F203 through weak van der Waals interactions. Meanwhile, G/A/S/V93 (to be denoted as X93 in short), F94, L100, M159, and A196 are those around DCP. Ab initio calculation results (Table 3) based on the molecular mechanics minimized geometry of the X-ray structures show that both the strong pcation–p interaction between NIC (positively charged aromatic p system) and MCP (6.0 kcal/mol in water) and the H-bond energy of MCP with the ribose of NAD+/ Y156 (4.5 kcal/mol in water) (drug-ENR binding) little affect the relative difference in the total drug binding energy between WT and the mutants. Since there is no significant difference in structure and hence in the interaction energy between WT and G93A, we here focus our attention on the energy change for the mutation of WT–G93V (Table 2). The H–p interactions of DCP are strengthened by V93 but weakened by M159 (and slightly by F94), resulting in substantial cancelation of their combined interactions. On the other hand, as compared with the WT complex, the H–p interactions of A196 with DCP and A197/I200 with MCP are significantly weakened in the G93V complex. Thus, the three residues (A196, A197 and I200) are largely responsible for the decrease in the binding affinity with TCL and they are located in the R192-F203 region having an a-helical turn. The significant difference in drug binding energy between WT and G93V (estimated
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Table 3 Ab initio interaction energies (in kcal/mol) in the gas (aqueous) phase from minimized geometries.a. Type
WT G93V
DE(MCP)
DE(DCP)
DEtotal (MCP+DCP)
NIC
A197
I200
X-93
F94
M159
A196
11.0 (6.0) 0.2 (0.1)
2.0 (0.8) 1.3 (0.5)
2.4 (1.8) 2.1 (1.6)
3.5 (2.3) 2.3 (3.3)
2.9 (2.3) 0.3 (0.2)
4.5 (4.5) 2.1 (2.1)
2.6 (2.5) 1.9 (1.8)
28.9 (27.0) 5.6 (3.0)
a Interaction energies between MCP/DCP and each key residue on the minimized geometries in the gas (aqueous) phase at the level of MP2/aug-cc-pVDZ theory [with 50% counterpoise correction]. The values in the aqueous phase are in parentheses. The relative interaction energies with respect to WT are given for mutants, while the total interaction energies are given in italic for WT. A part or the whole part of each residue interacting with TCL was optimized. Each moiety in the minimized geometry was replaced by the MP2 optimized structure.
Laboratory Program, subsidized MOST and MOICE of Korea, NRF (Global Research Lab project and National Honor Scientist Program). Computation was carried out at the KISTI Supercomputing Center (KSC-2008-K08-0002). References
Fig. 5. Average B-factor plots of WT and G93V mutant. L means loop, AH: a-helix, T: turn, BS: b-sheet, LAH: loop consisting of one complete a-helical turn.
to be 3 kcal/mol in water) is in good agreement with the relative free energy change (3.6 kcal/mol) based on the relative IC50 values. Molecular dynamics simulations of WT and G93V complexes have shown significant differences in the average B-factor for the atoms in residues around the R192-F203 region having an a-helical turn (Fig. 5). As anticipated before, the larger atomic fluctuation in this region is due to the weakened H–p interaction with DCP. The free energy perturbation calculation using the thermodynamic integration method for the mutation of V93–G93 shows the free energy change of 3.4 ± 1.0 kcal/mol by using a few structures selected from molecular dynamics simulations of G93V, in good agreement with the experiment. 4. Conclusions It should be noted that the substantial energy difference between WT and G93V is not due to their substantial structural differences, but due to only minimal structural changes of a rotated aromatic ring and slightly pushed conformation of a flexible loop. Therefore, this substantial energy difference arises solely from the H–p interactions; these insignificant structural changes depicting significant energy differences are not easily observable due to the unclear resolution of the H positions in X-ray analysis. This unusual ingenuous one-point mutation of ENR of the bacteria leads to the intriguing drug resistance against human-designed antimicrobial drugs. We believe that the molecular design which improves the binding strength around the flexible region, in particular, the region around A196 and A197 would improve the binding efficiency of the drug with ENR. Thus, the information provided here would be utilized to design new generation of antibiotics which will effectively act on the mutant ENR, helping avoid serious health concerns due to the TCL resistance. Acknowledgments We thank the staff at Pohang Light Source (PLS) for assistance with data collection and Dr. Kwang Jin Oh of KISTI for his help and discussion. This work was supported by the National Research
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Contents lists available at ScienceDirect
Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi
Structural basis of triclosan resistance N. Jiten Singh a,1, Dongkyu Shin b,1, Han Myoung Lee a,1, Hyun Tae Kim b, Ho-Jin Chang b, Joong Myung Cho b, Kwang S. Kim a,⇑, Seonggu Ro b,⇑ a b
Center for Superfunctional Materials, Department of Chemistry, Pohang University of Science and Technology, Pohang 780-790, Republic of Korea CrystalGenomics, Inc., Asan Institute for Life Sciences, 388-1 Pungnap-2dong, Songpa-gu, Seoul 138-736, Republic of Korea
a r t i c l e
i n f o
Article history: Received 16 September 2010 Received in revised form 22 October 2010 Accepted 4 November 2010 Available online 19 November 2010 Keywords: Enoyl–acyl carrier protein reductase ENR inhibitor Triclosan Protein–ligand interaction p-interaction Molecular dynamics
a b s t r a c t Triclosan (5-chloro-2-(2,4-dichloro-phenoxy)-phenol, TCL) is a well known inhibitor against enoyl–acyl carrier protein reductase (ENR), an enzyme critical for cell-wall synthesis of bacteria. The inhibitory concentration at 50% inhibition (IC50) of TCL against the Escherichia coli ENR is 150 nM for wild type (WT), 380, 470 and 68,500 nM for Ala, Ser and Val mutants, respectively. To understand this high TCL resistance in the G93V mutant, we obtained the crystal structures of mutated ENRs complexed with TCL and NAD+. The X-ray structural analysis along with the ab initio calculations and molecular dynamics simulations explains the serious consequence in the G93V mutant complex. The major interactions around TCL due to the aromatic(cation)–aromatic and hydrogen bonding interactions are found to be conserved both in WT and mutant complexes. Thus, the overall structural change of protein is minimal except that a flexible a-helical turn around TCL is slightly pushed away due to the presence of the bulky valine group. However, TCL shows substantial edge-to-face aromatic (p)-interactions with both the flexible R192-F203 region and the residues in the close vicinity of G93. The weakening of some edge-to-face aromatic interactions around TCL in the G93V mutant results in serious resistance to TCL. This understanding is beneficial to design new generation of antibiotics which will effectively act on the mutant ENRs. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Triclosan (5-chloro-2-(2,4-dichloro-phenoxy)-phenol, TCL) is a broad-spectrum biocide (Levy, 2001; Schweizer, 2001). Currently, its usage is expanded in consumer products such as toothpaste, mouthwashes, deodorants, hand soaps, and lotions. It is also incorporated in children’s toys, cutting boards, and plastic films to wrap meat products. Although several mechanisms of resistance to TCL were reported including activating efflux activities, capturing the TCL and increasing the expression of important regulators or metabolic enzymes (Yu et al., 2010), mutation of ENR gene showed dramatic change in MIC value. When triclosan-resistant strains were isolated from laboratories and hospitals, the mutations included G93S, G93V, M159T and F203L (McMurry et al., 1998; Chen et al., 2009). The ENR encoded by the fabI gene is an enzyme critical for cell-wall synthesis of bacteria. Subsequent studies show that TCL is an inhibitor of ENR (Heath et al., 1998, 1999; Levy et al., 1999; Qiu et al., 1999). Because TCL is broadly used in our daily life, the resistance may cause serious global health problems (Levy, 2001; Aiello and Larson, 2003; Russell, 2004). ENR is now one of the most important drug targets (Fidock et al., 2004; Kuo
⇑ Corresponding authors. 1
E-mail addresses: [email protected] (K.S. Kim), [email protected] (S. Ro). These authors contributed equally to this work.
1047-8477/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2010.11.008
et al., 2003; Wang et al., 2006) for methicillin-resistant staphylococcus aureus (MRSA) (Priyadarshi et al., 2010), Tuberculosis (TB) (Gagneux et al., 2006) and Malaria (Hall et al., 2005), three of major infectious problems of the world. Thus, the structural origin of TCL resistance should be understood to solve the resistance problems and discover new generation antibiotics for global infectious problems. Hence, it is vital to explore the structural changes of ENRs caused by mutation. Here we report the crystal structures of mutated ENRs complexed with TCL and NAD+ using X-ray crystallography along with the energetic analysis by molecular/quantum mechanical computation. We elucidate why the single G93V mutation of ENR raises serious consequences on the triclosan resistance. Among the identified single amino-acid mutations in ENR of Escherichia coli K12 i.e., G93V, M159T and F203L, the highest level of TCL resistance occurs in G93V mutants (McMurry et al., 1998). The G93A/S/C/V substitution greatly reduces diazaborine (an anti-TB drug) binding to E. coli ENR (de Boer et al., 1999). The X-ray data for structures of ENR of E. coli bound with TCL and cofactor NAD+ are reported (Levy et al., 1999; Qiu et al., 1999). Despite various mutational analysis of ENR of E. coli (Levy, 2001; McMurry et al., 1998; Qiu et al., 1999), P. falciparum (Kapoor et al., 2004), and MRSA (Takahata et al., 2007) as well as crystal structural studies of mutant Mycobacterium tuberculosis ENRNAD+ complexes (Oliveira et al., 2006) are already been reported, the structural data for mutant ENRs complexed with TCL and cofactor NAD+ are still lacking.
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2. Material and methods
the hexagonal space group P6122 with two molecules per asymmetric unit.
2.1. Protein expression and purification 2.4. Structure determination and refinement The full length of E. coli ENR (residue 1-262) was cloned into the Nde I/Xho I sites of pET21b expression vector (Novagen) with a C-terminal 6 His-tag. The sequence information is (1-262a.a + LE (restriction site) + His-6) MGFLSGKRILVTGVASKLSIAYGIAQAMH REGAELAFTYQNDKLKGRVEEFAAQLGSDIVLQCDVAEDASIDTMFAEL GKVWPKFDGFVHSIGFAPGDQLDGDYVNAVTREGFKIAHDISSYSFVA MAKACRSMLNPGSALLTLSYLGAERAIPNYNVMGLAKASLEANVRYM ANAMGPEGVRVNAISAGPIRMGFLSGKRILVTGVASKLSIAYGIAQAMH REGAELAFTYQNDKLKGRVEEFAAQLGSDIVLQCDVAEDASIDTMF. The His tag was not cleaved. The E. coli ENR mutants of Gly 93 to Ala (G93A), Ser (G93S) and Val (G93V) were generated by the QuickChange method (Stratagene) using the E. coli ENR as a template. The entire coding sequences of all the constructs were confirmed by DNA sequencing. The E. coli BL21 (DE3) transformed with the ENR expression constructs was grown and induced with IPTG for 15 h at 18 °C. Cells were resuspended in lysis buffer (50 mM Tris–HCl, pH 7.5, 200 mM NaCl, 5 mM b-mercaptoethanol, 5% (w/v) glycerol, 1 mM PMSF and complete EDTA-free protease inhibitor cocktail mixture) and disrupted by sonication. Cell lysate was clarified by centrifugation at 12,000 rpm for 1 h and the supernatant was loaded onto a Ni column (GE Healthcare). The column was washed with buffer A (50 mM Tris–HCl (pH 7.5), 200 mM NaCl, 5 mM BME, 5% (w/v) Glycerol) containing 50 mM imidazole and the protein was eluted with buffer A containing 250 mM imidazole. The ENR protein was further purified using a Superdex 200 gel filtration column (GE Healthcare) with buffer B (50 mM Tris (pH7.5), 5 mM b-mercaptoethanol, 5% (w/v) Glycerol). Peak fractions were pooled, concentrated to 25 mg/ml and stored at 80 °C before use. 2.2. Enzyme assay An enzyme activity of purified ENR proteins was determined in 50 mM sodium phosphate buffer (pH 7.5) containing 400 lM crotonyl-CoA and 100 lM NADH. Enzyme reaction was initiated with the addition of 500 nM of ENR followed by incubation for 1 h at RT and the oxidation of NADH–NAD was detected at 340 nM. IC50 values of TCL against the wild type ENR and two mutants (G93A and G93S) were determined using various concentrations of TCL when 50% of ENR activity is inhibited. 2.3. Crystallization and data collection The purified protein was incubated for 1 h at 4 °C with 5-fold molar excess of triclosan and NAD+. Cocrystallization experiments were set up using the hanging drop vapor diffusion technique. The crystals of G93A and G93S mutants were grown in 0.1 M HEPES pH 7.5, 2 M ammonium sulfate, and 5% (w/v) PEG400. The crystals of G93V mutant were grown in 0.5 M ammonium citrate/ammonium hydroxide, pH8.5, 15% (w/v) PEG 8 K. Diffraction data of G93A and G93S mutants were collected at room temperature to a resolution of 2.5 Å using RAXIS IV imaging plates coupled to a Rigaku X-ray generator with a copper rotating anode (k = 1.5418). The data were processed and scaled using CrystalClear (Pflugrath, 1999)20. The crystals of G93V mutants were cryoprotected in reservoir solution containing 20% (v/v) ethylene glycol for 3 min and flashfrozen in liquid nitrogen. Diffraction data were collected at 4A beamline in PAL (Pohang Accelerator Laboratory). The data were processed and scaled using HKL2000 (Otwinowski and Minor, 1997)21. The crystals of G93A, G93S and G93V mutants belong to
The structures of G93A, G93S and G93V were solved by molecular replacement with the E. coli ENR (PDB code: 1c14) as an initial model. Refinement of the structure was carried out with CNS. Several refinement cycles consisting of alternating cycles of simulated annealing, positional and B-factor refinement in CNS (Brünger et al., 1998) and model building with Quanta. Accelrys, Inc. (San Dego, USA) are performed. All three structures were refined in the same manner. The relevant data collection and refinement statistics are summarized in Table 1. 2.5. Data deposition The atomic coordinates structure factors are deposited in the Protein Data Bank (accession codes 3PJD for ENR G93A, 3PJE for ENR G93S, and 3PJF for ENR G93V). 2.6. Computational analysis Each of the X-ray structures of the E. coli ENR-NAD+-TCL (PDB ID: 1c14, 1d8a and new G93A, G93S and G93V mutant complexes) consists of two configurations A and B. Water molecules which are not hydrogen bonded to any of the protein residues are removed. The force field parameters and partial charges of NAD+ were taken from the work of Ryde and co-workers (Holmberg et al., 1999). The partial charges of triclosan were derived using the RED (RESP ESP charge Derive) program (Pigache et al., 2004). Amber atom types and force field parameters were assigned using the antechamber in Amber8 (Case et al., 2004). First of all, hydrogen atoms were added. The geometry was solvated with the truncated octahedron solvation method which gave a more uniform distribution of solvent around the solute, and the final system contained 9600 water molecules. The geometry was minimized with molecular mechanics. Residual decomposed molecular mechanics energies in the gas phase and free energies in water were obtained for the minimized geometries of WT/G93A/G93V mutant complexes using the Molecular Mechanics Generalized Born Surface Area (MM-GBSA) method (Massova and Kollman, 2000). Based on these results, important interactions were selected. Then, ab initio MP2/ aug-cc-pVDZ interaction energies were calculated by using the Gaussian suite of programs (Frisch et al., 2004). We performed the molecular dynamics (MD) simulations of WT (PDB ID 1c14 and 1d8a) and G93V for 2.5 ns MD run 25 ps constant volume molecular dynamics with the ENR-NAD+-TCL complex fixed, followed by constant pressure MD with cartesian restraint applied to NAD+ and TCL which was subsequently removed step by step. Then it is followed by 2.3 ns equilibration (800 ps) plus production (1.5 ns). We have also calculated the B-factor of the a-carbon atoms of the protein of each of the MD trajectories. Using a few selected geometries of G93V (with and without the TCL) from the production run of molecular dynamics (MD) simulations, we calculated the free energy change for the 93 V ! 93G mutation using thermodynamic integration method of free energy perturbation (Kollman, 1993). Calculations were done for both configurations A and B, and their average values are reported. 3. Results and discussion We have prepared the wild type (WT) and glycine 93 mutated (G93A, G93S and G93V mutants) E. coli ENRs. Inhibitory activity of TCL was assayed against the purified proteins. Compared with
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N. Jiten Singh et al. / Journal of Structural Biology 174 (2011) 173–179 Table 1 Data collection and refinement statistics. ENR G93A
ENR G93S
ENR G93V
Data collection Space group
P6122
P6122
P6122
Cell dimensions a, b, c (Å) a, b, c (°) Resolution (Å) Rsym or Rmerge I/r I Completeness (%) Redundancy
80.83, 80.83,328.40 90.0, 90.0, 120.0 30–2.50(2.59–2.50) 0.242(0.383) 2.5(1.4) 97.9(97.9) 9.2(8.9)
80.85, 80.85,328.40 90.0, 90.0, 120.0 30–2.50(2.59–2.50) 0.285(0.404) 2.1(1.6) 95.5(95.5) 7.5(10.9)
79.48, 79.48, 323.27 90.0, 90.0, 120.0 50–1.90(1.97–1.90) 0.083(0.276) 35.7(4.4) 94.7(90.1) 11.4(6.9)
Refinement Resolution (Å) No. reflections Rwork/Rfree No. atoms Protein Triclosan/NAD+ Water
30–2.5 22,600 0.210/0.234 4030 3885 34/88 84
30–2.5 21,986 0.220/0.248 4031 3887 34/88 83
35–1.9 46,530 0.219/0.247 4091 3814 34/88 216
B-factors Protein Triclosan/NAD+ Water
36.6 45.5/35.9 34.1
34.8 32.6/31.6 33.5
28.3 14.6/26.8 30.3
r.m.s. deviations Bond lengths (Å) Bond angles (°)
0.009 1.27
0.009 1.29
0.006 1.22
Diffraction data from one crystal were used to determine each structure. Values in parentheses are for the highest-resolution shell.
the activity against WT (IC50 = 150 nM), those against the Ala and Ser mutants are IC50 = 380 and 470 nM, respectively, while that against the G93V mutant is 68,500 nM. This result is well correlated with minimum inhibition concentration (MIC) of TCL against resistant strains containing the mutant ENRs (G93S: 4 times, and G93V: 60–500 times higher than WT) (Levy, 2001; McMurry et al., 1998; Qiu et al., 1999). Therefore, strains containing G93V mutant are highly resistant to TCL, while G93A and G93S show low resistance.
Additionally, we have collected the X-ray data for the corresponding G93A/S/V mutants (Table 1). The overall folding structures of the four proteins and NAD+ are almost fully overlapped (Fig. 1A). The major interactions around TCL which are found to be conserved in both WT and mutant complexes (Fig. 1B) are (i) the aromatic(cation)-aromatic [pcaion–p] interaction (Kim et al., 2000, 1994; Singh et al., 2009) between positively charged nicotinamide (NIC) and neutral TCL, (ii) the H-bond interactions (Pak et al., 2005) of the phenoxyl group of 5-(mono)Chloro-phenol
Fig. 1. (A) Overlapped crystal structures of the WT (the average geometry of PDB IDs (4,5) 1c14 and 1d8a) and mutant ENRs (G93A/S/V) complexed with NAD+-TCL. Protein backbones are superimposed (complexes for WT: blue, G93A: yellow, G93S: cyan, and G93V: red color). (X93: G/A/S/V93); (B) well conserved important interactions around TCL and NAD+ among the WT and mutant complexes. Only the G93A complex is shown for clarity. (C, D and E) Stereo views of the omit map contoured at 2.0r level of NAD and TCL respectively for G93A, G93S, and G93V mutant (left figure), electron density map contoured at 1.0r near the site of X93 (A93, S93 and V93) (middle figure), and electron density map contoured at 1.0r level in the flexible region (R192-F203) respectively for G93A, G93S and G93V mutant (right figure).
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Fig. 2. Overlapped crystal structures of the WT (PDB ID: 1C14) and mutant ENRs (G93A/S/V) complexed with NAD+-TCL (complexes for WT: blue, G93A: yellow, G93S: cyan, and G93V: red color).
(MCP) with both the ribose of NAD+ and the phenolic OH group of Y156 (2.6 ± 0.1 Å). The electron density maps of all three mutant complexes (G93A, G93S and G93V) reveal the mode of binding of TCL adjacent to the NIC ring of the nucleotide cofactor (Fig. 1C–E left figure), near the mutation site (Fig. 1C–E middle figure) and also with the flexible regions (R192-F203) (Fig. 1C–E right figure). Fig. 2 shows the overlapped crystal structures of the WT (PDB ID: 1C14) and mutant ENRs (G93A/S/V) complexed with NAD+-TCL in particular showing the difference in the loop area of the a-helix region. For p-stacked structure between aromatic rings, the interaction is maximal when two p-rings are displaced by forming a half overlapped sandwich with the plane-to-plane separation 3.5 ± 0.1 Å (Hunter and Sanders, 1990; Kim et al., 2002). The complexation of positively charged NIC with TCL in the WT and mutant complexes show similar distances and overlap patterns (Fig. 3). The face-to-face p+–p interaction (Singh et al., 2009) between the phenol ring of NIC and TCL ring is 3.5 Å. In addition, as shown
Fig. 3. Structural differences for the TCL and X93 (X = G/A/S/V) for WT, G93A, and G93V in crystal complexes – Side and Top views. G93V is distinctively different from others. The DCP ring in G93V is rotated by h = 45° with respect to WT to avoid the bump due to the bulky arms of V93. Complexes for WT: blue, G93A: yellow, G93S: cyan, and G93V: red color. (X93: G/A/S/V93).
in Fig. 1B the H-bonds between T194-OG1 and pyrophosphate (PP)-O1 N (2.9 ± 0.1 Å), the H-bond between I192-O and NIC-N7 N (3.4 ± 0.1 Å) and the H-bond between I192-N and NIC-O7 N (2.7 ± 0.1 Å) are well kept (Fig. 1B). An interesting difference is noted in the structure of TCL which is the ether linkage by two independent moieties: 5-(mono)chlorophenol (MCP) ring and 2,4-dichloro-phenyl (DCP) ring. The DCP rings of the G93A/S mutants are almost overlapped with that of WT (the DCP ring is rotated by only 10°/15°), whereas in the G93V mutant the DCP ring is rotated by 45° (Fig. 3). It seems to be well correlated to the inhibitory activities of TCL against the G93V mutant (Table 2). However, the differences in the inhibitory activity are not directly related to the configurational energy of TCL in the WT and mutants complexes, because the energy differences in the rotational conformers of the DCP ring are calculated to be negligible (<0.2 kcal/mol). The DCP ring rotates to avoid the bump due to the bulky arms [–CH(CH3)2] in the G93V mutant complex. These rotations are attributed from the configurational changes of residues in the active site around the DCP ring by virtue of the mutation. Therefore, these should reflect the interaction energies of TCL with residues including the loop part (R192-F203) of an a-helical turn (Fig. 4). Indeed, this is reflected in the differences in the hydrogen-aromatic ring (H–p) interactions (Burley and Petsko, 1985, 1986; Tarakeshwar et al., 2001; Lee et al., 2005) between alkyl hydrogen atoms of amino acids and aromatic rings of TCL among the WT and mutant complexes. For the edge-to-face conformation involving p rings, the optimal carbon-to-ring-center distance for the maximal H–p interaction is 3.5 Å when perpendicular to the facial ring center (or 4 Å when slightly slanted) (Lee et al., 2007). In the WT/G93A/ G93V complex, the distance between the methyl carbon of A196 and the DCP ring center is 4.32/4.37/5.94 Å and the average distance between the terminal carbon atom of I200 and that of DCP ring is 4.5/5.1/5.7 Å (Fig. 4). Thus, the H–p interactions of TCL with A196, A197 and I200 are strong in G93A/S mutants but weak in the G93V mutant. G93V also shows the longer H-bond distance
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N. Jiten Singh et al. / Journal of Structural Biology 174 (2011) 173–179 Table 2 Experimental inhibitory activity of TCL against ENRs and X-ray geometrical distances (Å) of important residues from TCL (MCP/DCP). Type
IC50 (nM)a
DDGb
hc
WT G93A G93V
150 380 68,500
0.0 0.5 3.6
0 10 45
MCPd
DCPe
NIC
Rib
Y156
I200
A197
X93
F94
M159
A196
3.47;1.39 3.47;1.37 3.47;1.40
2.65 2.60 2.56
2.52 2.78 2.65
4.75 5.10 5.72
5.05 5.06 5.47
5.51 4.73 3.87
5.27;7.84 5.13;7.57 5.45;7.94
4.58 4.03 5.94
4.03 4.32 5.94
a
Inhibitory activity of TCL against ENRs is expressed in terms of the concentration (nM) for the 50% inhibition of ENRs [IC50]. Relative free energy change [DDG in kcal/mol] was obtained based on the relative selectivity of IC50. Rotated angle [in degree] as shown in Fig. 1B. d The distances around MCP are described as follows. NIC: distance (left) between the NIC ring center to the MCP ring plane and distance (right) for the NIC ring displacement from the MCP ring center. Rib: distance between ribose O2 N and TCL-O17. Y156: H-bond distance. I200/A197: distance from the MCP ring center to the terminal methyl carbon of I200/A197. X93: distance from the MCP ring center to the C a (for G93)/terminal methyl carbon (for A93)/nearest terminal methyl carbon (for V93). e The distances around DCP (F94, M159, and A196) are from the DCP ring center to the C a/phenyl ring center of F94, the terminal methyl carbon atom of M159, and the terminal methyl carbon of A196, respectively. Reported distances are the average distance of A and B conformers in the crystal structures. For the WT, PDB ID 1c14 and 1d8a are the average geometrical distances. b
c
Fig. 4. pcation–p and H–p interactions around TCL (MCP + DCP) for (A) G93A and (B) G93V mutant complexes. Because of the bulky group in 93 V, M159 is pushed out from its original position and the DCP ring is significantly rotated, resulting in the a-helices pushed away from the original position in WT. This, in turn, reduces the H–p interaction of DCP with A196 and the H–p interactions of MCP with A197 and I200.
between A196-N and PP-O1A (3.27/3.40/4.45 Å for G93A/S/V), reflecting the weaker H-bonding (between the ENR and the cofactor NAD+). This weaker H-bond is the consequence of the weaker H–p interactions between TCL and A196/A197/I200, causing significant changes in the binding of TCL to the active site of ENR (Fig. 4B). The intriguing observation is that the R192-F203 region in the G93V mutant includes yet an a-helical turn, but is slightly pushed away. It is quite plausible that this region has more flexible backbones, due to the weakened H–p interaction with DCP. Therefore, the high TCL resistance of the G93V mutation of ENR is due to the weakening of p-involving interactions around TCL which are associated with both the flexible R192-F203 region and the residues in the close vicinity of V93 in the G93V complex. Thus, we note that while the strong pcation–p interaction and H bonds play the role in keeping the skeleton of the WT ENR, a few weakened H–p interactions are responsible for the high TCL resistance. As the overall structure does not change significantly (with all the binding sites still kept), it is the H–p interactions that play the key role in this unusual drug resistance. In order to obtain the structure-energy relationship, we analyze the residual energy decomposition of the total interaction energy using the molecular mechanics minimized geometry of the X-ray structures of WT and G93V complexes. It shows that NIC and ribose moieties of NAD+, Y146, Y156, P191, I192, T194, A197, I200, and F203 are the interaction sites around MCP. It is also weakly bound by Y146, P191, I192, T194, A197 and F203 through weak van der Waals interactions. Meanwhile, G/A/S/V93 (to be denoted as X93 in short), F94, L100, M159, and A196 are those around DCP. Ab initio calculation results (Table 3) based on the molecular mechanics minimized geometry of the X-ray structures show that both the strong pcation–p interaction between NIC (positively charged aromatic p system) and MCP (6.0 kcal/mol in water) and the H-bond energy of MCP with the ribose of NAD+/ Y156 (4.5 kcal/mol in water) (drug-ENR binding) little affect the relative difference in the total drug binding energy between WT and the mutants. Since there is no significant difference in structure and hence in the interaction energy between WT and G93A, we here focus our attention on the energy change for the mutation of WT–G93V (Table 2). The H–p interactions of DCP are strengthened by V93 but weakened by M159 (and slightly by F94), resulting in substantial cancelation of their combined interactions. On the other hand, as compared with the WT complex, the H–p interactions of A196 with DCP and A197/I200 with MCP are significantly weakened in the G93V complex. Thus, the three residues (A196, A197 and I200) are largely responsible for the decrease in the binding affinity with TCL and they are located in the R192-F203 region having an a-helical turn. The significant difference in drug binding energy between WT and G93V (estimated
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Table 3 Ab initio interaction energies (in kcal/mol) in the gas (aqueous) phase from minimized geometries.a. Type
WT G93V
DE(MCP)
DE(DCP)
DEtotal (MCP+DCP)
NIC
A197
I200
X-93
F94
M159
A196
11.0 (6.0) 0.2 (0.1)
2.0 (0.8) 1.3 (0.5)
2.4 (1.8) 2.1 (1.6)
3.5 (2.3) 2.3 (3.3)
2.9 (2.3) 0.3 (0.2)
4.5 (4.5) 2.1 (2.1)
2.6 (2.5) 1.9 (1.8)
28.9 (27.0) 5.6 (3.0)
a Interaction energies between MCP/DCP and each key residue on the minimized geometries in the gas (aqueous) phase at the level of MP2/aug-cc-pVDZ theory [with 50% counterpoise correction]. The values in the aqueous phase are in parentheses. The relative interaction energies with respect to WT are given for mutants, while the total interaction energies are given in italic for WT. A part or the whole part of each residue interacting with TCL was optimized. Each moiety in the minimized geometry was replaced by the MP2 optimized structure.
Laboratory Program, subsidized MOST and MOICE of Korea, NRF (Global Research Lab project and National Honor Scientist Program). Computation was carried out at the KISTI Supercomputing Center (KSC-2008-K08-0002). References
Fig. 5. Average B-factor plots of WT and G93V mutant. L means loop, AH: a-helix, T: turn, BS: b-sheet, LAH: loop consisting of one complete a-helical turn.
to be 3 kcal/mol in water) is in good agreement with the relative free energy change (3.6 kcal/mol) based on the relative IC50 values. Molecular dynamics simulations of WT and G93V complexes have shown significant differences in the average B-factor for the atoms in residues around the R192-F203 region having an a-helical turn (Fig. 5). As anticipated before, the larger atomic fluctuation in this region is due to the weakened H–p interaction with DCP. The free energy perturbation calculation using the thermodynamic integration method for the mutation of V93–G93 shows the free energy change of 3.4 ± 1.0 kcal/mol by using a few structures selected from molecular dynamics simulations of G93V, in good agreement with the experiment. 4. Conclusions It should be noted that the substantial energy difference between WT and G93V is not due to their substantial structural differences, but due to only minimal structural changes of a rotated aromatic ring and slightly pushed conformation of a flexible loop. Therefore, this substantial energy difference arises solely from the H–p interactions; these insignificant structural changes depicting significant energy differences are not easily observable due to the unclear resolution of the H positions in X-ray analysis. This unusual ingenuous one-point mutation of ENR of the bacteria leads to the intriguing drug resistance against human-designed antimicrobial drugs. We believe that the molecular design which improves the binding strength around the flexible region, in particular, the region around A196 and A197 would improve the binding efficiency of the drug with ENR. Thus, the information provided here would be utilized to design new generation of antibiotics which will effectively act on the mutant ENR, helping avoid serious health concerns due to the TCL resistance. Acknowledgments We thank the staff at Pohang Light Source (PLS) for assistance with data collection and Dr. Kwang Jin Oh of KISTI for his help and discussion. This work was supported by the National Research
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