Refinement of a Low-resolution Crystal Structure to Better Understand Erythromycin
Interactions on Large Ribosomal Subunit
http://www.jbsdonline.com
Abstract
Macrolides are a group of diverse class of naturally occurring and synthetic antibiotics made of macrocyclic-lactone ring carrying one or more sugar moieties linked to various atoms of the lactone ring. These macrolides selectively bind to a single high affinity site on the prokaryotic 50S ribosomal subunit, making them highly effective towards a wide range of bacterial pathogens. The understanding of binding between macrolides and ribosome serves a good basis in elucidating how they work at the molecular level and these findings would be important in rational drug design. Here, we report refinement of reconstructed PDB structure of erythromycin-ribosome system using molecular dynamics (MD) simulation. Interesting findings were observed in this refinement stage that could improve the understanding of the binding of erythromycin A (ERYA) onto the 50S subunit. The results showed ERYA was highly hydrated and water molecules were found to be important in bridging hydrogen bond at the binding pocket during the simulation time. ERYA binding to ribosome was also strength-ened by hydrogen bond network and hydrophobic interactions between the antibiotic and the ribosome. Our MD simulation also demonstrated direct interaction of ERYA with Domains II, V and with C1773 (U1782EC), a residue in Domain IV that has yet been described of its role in ERYA binding. It is hoped that this refinement will serve as a starting model for a further enhancement of our understanding towards the binding of ERYA to ribosome.
Key words: Molecular dynamics simulation; Refinement; Erythromycin A; 23S rRNA; Large ribosomal subunit; and Water-bridged hydrogen bond.
Introduction
The macrolides are clinically well-established and highly prescribed antibiotics, ranging from the erythromycins to the newer analogues such as ketolides deriva-tized in numerous ways to improve their pharmacological properties (1). These antibiotics which consist of a 12- to 16- membered lactone ring, to which one or more sugar substituents are attached, mainly target exclusively at 50S prokaryotic ribosomal subunit. Biochemical and genetic data have revealed that the macrolides interacted with the loop of helix 35 in Domain II of the bacterial 23S ribosomal RNA (rRNA) and with the peptidyl transferase center (PTC) in Domain V (2-7). The X-ray structure (8) also confirmed similar location for the binding site for mac-rolides (erythromycin, clarithromycin, and roxithromycin), chloramphenicol, and clindamycin where the binding site was found to be near to the PTC, specifically to nucleotides in the Domain V of 23S rRNA. The crystallographic study also showed that the binding of the antibiotics has no significant interactions with nearby ribo-somal proteins and did not cause major conformational change to the PTC (8).
Erythromycin A (ERYA), produced from Saccaropolyspora erythraea is the first macrolide antibiotic introduced in 1952 (9). It is often used to treat patients who are
1Pharmaceutical Design and Simulation (PhDS) Laboratory2School of Pharmaceutical Sciences3School of Biological SciencesUniversiti Sains Malaysia11800 Minden, Pulau Pinang, Malaysia
allergic to penicillin. Despite the discovery and the use of ERYA since half a cen-tury ago, it is still one of the most important antimicrobial agent as it is effective to-wards many common bacterial pathogens and some non-typical pathogens (10-12). ERYA has a 14-membered lactone ring (L), attached with a desosamine sugar (D) and a cladinose sugar (C) extended from C-5 and C-3 position of the lactone ring, respectively (Figure 1). ERYA showed high specificity and affinity for the bacterial 50S subunit with Kd ~ 10-8 M (3, 7, 13-16). However, the actual binding mode of ERYA or its inhibition mode to the bioactivity of ribosome is merely understood to the most general idea. A comprehensive understanding of the drug binding sites is therefore needed to understand the mechanisms of drug action, and hence enabling a rational approach for antibiotic development.
Ribosomes are macromolecules that are responsible for the production of protein chains. They are complex structures made of two subunits; the large and small subunits, and both subunits comprised enormous amount of rRNA and ribosomal protein. Both subunits have different features in strengthen the transition between various catalytic sites that leads to efficient protein biosynthesis process. The small subunit is responsible for the formation of initiation complex and contains messen-ger decoding site. On the other hand, the large subunit contains PTC that catalyzes peptide bond formation and provides exit tunnel for nascent polypeptide chain to leave ribosome. Due to its prominent role in this important bioprocess and the fact that prokaryotic ribosome differ significantly from the eukaryotic counterpart, ribo-some has become favourite target for many natural or synthetic antibiotics to inhibit the bacterial protein biosynthesis process, thus suppressing the bacterial growth.
The availability of X-ray structures of ribosome (8, 17-28) has opened up the possi-bility of performing computational calculation like molecular dynamics (MD) sim-ulations for further understanding of the structure and functions of ribosome as well to design novel inhibitors. Yet, seven years after the first ribosome structure being elucidated at the atomic level using X-ray crystallography, very few MD simula-tions on ribosome have been carried out as compared to other biological molecular system such as protein, nucleic acid, lipid bilayer, and so forth. One possible ex-planation for this is the enormous structure of ribosome could easily yield a system containing massive number of atoms that is prohibitive to be simulated on a typi-cal workstation. Nevertheless, MD simulation of 70S ribosome was successfully done by Trylska et al. (29) in 2005. In their work, the coarse-grained method was adapted as an approach to shrink the simulation size to a reasonably size, feasible enough to be simulated to half a microsecond. These findings have important im-plication for understanding ribosome’s movement in translocation of tRNA during the translation process. The use of coarse-grained model has reduced the number of atoms in a simulation significantly, however it also gives less information and re-parameterization of force field might bring less reliability in simulation accuracy.
Sanbonmatsu et al. (30) overcome the coarse-grained limitation in understanding the translation machinery by producing the first all-atom targeted MD simulation of the accommodation of tRNA into the 70S ribosome in explicit solvent. This large scale all-atom simulation yielded 2.6 million atoms and some 106 computer hours were used to simulate seven systems of 2 ns time frame in order to understand the critical step of identifying cognate tRNA selection in the decoding stage. The simulations have enlightened the understanding of tRNA movement into ribosome during decoding stages in atomic level. However, these MD studies (29, 30) in-vestigated the role of ribosome in protein biosynthesis process and so far no MD simulation is used to study ribosome’s function as a drug target.
Investigation on the ribosomal subunit crystal structures in the Protein Data Bank (PDB) revealed majority of them are lacking structural information due to the low percentage completeness of the structure. In the case of the large ribosomal subunit, some rRNA and ribosomal proteins were not able to be captured due to
Figure 1: Schematic representation of erythromycin A. It consists of a 14-membered lactone ring, a desosamine sugar and a cladinose sugar.
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high density and their complicated structural organization in this macromolecule. The coarse-grained model allows for much longer simulation than the simulation with all-atom resolution. The latter has an advantage of high accuracy due to specific interactions and is more suitable for drug design. Based on this, we did a reconstruction of the missing residues in the crystal structure and then followed by refinement stages using MD simulation. In the process, we were able to obtain some valuable information from this short simulation with regards to the binding of ERYA to the large ribosomal subunit.
Here, we report the results of MD simulation of ribosome-ERYA complex, with the aim to elucidate the mechanism of action of this macrolide on the 50S ribo-somal subunit. To date, this is the first kind of MD simulation on such system and we hoped the results could provide both qualitative and quantitative information to explain the molecular basis of the antibacterial action at the molecular level. This simulation has enabled us to investigate water molecule’s involvement and their contribution in mediating the binding of ERYA with the binding pocket. Our study is also consistent with the experimental results (8) in terms of the existence of hydrogen bond network in the ERYA binding at the large subunit; and also the non-involvement of ribosomal proteins in the ERYA binding. In addition, we were able to identify nucleotides from domains II and IV that were involved in the opti-mum binding of ERYA to the large subunit.
Methods
Construction of Simulation System
The starting structure was based on a 3.50 Å resolution X-ray structure of the 50S large ribosomal subunit, taken from the PDB: PDB code, 1JZY (8). This structure contained one chain of 23S rRNA and three chains of ribosomal proteins namely L4, L22, and L32, in complex with ERYA including two magnesium ions. This crystal structure is 79.5% complete and 106 nucleotides were found missing from rRNA in domains I, II, V, VI, and 14 missing amino acid residues from L4 and L32 protein chains. The missing side chains for the ribosomal proteins were built based on the carbon alpha atoms found in the PDB structure using LEAP module of AMBER 8 package (31). Missing RNA residues were constructed using the following proce-dure: (I) Missing residues were identified and their corresponding contiguous resi-dues were extracted to Hyperchem7.5 (Hypercube Inc.). (II) The missing residues were built according to their sequence using Hyperchem7.5. (III) These newly con-structed part from the Hyperchem7.5 were then inserted into the starting structure to form a complete structure of the large ribosomal subunit (It is worth noted here that all of these missing residues were not located in the binding pocket). Subsequently, the structure was refined for a total of 10,000 steps each of Steepest Descent (SD) and Conjugate Gradient (CG) minimizations to relieve possible steric clashes and overlaps of side chains. The structure was examined at every 5000th step of minimi-zation, in terms of its energy, RMS values and atoms that were overlapped or made unfavorable contacts. The final structure was taken when all of the above criteria were satisfied and used as the starting structure for subsequent MD simulation runs.
The geometries of ERYA were fully optimized and their electrostatic potentials were computed, both at the B3LYP level with the 6-31G(d,p) basis set using GAUSSIAN 03 (32) program and their partial charges were obtained by restrained electrostatic potential (RESP) using ANTECHAMBER (33). The Amber 99 force field (34) was used to describe the molecular mechanics of rRNA and ribosomal proteins, while the general amber force field (GAFF) (35) was used to describe ERYA. Hydrogen atoms for the entire complex were added explicitly using LEAP. The complex (50S ribosomal subunit and ERYA) was immersed in a TIP3P (36) waterbox of edge lengths of 233.736 Å × 270.195 Å × 198.736 Å containing 296,254 water molecules. The complex was found to be largely anionic due to the presence of
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phosphates in the RNA. Therefore, to ensure the neutrality of this system, potas-sium and sodium counterions were added almost equally to the system and they were placed by LEAP at the most negative position of the complex. The details of the simulation system including number of counterions and number of atoms for each molecule are shown in Table I. The parameters of magnesium, sodium, and potassium ions were taken from the standard AMBER database.
Minimizations and Molecular Dynamics Simulations
Minimizations and molecular dynamics (MD) simulations were carried out using the SANDER module of AMBER 8. Prior to MD equilibration runs, the system was subjected to a total of 4,500 steps of SD and 10,500 steps of CG minimizers on three different minimization stages where the complex was fixed on place with a positional restraint force of 300 and 150 kcal mol-1 Å-2, respectively, in the first and second stages of minimization, and the entire system was permitted to move freely after that. The relaxed structure was then subjected to heating stages, each with 20 ps from 0 to 150K then to 300K with positional restraints of 150 kcal mol-1 Å-2 on the complex. It was then followed by 20 ps of fully unrestrained equilibration at constant temperature of 300K, controlled by the Langevin thermostat with collision frequency of 1.0 ps-1 (37). A 40 ps MD simulation was done using the canonical ensemble before switching over to the isobaric-isothermal ensemble. Pressure of the system was regulated at 1 bar with isotropic position scaling of 1 ps pressure relaxation time. SHAKE (38) method was applied throughout the MD simulation to allow the integration of force equation at 2 fs and Particle Mesh Ewald (PME) (39) was turned on for proper treatment of long range electrostatic interactions and the non-bonded cutoff was set to 8.0 Å. The translational and rotational around the center of mass were removed every 1000 steps, and the non-bonded pair list was updated every 25 steps. Trajectories were saved every 0.1 ps during the simulation for later analyses. Approximately 700 hours were used on a 16-CPU Linux cluster upon the completion of the current 1.3 ns of MD simulation.
Table IMD system setup details.
Simulation system 23S rRNA Ribosomal proteins Ligand / Ions Water molecule Total
Figure 2: Thermodynamics properties of (A) potential energy (B) simulation box volume for the 1.3 ns simula-tion showed equilibration from 500 ps onwards. The first 100 ps that are intended for heating are omitted.
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Analysis
Trajectories were analyzed using PTRAJ from AMBER 8, while snapshots from the trajectories were visually examined and illustrated using VMD (40). Hydrogen bond analysis was calculated using PTRAJ and represented in the schematic form generated by Ligplot (41). Hydrophobic interaction was computed using HBPLUS (42), where HBPLUS generates a list of non-bonded interactions by computing all possible positions for heavy atoms that are less than a specified distance apart. Those interaction/forces that were exerted 3.90 Å or less for paired carbon-carbon were then extracted. The distribution of water molecules in the hydration shell of ERYA and radial distribution function (RDF) was calculated using PTRAJ. Analy-sis of MD trajectories was focused on its production stage (500-1300 ps) while thermodynamics properties were monitored throughout the simulation.
Results and Discussion
Stability of the Trajectories
Stability of the trajectory was demonstrated by thermodynamics properties versus simulation time as shown in Figure 2A-B. In general, the plots were stable through-out the simulation, but sharp increase could be observed in the beginning of the simu-lation (data not shown) due to heating stages. Consequently, the raise of temperature and release of positional restraints have increased the potential energy of the system and finally, causing the system to reach equilibration from 500 -1300 ps. Figure 2B showed simulation box volume versus simulation time. It can be seen that the simu-lation box reached an average value of 1.05 × 107 Å3 and it did not change much dur-ing the production stages, which also signified the system has reached equilibration.
Convergence and stability of the simulation could be further reflected from its de-viation from a reference structure using the root mean square deviation (RMSD). The mass-weighted RMSD of the erythromycin binding pocket (taken as 15 Å from the center of mass of ERYA, as shown in Figure 3) over 1.3 ns of MD simulation was calculated with reference to the initial structure. The RMSD plots (Figure 4A-C) showed structure reached a stable state only after 500 ps where average displace-ment of the binding site was 3.38 ± 0.14 Å. For the ligand, it only has an average displacement of 1.57 ± 0.09 Å from the initial structure during the production stage. The low RMSD values showed ERYA was well maintained in the pocket through-out the 1.3 ns simulation. The RMSD of ribosomal proteins (Figure 4C) inside the pocket was also calculated based on their backbone with respect to the initial struc-ture and it was found to have an average value of 2.80 ± 0.19 Å.
To have better insight on the flexibility of each nucleotide residue in the binding pocket, a root mean square fluctuation (RMSF) analysis was evaluated on the ribo-
Figure 3: Line and tube representation of rRNA in 50S ribosomal subunit (left). Red arrow showed location of ERYA binding pocket (taken as 15 Å from mass center of ERYA) with yellow surface representation (right). The backbone of the pocket is indicated by light blue tube and ERYA is represented in dark blue licorice.
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some backbone with reference to the average structure. Several key residues in-volved in the interaction of ERYA with the binding pocket, including C765, C1773, A2041, A2042, A2045, A2418, A2482, G2484, U2564, U2588, and U2589 [here and throughout the entire manuscript, nucleotides and amino acids are numbered according to D. radiodurans, unless stated in parentheses with E. coli (EC) num-bers or H. marismortui (HM) numbers] showed low flexibility as indicated by their relatively smaller RMSF values (Table II). These important residues were mainly located in Domain II and V (except for C1773 in Domain IV) and formed the bind-ing site for ERYA. They also appeared to be the residues that were responsible for the binding of ERYA, either through H-bonds, hydrophobic interactions, etc. These interactions indeed restricted the motion of these residues resulting in low fluctua-tion and less mobility compared to other residues that were not at the vicinity of the binding pocket. Some residues (those of not in the binding pocket) were found to have high fluctuation (> 10 Å) as shown in Table II. Visual inspection showed these residues were located at the peripheral and exterior of the large ribosomal subunit and therefore experienced high fluctuations as the simulation progressed. We have also measured their distances to ascertain that these regions indeed were located very distant from the binding pocket (ranging from 80-150 Å). Therefore, it is expected that these residues (which fluctuated remarkably) did not exert any significant and direct effects to the binding of ERYA in the binding pocket.
Solvent Effects and Water-bridged Hydrogen Bond in the Binding Pocket
The characteristics of solvation effects and hydration shell are difficult to be stud-ied by experimental studies due to the high mobility of water molecules (43, 44). Therefore, computational simulation such as MD simulation on the other hand, are often used to study details on water networks within the molecule, their dynamic movements, and contribution in receptor-ligand binding (45).
In our study, the distribution of water molecules in ERYA binding pocket was inves-tigated using radial distribution function (RDF), also known as the pair distribution function or g(ij, r). It was calculated by dividing the space around the two atoms (i, j) into spacing-bins at intervals of 0.05 Å, ranging between 0 to 10 Å. The water molecules that were found in each shell were counted and averaged over configu-rations that were generated by the 500-1300 ps window of MD simulation. The average number was then divided by the volume of each shell to obtain the average density of water as a function of the distance r from a reference atom in the ligand.
Figure 4: Root mean square deviation (RMSD) plots for (A), backbone of erythromycin binding pocket (B) ERYA, and (C) backbone of ribosomal proteins in the binding pocket, with reference to the initial structure. The first 100 ps that are intended for heating are omitted.
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In this study, RDF was calculated based on selected acceptor atom in ERYA (see below) to an oxygen atom of water molecule (Table III). In order to have a clearer insight of the distribution of water molecules around the acceptor atoms, the esti-mated number of water molecules that were found within the first solvation shell (spherical radius of <3 Å) and second solvation shell (spherical radius between 3-5 Å) were also counted and averaged in the 500-1300 ps window as shown in Table IV. Figure 5 showed the general pattern for RDF plot that was obtained for oxygen of water (Ow) and O6 (4ʹʹ-OH group of cladinose). RDF plots of other calculated ligand atom also showed similar pattern (not shown here) and their first peak of the plot together with their intensity values are also recorded in Table III.
Table IIRoot mean square fluctuation (RMSF) and types of interactions involved for residues found 4 Å or less to ERYA in the binding pocket. Selected reconstructed residues that were distant from the binding pocket are labeled with #.
Residue Number Domain RMSF
(Å)Types of interactions predicted in this work
Types of interactions predicted in crystal structure (8)
U727# II 14.35 - -G728# II 14.46 - -A729# II 12.35 - -C759 II 0.42 HP -C765 II 0.86 H-bond -
A1059# II 12.10 - -C1090# II 16.05 - -C1120# II 18.41 - -C1506# III 17.27 - -A1511# III 11.86 - -U1521# III 12.72 - -C1773 IV 0.52 H-bond and HP -A2041 V 0.44 - H-bondA2042 V 0.61 HP H-bond and HPA2045 V 0.70 HP H-bondC2046 V 0.80 HP -
G2286# V 10.29 - -U2298# V 10.49 - -A2418 V 0.78 H-bond -A2482 V 0.47 H-bond and HP -G2484 V 0.52 HP H-bond and HPU2564 V 0.47 H-bond and HP -C2565 V 0.43 HP -U2588 V 0.57 H-bond H-bond and HPC2589 V 0.63 HP HPU2590 V 0.52 HP HP
Hydrophobic (HP) interaction was calculated using HBPLUS (42), accounted for interactions by paired carbon-carbon that were those of 3.90 Å away.
Table IIIRadial distribution function (RDF) calculated for selected ligand atoms in ERYA.
RDF First Solvation Peak (Å)
RDF Intensity at First Peak
Ow-O13 2.68 0.13Ow-O6 2.78 1.28
Ow-O10 2.78 0.25Ow-O5 2.78 0.62
Ow-O11 2.83 0.24Ow-O8 2.88 0.23
Ow-O12 2.98 0.22Ow-N 3.43 0.32Hw-O6 1.78 0.60
Table IVEstimated number of water molecules from ERYA and selected acceptor atoms of ERYA.
1st signified first solvation shell with spherical radius of <3 Å from acceptor atom and 2nd is second solvation shell with spherical radius between 3-5 Å.
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RDF of Ow and O6 showed first and highest sharp peak at 2.78 Å with the coordination number of 1 when integrated up to the first minimum at 3.48 Å. This RDF profile also showed the highest RDF intensity, indicat-ed most water molecules clustered around O6 (refer to Table IV) as com-pared to the other acceptor atoms. RDF of Ow-O13 (oxygen of 12-OH of lactone ring) and Ow-O10 (oxygen of 6-OH of lactone ring) showed the first peaks at 2.68 and 2.78 Å, respectively. The low intensity indicated less number of water molecules surrounded these acceptor atoms. This is also agreed with the low number of water molecules found in the first and second hydration shells (Table IV). Comparing to the other RDF profiles (with the exception of Ow-O6), Ow-O5 has the highest RDF intensity values and more water molecules (totaling 35) at its first and second solvation shells. In contrast, Ow-O11 has only a total of 8 water
g(r)
Distance (Angstrom)
Figure 5: Radial distribution function (RDF) between oxygen of water molecule (Ow) and O6 of ERYA.
Desosamine
Lactone1.70
1.88
O6
Cladinose 2.03
U2564
O52.61
2.83
1.86
2.982.37
G2562
1.92WAT79259C2589
C 2046A 2045 A 2482
U 2564
O1P
P
O2P
A 2042
ERYA
C 2565
U 2590
C 2589
G 2484
C 759C 1773
U 2588
Figure 6: Water-bridged H-bonds at the binding site between O5 and O6 of ERYA and nucleotides G2562, U2564, and C2589. H-bond distances (in Å) are also shown.
Figure 7: Ligplot (41) representation of average structure of 500-1300 ps window, revealing hydrogen bonds (green dotted lines) and hydrophobic contacts (red spoked arcs pointing towards ERYA) between ERYA and bases C765, U2588, A2045, A2482, G2484, C2565, C2589, and U2590.
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molecules in the first and second solvation shells. The RDF plots for Ow-O8 (2ʹ-OH of desosamine sugar) and Ow-O12 (11-OH of lactone ring) showed their first peaks at 2.88 and 2.98 Å, respectively. The average number of water molecules was found to be the lowest for O12 (only 1 in the second solvation shell). In the case of N atom, the first peak occurred only after 3 Å and 39 water molecules were found in the second solvation shell (no water molecule found in the first solvation shell) indicating that this atom was less hydrated than the other oxygen atom in the sugar moiety.
To confirm whether O6 is making any H-bonding with water molecules, RDF and H-bond analysis were also performed for Hw (hydrogen atom of water molecule) and O6 (Table III). Hw-O6 plot showed the first peak formed at 1.78 Å. H-bond analysis (Table V) also showed that 86.12% of occupancy when water molecules acted as H-bond acceptor to O6, with the average length of 2.84 Å and angle of 160.88º (see below Hydrogen Bond Analysis and Hydrophobic Interactions for cutoffs used). On the other hand, when water molecules acted as H-bond donor
Figure 8: Distance for selected H-bonds suggested by Schlünzen and co-workers (8) as seen in this 1.3 ns simulation. Majority H-bonds had its distance for >3.50 Å when approaching equilibration and production stag-es. As a result, H-bonds between them were no longer formed and this result did not show agreement with the pattern found in crystal structure.
Figure 9: (A) (Left) Distance of ribosomal proteins (in Å) L4, L22, and L32 from mass center of ERYA is shown here. New cartoon representation of backbone indicated three nearest ribosomal protein: L4 (yellow), L22 (cyan), and L32 (purple), together with ERYA in licorice representation. Other nucleotide residues are omitted for clarity. (Right) Time dependent distances between the backbone of selected amino acid from L4, L22, and L32 to the mass center of ERYA. (B) (Left) Time dependent distance for magnesium ions from nearest atoms of ERYA. (Right) Figure showed two magnesium ions (MG3281 and MG3282), licorice rep-resentation of ERYA (all H atoms were omitted) and nucleotides A806, C2420, C2421, C2431, and U2485. Both magnesium ions are found far from ERYA; how-ever, they are mainly coordinated to the phosphate ox-ygen of these nucleotides. Other nucleotide residues were omitted for clarity.
24
22
20
18
16
14
Dist
ance
(Ang
stro
m)
0 200 400 600 800 1000 1200Time (ps)
14
12
10
8
6
4
Dist
ance
(Ang
stro
m)
0 200 400 600 800 1000 1200Time (ps)
L4
L22
L32
ERY-A18.37
21.55
14.72
R109-S113 of L22M1-K8 of L32Y59-A67 of L4
MG3281-O11@ERY-AMG3282-C28@ERY-A
A806
MG3281
10.11
21.55
7.88
C2421
C2420
O11
Lactone
Cladinose
Desosamine
C28
MG3282C2431
U2485
A
B
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to O6, occupancy of 52.25% was found with 3 Å and 145.99º, respectively. It is interesting to note that O5 also formed H-bond with water molecule with high occupancy of 66.62% (length of 2.97 Å and angle of 144.05º).These observations showed strong and persistent H-bonds formed between water molecules and cla-dinose sugar, thus maintaining the cladinose sugar at the binding pocket. These H-bonds were also found at appropriate geometry to bridge H-bonds between cla-dinose sugar and nucleotides in the binding site.
It is interesting to note that there were many water molecules clustered in between U2564 (U2585EC) and O6. However, when H-bond cutoff were applied to classify them, water-bridged H-bonds were found to occur only in 30% of the simulation time. These water-bridged H-bonds were formed between O6 and mainly with N3 and H3 of U2564. However these water-bridged H-bonds were found to be not per-manent, as when O6 and U2564 came closer together, water molecules moved aside to make way for direct H-bond. It is also worth noting here that the water molecule that bridged H-bonds between O6 and U2564 was not always the same molecule. Another water-bridged H-bond was found between O5 and G2562 (G2583EC) and C2589 (C2610EC). Unlike the former H-bond, water molecule that bridged H-bond between O5 and G2562 was found to be the same molecule (WAT79259) from 600 ps onwards. A snapshot of these water-bridged H-bonds is shown in Figure 6. In contrast to this finding, Mao and co-worker (46) in their Structure Activity Relationship (SAR) studies previously showed that O6 was not able to mediate any H-bonds due to its dispensability for macrolide binding. The crystal structure (8) also showed that no H-bonds were mediated from this sugar. The analysis of our trajectory gives, however, some indication that this sugar moiety might involved in the binding of ERYA to ribosomal subunit through water molecules.
We also observed that there were many water molecules at the binding site and in the first solvation shell of ERYA, but not all of them were involved directly in bridging ERYA with the binding pocket. These water molecules might possibly contributed to the overall stabilization of ERYA binding pocket by holding them in the right position via interconnecting network of H-bonds. Some crystallography and MD simulation studies have previously shown the importance of water and water-bridged H-bond in many receptor-ligand system such as protein-ligand (47-50), DNA-ligand (51-53), RNA-ligand (54-57), etc.; therefore, we believe this finding is important and should be taken into consideration in the understanding of their binding interaction.
Hydrogen Bond Analysis and Hydrophobic Interactions
Hydrogen bond (H-bond) is one of the main interaction for the binding of ligand to the binding pocket and there were at least six H-bonds observed from the X-ray structure (8) that kept ERYA in the pocket. H-bonds were assumed to be present if distance of H-bond donor-acceptor was <3.5 Å and the angle formed between donor-H-acceptor was >120º. The classification of H-bond occupancies used are as follows: persistent H-bond if occupancy is >60%, medium H-bond if occupancies are 30% to 60% and weak H-bond if <30% (58). Due to the enormous amount of H-bonds found during this simulation, only H-bond with >20% of occupancy are discussed here.
It was found that C1773 (U1782EC), U2588 (U2609EC), C765 (A752EC), A2418
Table VH-bonds formed between water molecules and ERYA in the 500-1300ps window.
H-bond distance and angle cutoff used in this analysis are 3.50 Å and 120º, respectively. Occupancy <50% are not shown here.
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(A2439EC), and A2482 (A2503EC) played an important role in the binding of ERYA. C1773 formed at least five different H-bonds with O12 and O13 of the lactone ring for ERYA throughout the 1.3 ns simulation (Table VI). The H-bond between N3 of the pyrimidine base of C1773 and hydrogen of O13 of ERYA were found to occupy 50.85% of the total simulation with an average distance of 2.93 Å and mean angle of 145.25º. This H-bond was found with medium occupancy during the equilibration stage but became persistent until the end of simulation. C1773 also formed medium H-bond contacts with hydrogen of O12 and O13 of ERYA through its N3 and O2 at the pyrimidine base, with the percentage occupancy of 31.00% (average distance and angle of 3.11 Å and 152.32º, respectively) and 32.46% (3.14 Å and 149.21º) correspondingly. There were also H-bonds formed between hydrogen of O12 of ERYA with O2@C1773 and between oxygen O13 to N4 of C1773. The dynamic features of these two bonds were correlated with the percentage occupancy of 28.77% and 24.85%, with average bond length and angle of 2.99 Å and 139.94º, 3.16 Å and 136.31º, respectively. These two H-bonds were seen with low occupancy in the early stage of MD but strengthened during MD production stage before it weakened in the final stage of simulation.
U2588 formed a medium H-bond at O4 of its pyrimidine base with hydrogen of O12 of ERYA, with mean distance of 2.80 Å and angle 142.97º. This H-bond was found to be persistent in early stage of MD, but was deformed during 700 ps to 1000 ps. However, it was reformed after 1000 ps and stayed on moderately as the production time evolved. This H-bond was 44.38% occupied during the 1.3 ns simulation, indicated its significance in keeping ERYA at its pocket. C765 formed H-bond with oxygen of O13 at ERYA via N4. This H-bond has an occupancy per-centage of 34.69% and with average bond distance of 3.07 Å and a mean angle of 137.10º. This bond interacted strongly and persistently even from the start of the simulation but became weaker after 500 ps. Other residues in Domain V that were found to be involved in H-bonds interaction included A2418 and A2482. A2418 formed a weak H-bond at N6 of its purine ring with oxygen of O10 of lactone ring with the occupancy of 22.46%, average distances and angle of 3.21 Å and 138.27º, respectively. H-bond between N6 of A2482 and oxygen O9 of desosamine sugar was found in the early stage of the simulation but was weakened after 500ps. The average distance and angle formed was 3.18 Å and 145.08º, respectively.
Hydrophobic interaction was computed using HBPLUS, where the interactions/forces exerted were those of 3.90 Å away for paired carbon-carbon and it is il-lustrated in Ligplot representation. Figure 7 showed H-bonds and hydrophobic interaction found in the average structure with its interacting nucleotide residues. There were a total of four H-bonds found in the average structure, namely H-bonds between oxygen O12 of ERYA and O4@U2588 with distance of 3.26 Å, nitrogen N3 of U2564 and O6@ERYA with distance of 2.96 Å, oxygen O12@ERYA and O2@C1773 of 3.02 Å and lastly, oxygen O13@ERYA and N3@C1773
Table VIH-bonds formed between ERYA and binding pocket during the 1.3 ns MD simulation.
H-bond distance and angle cutoff used in this analysis are 3.50 Å and 120º, respectively. Occupancy <20% are not shown here. +, predicted H-bond; -, H-bond not predicted.
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with 2.90 Å. There were also 20 hydrophobic interactions found between carbon atoms in ERYA with carbon atoms of interacting residues in the average structure. Of all the 20 hydrophobic interactions, some of these interacting residues made edge to face π-π interaction to ERYA. For example, the hydrophobic interactions between C30 of ERYA to C6@G2484, and C32 of ERYA to C2 and C6 of A2042 showed lactone ring of ERYA was located perpendicular to the base sugar of these purine bases, forming the T-shape π-π interaction. Comparing to the crystal structure, we observed six H-bonds and 11 hydrophobic interactions between the ERYA and the nucleotides in the binding pocket.
The crystal structure (8) showed all the six H-bonds between erythromycin and its binding pocket were mediated through the reactive group of desosamine sugar and lactone ring of the ERYA. Our MD simulation did not showed consistencies with the observed pattern in crystal structure as the observed H-bonds were mainly mediated from O12 and O13 of lactone ring. H-bonds that were found in the crystal structure were H-bonds between 2ʹOH group of desosamine sugar with N1 and N6 of A2041 and to N6 of A2042, 6-OH group of lactone ring with N6@A2045, 11-OH group of lactone ring with O4@U2588 and 12-OH group of lactone ring with O4 at U2588. In our simulation, all of these H-bonds (except H-bond between O12 and O4@U2588) were found to have bonds length >3.50 Å in most of the simulation time and none of them were able to form permanent H-bonds (Figure 8). The importance of the H-bonds between nucleotides A2041 (A2058EC) and A2042 (A2059EC) and mac-rolides were previously shown by crystallography studies (8, 59, 60). Schlünzen and co-workers found three H-bonds that connect erythromycin to A2041 and A2042. Tu and co-workers (59) found a H-bond between 2ʹOH of its desosamine sugar with the N1 of the mutated G2099A,HM (A2058EC) from the crystal structure of Haloarcula marismortui large subunit with MLSBK antibiotics (including erythromycin). Hansen and co-workers (60) on the other hand found in the crystal structure of H.marismortui 50S subunit complexed with the 15- and 16-membered macrolides, that the myca-minose of 16-membered macrolide (or desosamine sugar from 15-membered mac-rolide) formed a H-bond from its 2ʹOH to G2099HM (A2058EC). Although these H-bonds were known to be important in the overall binding of macrolides, we did not found these H-bonds in our simulation. The only interaction that we were able to associate with A2041 and A2042 is the H-bond between 2ʹOH of desosamine sugar and N1@A2041 that was found in the very early stage of the simulation. However, as simulation time evolved, this H-bond slowly disappearing during the equilibration stage and it was totally distorted shortly after this stage (Figure 8).
One of the possible explanation for these observations could be the orientation of the system was different from the orientation found in the crystal structure. This might caused by minimizations done prior the start of this simulation and therefore, causing their interactions to be varied in accordance with their chemical nature. Other explanations might be the criteria used to define H-bonds differ from the one used in the crystal structure and it should also be noted that the classification of these H-bonds were based on their occupancies (total time of existence divided with total simulation time) and therefore those with low occupancies were not discussed and shown here. In a static system, like in a crystal structure, only one snapshot was able to be captured while on the other hand; a dynamic system like the MD simula-tion offered a trajectory of many snapshots capturing many different conformations, thus giving a different pattern of H-bond network when comparing with the crystal structure. Nevertheless, the H-bond pattern that showed here has provided another perspective of H-bond pattern as explicit analysis of H-bond properties enabled us to look at the occupancies and assignment of each H-bond that was formed.
Involvement of Ribosomal Protein and Magnesium Ion
Figure 9A showed distances between the center of mass for the backbone of se-lected nearest amino acid residues to ERYA’s center of mass. These plots demon-
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strated that the nearest ribosomal protein chain, specifically the Met1 to Lys8 of L32, were about approximately 15 Å away from ERYA at the start of the simula-tion, and fluctuated around the mean value of 15.50 Å until the end of the simula-tion. The second nearest protein chain, the ribosomal protein chain of L4 (from residue Tyr59 to Ala67) was ~17 Å distance away from the ligand at the start of the trajectory. It oscillated ~16.50 Å during production stage and the distance was maintained at ~16 Å in the last few hundred ps. The most distant protein chain from ERYA, L22 as demonstrated by its nearest amino acid to the ligand, namely Arg109 to Ser113 were ~19 Å away at the start of the MD simulation. The distance increased to ~22 Å during production stage, and fluctuated around 1-2 Å after 500 ps until the end of simulation. On the average, these three nearest ribosomal protein chains, namely the L4, L22, and L32 were found to be at least 15 Å away from the ERYA, as seen in this MD simulation and also in the crystal structure (8). These finding indicates that the ribosomal proteins were too distant for any significant chemical interactions to occur, and hence supporting the claim that erythromycin binding site is made of only rRNA and ribosomal proteins have no direct implication to the binding of ERYA (8).
Figure 9B showed distances of the two magnesium ions that were found in crystal structure from ERYA. Two magnesium ions (MG3281 and MG3282) were found to be located distant from ERYA. On average, the nearest atom from ERYA to MG3281 was found to be 10.96 Å from O11 of the lactone ring of ERYA and on the other hand, MG3282 was 7.03 Å from C28 of desosamine sugar of erythromy-cin. Based on these distances, both magnesium ions were far for any significant direct interaction to the binding of erythromycin. Both magnesium ions were found near to the phosphate oxygen of A806, C2420, and C2421 (for MG3281); C2431 and U2485 (for MG3282) although no restraints were applied in this simulation to maintain this interaction. Based on these, we believe magnesium ions were not directly involved in the binding of ERYA; instead they might be involved in main-taining the stability of the binding pocket.
Further Discussion
Our MD simulation showed direct interaction of ERYA to Domain II of the 23S rRNA. An H-bond with occupancy of 34.69% was found between N4 of C765 and hydrogen of 12-OH group of the lactone ring of ERYA. Nucleotide C759 formed hydrophobic interaction with ERYA in most of the simulation time. These observa-tions therefore agree with the protection effect found on hairpin 35 of Domain II in footprinting experiments (2, 3, 7) and supported the speculation that the Domain II has direct and significant interactions with ERYA. Nucleotides A2041 (A2058EC), A2042 (A2059EC), and G2484 (G2505EC) were previously demonstrated to be important, as it was shown to be strongly protected by erythromycin from chemical modification (2, 3, 7, 14, 61). Although our simulation was not able to prove the in-volvement of these residues in H-bond analysis (as mentioned above), hydrophobic analysis showed these nucleotide exerts its hydrophobic interaction to ERYA by π-π interaction between its purine bases and desosamine sugar of ERYA.
Our MD simulation also showed the significance of residue C1773 (U1782EC) to the binding of ERYA. C1773 is located in Domain IV, a Domain that has never discussed before to have any direct interaction with the binding of erythromycin. However, C1773 was previously associated to the binding of quinolyallyl group of ketolide ABT-773 (62) through hydrophobic interaction and other than that, it was never reported to have contribution in the binding of other macrolides to 23S rRNA. Visual inspection showed that C1773 was folded in a proximal distance to C765 (from Domain II) and the exposure of this residue to ERYA became apparent in the equilibration stage. H-bond analysis also revealed that C1773 formed at least five different H-bonds with ERYA at different locations and yet, most H-bonds occurred for >20% of the simulation time. This nucleotide residue might be as important as
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other residues that interacts with ERYA, considering it is a cytosine in Deinococ-cus radiodurans (prokaryotic ribosome) and is a uracil in Haloarcula marismortui (eukaryotic ribosome), where the difference in nucleotide sequences between a pro-karyote and eukaryote might potentially affect the drug affinity and selectivity of ERYA to the ribosome. With this, we believed that this residue might have some important implications in the binding of ERYA and further research should be done to investigate the importance of this residue.
Conclusions
In the process of refining a low-resolution structure, enormous amount of valu-able information were obtained in understanding ribosome’s role as a drug target. Based on our study, it was found that the drug binding site was composed only by rRNA and no ribosomal proteins were found to be involved directly as they were too distant from the binding pocket such that direct interaction is irrelevant. Similar observation was seen for the putative magnesium ions as discussed in (8) and it was found that the two ions, were also far from the drug. Therefore, they would also not be able to be involved directly in the overall binding of ERYA-ribosome.
Our study also showed the importance of water molecules as ERYA was highly hy-drated during the course of the simulation. Water molecules were found to form H-bonds with ERYA with high occupancies and interestingly, water-bridged H-bonds were found in maintaining the strong interaction of ERYA to the binding pocket. Water molecules at the binding site have played important role by forming multiple H-bonds, bridging ERYA with binding site and possibly stabilizing the entire drug-binding site interactions. This study should also be useful in understanding hydra-tion sites at ERYA binding site and would be applicable in methods for de novo drug designing. Besides that, H-bond analysis and hydrophobic interactions also contributed to the overall ERYA binding. Although some of H-bond formed did not show consistencies with the experimental results, in general H-bond still exist between ERYA and their contribution is conclusive.
We believe this simulation served as the stepping stone of studying more rigor-ously into the detail mechanism of action of the ERYA to 50S large ribosomal subunit that currently is not able to be offered by the experimental studies. A lon-ger simulation (which is now in progress) would ensure a higher reliability, and insight obtained from it would definitely be needed to assist in the development of safer macrolide antibiotics in the future.
Acknowledgements
This work was supported by the Ministry of Science, Technology and Innovation (MOSTI) grant (grant number: 304/PFARMASI/640038/K105). The generous supply of computational time by MIMOS Berhad for this research is gratefully acknowledged. We thank National Biotechnology Network for providing our lab with basic computational facilities. We also thank Prof. Janez Mavri for criti-cally reading this manuscript.
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