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* Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, Texas 77030; Department of Chemistry and Department of Biology and Biochemistry, University of Houston, Houston, Texas, 77004; and Department of Biochemistry, University of Otago, Dunedin, New Zealand 9001
Correspondence: Address reprint requests to B. Montgomery Pettitt, E-mail: pettitt{at}uh.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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). T. vaginalis is pervasive and cases are often asymptomatic. Fortunately, infections are usually curable with a single dose of the drug metronidazole, a nitroimidazole (trade name Flagyl). T. vaginalis has recently come under closer scrutiny, as studies have shown that infection can increase the transmission rate of human immunodeficiency virus (HIV) (2–5). This fact, coupled with the often subclinical nature of an infection, points to T. vaginalis as an important public health risk. In addition, there is an increasing prevalence of metronidazole-resistant T. vaginalis strains (6).
For more than 30 years, T. vaginalis has proven to be an excellent organism to use to study the mechanism of action of the important antibiotic metronidazole. T. vaginalis' exquisite sensitivity to metronidazole sparked extensive research in the 1980s that uncovered an important role of hydrogenosomal ferredoxin in the activation of this prodrug. Within T. vaginalis, the hydrogenosomal protein T. vaginalis ferredoxin (Tvfd) activates the drug metronidazole. Tvfd is a small protein at 9.8 kD and 93 residues with a [2Fe-2S] cluster that participates in electron transfer reactions (7). Physiologically, Tvfd functions to shuttle electrons between pyruvate:ferredoxin oxidoreductase and hydrogenase in the metabolic cycle of T. vaginalis (8). When metronidazole is present, an electron is transferred from Tvfd to the drug. Interestingly, Tvfd has a redox potential of –347 mV, which is more positive than photosynthetic [2Fe-2S] ferredoxins (e.g., –440 mV for vegetative Anabaena ferredoxin) (9) but less positive than most oxidase ferredoxins. Unfortunately, no cocrystal with both Tvfd and metronidazole exists.
The crystal structure for this protein was solved to 2.2 Å by x-ray crystallography (10). Overall, the fold was similar to that found for [2Fe-2S] vegetative and oxidase classes of ferredoxins. The average B-factors from the crystal structure were 35, 36, and 47 Å2, respectively, for the three molecules A, B, and C in the asymmetric unit (10). In addition, electron density for the third molecule in the asymmetric unit showed main chain breaks between residues 31 and 42 (10), and all molecules showed increased B-factors for this loop of residues, >80 Å2 for Asp-35 (Protein Data Bank structure 1L5P). These data suggested to us that the [2Fe-2S] active site loop was dynamic and that one of the molecules of Tvfd in the unit cell might have a different structural conformation for the loop than the other two molecules of Tvfd in the asymmetric unit. Since strict noncrystallographic symmetry restraints were used in the solution of the structures, no variations would have been seen between the backbones of the molecules in the crystal structure (10). Few waters in the interior of the protein and none within 6 Å of the active site were resolved (10). The crystal structure revealed an interesting feature of Tvfd: one side of the iron-sulfur cluster was more exposed to solvent than other similar ferredoxin structures, with a small cavity allowing one of the active site sulfurs to be just accessible. These results prompted the hypothesis that direct access to the active site cluster was responsible for the fast kinetic rates seen for the Tvfd to metronidazole electron transfer reaction: first, from the crystal structure showing an opening into the active site (10) and second, from the kinetic data that showed extremely fast metronidazole active by Tvfd (11).
The exact role of Tvfd in metronidazole activation is an object of debate and continued study. Tvfd has been found to activate metronidazole in a variety of different experiments. Work carried out in vitro showed rapid activation of metronidazole by Tvfd (11), and some studies have also shown lowered expression of Tvfd mRNA in metronidazole-resistant strains of T. vaginalis (12). Expression of Tvfd in a metronidazole-resistant Trichomonas fetus cell line that was lacking native ferredoxin expression lead to metronidazole susceptibility within the cell line (13). In addition, kinetic studies of the ferredoxin to nitroimidazole electron transfer reaction showed that Tvfd reduces metronidazole and other nitroimidizole drugs one to two orders of magnitude faster than [2Fe-2S] photosynthetic Anabaena ferredoxin, even given that the Anabaena ferredoxin has a more negative redox potential (11).
Interestingly, however, a Tvfd knockout was created that did not show resistance to metronidazole, leading to the hypothesis that there may be other ferredoxins or flavins in T. vaginalis in addition to Tvfd that can activate the drug (14). Review of the recently completed Tvfd genome reveals seven hydrogenosome targeted ferredoxin genes (15), explaining why a single knockout would be unlikely to result in nitroimidazole resistance. Although these other redox proteins may play a role, the preponderance of evidence points to Tvfd as a major activation protein of metronidazole in T. vaginalis.
The two iron atoms in plant-type ferredoxins are high spin, antiferromagnetically coupled to each other (16). Preceding the reaction with metronidazole, the [2Fe-2S] cluster in Tvfd is in its reduced state, Fe+2/Fe+3. The extra electron was shown in plant-type ferredoxins to be localized to the iron closest to the exterior of the protein (labeled Fe1) by NMR spectroscopy using the nuclear Overhauser effect (17). The iron sulfur site is coordinated with the protein through the sulfur groups on four cysteines (7). Three of these cysteines are part of a loop of residues 31–48 that wraps around the iron-sulfur cluster shielding it from the solvent. In the process of antibiotic activation, an electron is transferred from the reaction center to metronidazole, oxidizing the [2Fe-2S] cluster. Once the electron is transferred to metronidazole, electron spin resonance studies show a characteristic nitro radical species for metronidazole (18–22). This radical anionic species of metronidazole causes oxidative damage to the organism, mainly through inhibition of nucleic acid synthesis (23) caused by direct damage to DNA (18,24). Although metronidazole has a redox potential of –345 mV, comparable to many ferredoxins from anaerobes, it is more negative than most aerobes. Various mechanisms are postulated for metronidazole being used to treat human anaerobic infections without damaging human DNA such as the redox potential difference of TvFd verses aerobes reduction potentials and metronidazole (14) or by oxygen inactivation of metronidazole radicals (25).
Two [2Fe-2S] ferredoxins have been crystallized in both their reduced and oxidized forms. Anabaena ferredoxin structures differed by a flip of the backbone between Cys-46 and Ser-47 (26). Those authors labeled the flip "CO out" in the reduced form and "CO in" in the oxidized form. There were no other structural changes between the two forms of the protein. Putidaredoxin has also been crystallized in the reduced form and compared to the oxidized x-ray crystal structures (27). The reduced form also showed an analogous backbone flip, between Cys-45 and Ala-46. In addition, other small structural changes were seen within the region of the iron-sulfur cluster.
In this study we set out to explore the role of flexibility in the [2Fe-2S] loop region of the protein and its potential implications in metronidazole activation. We hypothesize that the structural flexibility in the loop composed of residues 31–48 and the additional access to the active site sulfur seen in the crystal structure may play a role. Since no cocrystal has been solved, we look solely at the dynamics of the Tvfd protein. Starting with the crystal structure of Tvfd solved to 2.2 Å by multiple anomalous dispersion phasing (10), we explore the dynamics of solvation and the structure of the active site region to better explain Tvfd's exceptionally fast kinetics and selective activation of metronidazole. We use molecular dynamics (MD) simulations to study the movement and solvation of a model of Tvfd. Two simulations of equal length were performed, one with the protein in the reduced state, and the other in the oxidized state. These simulations were run for a period of time (20 ns) sufficient to observe aspects of conformational equilibrium in the protein.
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MATERIALS AND METHODS |
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). Because the iron-sulfur parameters were not included as part of the Charmm27 set, they were derived from experimental and computational sources. The bond and angle force constants were obtained from resonance Raman spectra (29), the partial charges from density functional calculations (30), and the angles and dihedrals from those used in the refinement of the crystal structure (10). Other ferredoxin simulation studies have used the force constants from similar sources (31,32).
The initial configuration was the structure labeled A in the asymmetric unit from the crystal structure of Tvfd (1L5P), but with added hydrogens. This structure was chosen as it had the lowest average B-factor and the best electron density throughout the main chain (10). The protein was solvated with 9447 Charmm TIP3P water (33) molecules within a cubic box of length 66.4 Å. No water was placed for which the oxygen from the water was within 2.5 Å of a heavy atom in the protein. This setup produced at least three solvation shells around the protein. Nine sodium counterions were added to the box to neutralize the charge on the protein, and an additional 32 Na+ and 32 Cl– were added to make a final salt concentration of 0.18 molar NaCl. The ions were added by replacing a water molecule randomly in the box. The same box was used to set up the oxidized structure; however, the appropriate parameters were used and a sodium ion was removed to neutralize the charge in the box. This initial box went through three rounds of steepest decent minimization: the first round minimized the water holding the protein rigid, the second minimized the protein while holding the water rigid, and the third minimized the whole system.
MD simulations were run using the microcanonical ensemble with periodic boundary conditions in the Extended System Program (34). Electrostatics were calculated using the Ewald sum (35), with the Lennard-Jones real space cutoff set to 16.2 Å. Ewald parameters were optimized using the guidelines set by Fincham (36) with rcut set to 16.2 Å. The RATTLE algorithm (37) was used to constrain all the bond lengths with a tolerance of 1 x 10–7 Å, and the Velocity Verlet algorithm was utilized with a time step of 2 femtoseconds. The system was initially relaxed by running a series of 10 10-ps simulations with velocities being reassigned at increasing intervals. Another 900 ps of simulation was run with no modifications during equilibration.
All root mean-squared deviation (RMSD) and dihedral calculations were calculated using visual molecular dynamics (VMD) (38). Water and ion density profiles were calculated by dividing the simulation box into 81 bins per side of the box, which translates to a bin length of 0.82 Å. The oxygen nucleus in a water molecule was used to form the water density histograms. This was done over the last nanosecond of trajectory data and normalized (39). The same calculation was done for ions. The system was equilibrated for 1 ns as described above. After equilibration, 20 ns of production were run with trajectory snapshots saved every 100 femtoseconds. The total energy of the two simulations after equilibration remained constant; both had an internal root mean-squared difference of <0.956 kcal/mol.
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RESULTS |
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). Mean-squared atomic fluctuation (MSF) showed strong variations with respect to different time windows. These calculations were done for the 3rd, the 10th, and the 19th nanosecond of trajectory data and also compared to the B-factors for each molecule in the asymmetric unit of the crystal (10) (Fig. 2). The time periods over which the trajectories were averaged for later comparison were chosen for having stable equilibrated structures over at least an entire nanosecond of data. They correspond to a time period after a conformational change that also includes relaxation time for the other molecules in the system to respond to the protein's changes. From this point on, the time period before the 5th nanosecond will be labeled T1, the 5th nanosecond to the 15th nanosecond labeled T2, and the time period after the 15th nanosecond T3. Also, averaged structures over the 3rd, 10th, and 19th nanoseconds will be labeled S1, S2, and S3, accordingly. The calculations were completed over 1-ns windows to provide enough data to represent the local minimum surface.
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2) (40).
Although it is difficult to quantitatively relate the B-factors to the MSF calculations due to errors in the model, noncrystallographic restraints in the refinement, and poor density in some regions (10), a clear relation between the two is seen in the trends of each graph. The differences in MSF between the different nanoseconds calculated showed only small differences in the fluctuations occurring in the residues, which indicate stability. There are, however, small changes that have occurred in the structure that were seen from the backbone overlay of three averaged structures and the crystal structure (Fig. 3). Some movement in the loop composed of residues 31–48 that contains the active site was seen. Examination of the crystal contacts in the crystal structure (Fig. 2 B) shows contacts in the loop region for all molecules. However, molecule A has more than C, and B has more than C (41). This could be one explanation for the improved order of the loop in molecule A in comparison to B and C. Note that B and C are quite similar to A in the rest of the protein (not the loop) despite having fewer contacts.
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atoms. The major feature of this opening event was seen in the increase in heavy atom RMSD from <2 Å to >2.5 Å at the fifth nanosecond (Fig. 1).
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). It is the deviation from the crystal structure of this loop portion of the protein that increases the heavy atom RMSD, whereas the rest of the protein is essentially stable. When viewing the loop in S1 and S2, the top and edge of the loop that was originally tilted up shielding the [2Fe-2S] cluster has moved away from the cluster and is instead extended into the solvent region, leaving a sizable accessible pocket near the [2Fe-2S] cluster. This provides different and larger access to the iron sulfur cluster than seen in the crystal structure. There are two areas in the structures of S2 and S3 that deviate. The first is the same loop as between S1 and S2 made of residues 31–49; the second is between residues 81–89, in which the third helix in the protein has relaxed. Dynamically, the iron-sulfur cluster moves very little and has an RMSD compared to the crystal structure after the loop movement of 0.85 Å.
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Analysis of loop-cap opening transition
The conformational change during the fifth nanosecond of simulation occurred over 
1 ns. The first step in this structural change was seen at 4.8 ns, during which the dihedral angles of Lys-42 and Ala-43 flip (Fig. 7). At 5.4 ns on the opposite side of the loop, residues 34–39 move away from the [2Fe-2S] cluster. This loop movement was also observed in the dihedral angles of Ala-34, Asp-35, Asp-36, and Gln-39 (Fig. 7). Further movement of the loop occurs after the 16th nanosecond to move the tip of the loop farther from the protein close to 90° from where it was originally located in the crystal structure, further enlarging the exposure of the [2Fe-2S] cluster. This was shown in the additional dihedral movements of Gln-39 and Lys-42 near the end of the simulation (Fig. 7).
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The nonbonded interactions between the protein and the [2Fe-2S] cluster changed significantly after the fifth nanosecond. The movement of the loop away from the iron-sulfur cluster disturbed many of the nonbonded interactions between the backbone of the protein and the cluster. In addition, a structurally significant salt link was broken; this interaction was between Lys-42 and Glu-65. This connection "held" the top of the loop closed against the second helix in the structure which was not directly connected to the redox cluster. As the loop moved away from the cluster, the noncovalent bond was broken between this pair, with the residues then becoming solvated. The analysis of the dynamics of the active site during the fifth nanosecond showed that residues 31–49 have opened up to create a crevice that is large enough to allow easy access to the [2Fe-2S] cluster to both dynamic water and ions.
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DISCUSSION |
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). This loop-cap transition seen to a lesser extent in the oxidized state can also explain the refinement difficulties in this region for the original x-ray data; it implies conformational heterogeneity in the structure. It is known that redox centers of metalloproteins can undergo photoreduction during synchrotron data collection (42,43). It is possible that this phenomenon could have occurred during the data collection for the structure; but no direct proof is available.
Computational studies on both the reduced and oxidized states of a rubredoxin have also shown increased solvation of the active site in the reduced state in relation to the oxidized state (44). In addition, loop-cap transitions of this type have been described before. In triosephosphate isomerase a loop-cap transition plays a key kinetic role in the mechanism (45). Studies on the oxidized and reduced state of both Anabaena ferredoxin (26) and putidaredoxin (27) showed that conformational changes near the active site have occurred between the two redox states. This implies loop movement near the redox center is associated with redox state.
Marcus and Sutin showed that the electron transfer rate is dependent on the coupling between the electron acceptor and donor, which is mainly due to distance, the redox potential, and the so-called rearrangement energy cost of the reaction (46). If the electrostatic thermodynamic driving force were the main contribution to the electron transfer rate (47), then Anabaena ferredoxin would be expected to show faster kinetics than Tvfd due to its higher reducing potential, –440 mV for Anabaena ferredoxin versus –347 mV for Tvfd. However, this is not the case, implying that either the electronic coupling or the rearrangement energy must be the primary determining factor of the kinetic rate.
The crystal structure of Tvfd showed a crevice in Tvfd that modestly exposes the iron-sulfur cluster of the active site to solution (10). Our simulation showed a different but nearby large active site exposure in the reduced state through the movement of the active site loop away from the iron-sulfur cluster. This clearly allows for molecular contact with solvent and small molecule drugs like metronidazole. A distance decrease of 20 Å between reacting centers has been previously shown to increase to 1012 in the electron transfer rate (48). Interestingly, it has also been theoretically shown that the solvation of iron-sulfur clusters may also affect the redox potential, with more highly solvated clusters having a more positive redox potential (49). Although we cannot make direct comparisons with kinetics from just this one simulation, we can hypothesize that the opening of the loop of residues to create an opening into the active site would allow metronidazole easier access to the [2Fe-2S] cluster, and thereby faster reaction kinetics.
The structural changes shown here also give a plausible explanation of the experimental crystal B-factor data. This is an indication that the experimental density that was seen in the structure was related to the actual dynamics of the protein. Our simulation also predicts that changes to certain amino acids in the protein will affect the kinetics. Changes to Lys-42 and Asp-62 or Glu-65 should affect the kinetics since this nonbonded link may keep the loop closed. In addition, other residues could be changed within the [2Fe-2S] binding region to investigate steric affects. We hope to test these hypotheses in future work.
Due to both the increase in drug-resistant strains and to the link between Trichomoniasis and increased HIV transmission rates, T. vaginalis is a serious public health risk. Greater understanding of the mechanism of current therapy can aid efforts to counteremerging resistance as well as aid in our understanding of electron transfer reactions of high relevance to biology. To this end, our results have shown a loop-cap transition that not only explains the high B-factors seen in the crystallographic data, but also offers an explanation of the exceptionally fast and selective electron transfer kinetics seen for Tvfd and nitroimidazoles.
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SUPPLEMENTARY MATERIAL |
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ACKNOWLEDGEMENTS |
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The authors acknowledge the use of VMD for making the molecular graphics plots and CCP4 for calculation of the crystal contacts in Fig. 2.
Submitted on April 27, 2006; accepted for publication November 27, 2006.
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REFERENCES |
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(solid) and 
(shaded) of specific residues in the active site loop.