Targeting Divalent Metal Ions at the Active Site of the HIV-1 RNase H Domain : NMR Studies on the Interactions of Divalent Metal Ions with RNase H and Its Inhibitors

HIV-1 reverse transcriptase (RT) RNase H (HIV-RH) is a key target of anti-AIDS drugs. Metal-chelating compounds are an important class of chemicals in pharmacological drug discovery, especially in relation to HIV-RT and the highly-related HIV-integrase. The correlation between the metal-chelating properties and enzyme activities of the metal chelators is always of high scientific interest, as an understanding of this may accelerate the rational optimization of this class of inhibitors. Our NMR data show that Mg and Ca bind specifically to the active site of the RNase H domain and two Mg ions sequentially bind one molecule of RNase H. We also demonstrate here, using saturated and unsaturated tricyclic N-hydroxypyridones designed to block the active site, that the primary binding sites and affinities of divalent metal ions are correlated with the structures of the chelating motifs. Chemical shift perturbation studies of protein/metal-ion/compound ternary complexes also indicate that divalent metal ions play important roles for the specific interaction of the compounds with the RNase H active site.


Introduction
HIV-1 reverse transcriptase [1] converts single-stranded retroviral RNA into double-stranded DNA, which is integrated into the cellular genome [2][3][4].HIV-1 RT is a multifunctional enzyme that has RNA-directed DNA polymerase, DNA-directed DNA polymerase and ribonuclease H (RNase H) activities [1].HIV-1 RT is a heterodimer composed of two peptide subunits, p66 and p51.The polymerase active site is located at the N-terminus of the p66 subunit, whereas its C-terminal end contains the RNase H active domain [5].The RNase H domain of HIV-1 RT (HIV-RH) plays a role in many steps of reverse transcription, such as the generation of an RNA primer for synthesis of the (+)-strand DNA, the degradation of the viral genomic RNA in the intermediate RNA•DNA hybrid, and the removal of host tRNA and plus-strand primers [2,6,7].HIV-RH forms a central five-stranded β-sheet surrounded by four α-helices.The core domain of the RNase H active site contains a highly-conserved DEDD motif that consists of four acidic residues, D443, E478, D498, and D549 [5,7].The hydrolysis of the scissile phosphodiester bonds catalyzed by RNase H requires divalent metal ions, preferably Mg 2+ [6,8].However, there has been some controversy regarding the number of metal ions (one or two) involved in the catalysis.Crystallographic studies of both E. coli RNase H and HIV-RH have shown that the active site can bind two Mn 2+ ions separated by approximately 4 Å and the authors proposed a two-metal-ion catalytic mechanism [9,10].The ability of HIV-RH to bind to two metal ions, either two Mn 2+ or two Mg 2+ , has also been confirmed by calorimetry and NMR, respectively [9,11].However, this result was not supported by the crystal structure of the E. coli enzyme, in which only a single bound Mg 2+ ion was identified (even though the crystal was obtained in high concentration of MgSO 4 ) [12].Recently, two Mg 2+ ions were also observed ~4 Å apart in Bh-RNase HC-substrate complexes by Nowotny et al. [6,8].Later analysis by the same group also strongly supported the two-metal-ion catalytic mechanism [6,13].
The RNase H activity of HIV-RH plays a crucial role in the retroviral life cycle [14].Defective mutations of two key residues (E478Q and H539F) in the RNase H domain induce a marked reduction in viral proliferation [15], thus making HIV-RH an attractive chemotherapeutical target for anti-HIV drugs [16,17].Designing metal-chelating compounds that are able to bind two divalent metal ions at the active site is one strategy that enables the direct blocking of the active site.Small N-hydroxyimide analogs that are optimized to bind two divalent metal ions at a 4 Ǻ distance between the ions inhibit HIV-RT activity in vitro with an IC 50 < 1.0 uM [10].Metal-chelating compounds are an important class of chemicals in pharmacological drug design, especially when targeted against proteins with metal ions at their active sites [4].Metal-binding property of the metalchelators affects the potency of enzyme inhibitors, and the correlation between metal-chelation affinities and enzyme inhibition is always of interest in the design of metal-chelating inhibitors.
In this report, we applied NMR analytical methodology to study the interactions of the divalent metal ions Mg 2+ and Ca 2+ with HIV-RH and chemically-engineered metal-chelating compounds.When the pH and ionic strength are carefully maintained, we observed clean chemical shift perturbations at the active site of RNase H with both Mg 2+ and Ca 2+ .We also used 1D 1 H-NMR to characterize the interactions between the divalent metal ions and HIV-RH inhibitors.Two chemically similar series, saturated and unsaturated tricyclic N-hydroxypyridones have different chelating affinities depending on the structure of the primary metal-binding sites.To understand the correlation between the metal-chelating properties and the inhibition of the enzyme by metalchelating inhibitors, we also studied the HIV-RH/Mg 2+ / inhibitor ternary complexes using a saturated tricyclic N-hydroxypyridone and -thujaplicinol as the tool compounds.

Backbone Resonance Assignment of HIV-RH
All of the experiments for the resonances assignment were recorded at 30˚C on a Bruker Avance 700 MHz spectrometer equipped with a TCI cryo-probe.The NMR sample contained 0.9 mM 13 C, 15 N-labeled HIV-RH in 25 mM bis-Tris-d 14 (pH 6.5), 150 mM NaCl, 1 mM dithiothreitol-d 6 (DTT) and 5% 2 H 2 O.The backbone resonances were assigned using standard triple resonance experiments, including CBCA(CO)NH, HNCACB, HNCA, [18] and HN(CO)CA [19].Sequential assignments were obtained using TopSpin 2.0 (Bruker Biospin) for spectral processing and SPARKY for sequential analysis [20].

Metal Titration with HIV-RH and Its Inhibitors
In the HIV-RH titration experiments, 0.5 M MgCl 2 or Ca(NO 3 ) 2 was titrated into the protein solution.HIV-RH protein (70 uM) was prepared in buffer containing 25 mM bis-Tris-d 14 (pH 6.5), 150 mM salt (NaCl for Mg 2+ titration and NaNO 3 for Ca 2+ titration, respectively), 1 mM DTT, and 5% 2 H 2 O.The pH of all reagents used in the titrations was carefully monitored and readjusted to 6.5 (if required) at each step of the titration. 1H- 15 N HSQC spectra were acquired on an Inova 600 MHz spectrometer (Varian Inc.) at 25˚C to monitor the chemical shift changes as the metal ion concentrations increased.
In the titrations with the inhibitors, each sample was prepared independently, using identical buffer conditions (except for the concentration of the metal ions).The final observed, most likely as a result of regional flexibility and/or signal overlapping.Most of those amino acids, i.e., N474-K476, E514-L517, A538-K540, and V548-L560, were located in loop regions and at the C-terminus.We observed and assigned three amino acids (D443, E478, and D498) of the DDED motif involved in metal binding, except D549 which is located in the flexible C-terminus of the protein.This is consistent with previous reports [22] that the C-terminus of isolated HIV-RH is highly dynamic and can adopt either an α-helical or random coil conformations in crystal structures, depending on the crystallization conditions and the space group [23].In solution, the C-terminus is usually disordered, but can be stabilized at high concentration of Mg 2+ (80 mM) [11,24].concentration of the compound was 0.20 to 0.50 mM.1D 1 H-NMR experiments were performed using an Inova 600 MHz spectrometer (Varian Inc.) at 25˚C.All spectra were recorded with 128 transients, a 16-ppm sweepwidth using presaturation for water suppression.
The NMR data were analyzed using ACD NMR Processor (ACD-Labs, Inc) and the total chemical shift change Δ obs of the 1 H-15 N cross peak was calculated according to the formula, 0.17 where were the chemical shift changes in 15 N and 1 H dimensions, respectively.The co-crystal structure of HIV-RH domain protein [21] was used for chemical shift mapping.
We observed that the conformation, stability and activity of HIV-RH are very sensitive to pH, ionic strength and divalent metal-ion concentration.A drop in the pH of the buffer from 7.0 to 5.0 induced dramatic and global shifts in the 1 H-15 N HSQC spectrum, and more HSQC signals were detectable at lower pH.Interestingly, most early structural studies of HIV-RH by NMR [25][26][27][28] and x-ray crystallography [29][30][31] were carried out in acidic conditions, suggesting that the isolated HIV-RH domain is more stable at lower pH.

Calculation of the Dissociation Constants (K d )
In the single binding mode, one metal ion binds with one molecule of protein or metal-chelating inhibitor.When the reaction occurs under the fast exchange conditions, the observed chemical shift ( obs ) is the weighted average of the chemical shifts of the free and bound species.Therefore, the observed chemical shift change (Δ obs ) is a function of the dissociation constant K d [2] (Equation ( 2)),

Determination of the Binding Constants (K d ) of Divalent Metal Ions with HIV-RH
where  f and  b are the fractions and  f and  b are the chemical shifts of the free and bound protein or compound, respectively.M 0 and L 0 are the initial concentrations of the metal ion and the chelator (protein or ligand).The data were fit using GraphPad Prism (GraphPad Software) to determine the K d from the chemical shift changes.
Divalent metal ions are crucial for RNase H activity and contribute to the conformation and stability of the protein, especially at the C-terminus [11,28].In our NMR binding study of the divalent metal ions to HIV-RH, we chose 25 mM bis-Tris (pH 6.5) containing 150 mM NaCl as our buffer to maintain pH and ionic strength, therefore to mitigate the effects on conformation, stability and activity of HIV-RH.The buffer was also compatible with those used in the biochemical assays.Every reagent used in the experiment was prepared in the buffer described, and the pH was checked in each step and adjusted when necessary.

NMR Assignments of the 1 H-15 N HSQC Spectra and Flexibility at the C-Terminus of HIV-RH
An isolated, C-terminal His 6 -tagged protein that contains the C-terminal domain of HIV-RT p66 protein (W426-L560) was expressed and purified in its 15 N-labeled or 13 C/ 15 N-labeled forms for our NMR studies.The protein was mostly well-folded as the 1 H-15 N HSQC peaks were well resolved and dispersed.Backbone resonance assignments of most amino acids were obtained from the standard triple resonance experiments (data available if required).The 1 H-15 N HSQC peaks of 26 amino acids, not including those of the His 6 -tag and prolines, were not In the metal titration experiments, we dialyzed 15 Nlabeled HIV-RH in the described buffer.MgCl 2 (1.0 M, prepared in the same buffer) was titrated into the protein solution to increase the Mg 2+ concentration from 0 to 80 mM.Approximately 24 peaks showed obvious shifts on the 1 H- 15 N HSQC spectra (Figure 1(a)).The Mg 2+ dosedependent chemical shift changes (∆), calculated as weighted sums of the changes in both the 15  and 1 H dimensions (Equation ( 1)), were fitted by Equation (2) using GraphPad Prism (GraphPad Software) as shown in Figure 1(b).It was clear that the titration curves reached a maximum plateau at about 80 mM of Mg 2+ .The binding constant (K d ) of Mg 2+ /HIV-RH were determined by the titration data of 17 amino acids that showed ∆ > 10 Hz with at least 6 data points.The calculated K d ranged from 8.4 to 16.6 mM with a goodness of fit (R 2 ) from 0.984 to 0.999.The averaged K d was 13  4 mM, very similar to the value reported previously [11].
We also carried out titration experiments with stepwise increases in Ca 2+ concentrations from 0 to 15 mM in a similar fashion to those using Mg 2+ .We obtained a very similar pattern of chemical shift perturbations.In the plots of ∆ vs. the Ca 2+ concentration, the titration curves reached plateaus at 15 mM Ca 2+ .The K d values of 21 amino acids were calculated to be in the range from 1.5 to 3.3 mM, yielding reasonable values of R 2 from 0.992 to 1.000.The average K d for Ca 2+ to HIV-RH is 2.9  0.5 mM, indicating that the binding of Ca 2+ to HIV-RH is four times tighter than that of Mg 2+ .
Figure 1(c) showed the chemical shift perturbation map of Mg 2+ on the HIV-RH domain protein.Amino acids at the catalytic center showed the greatest chemical shift perturbations.All three observable residues in the metal chelating DDED motif, i.e., D443, E478, and D498 stood out in the chemical shift perturbations induced by Mg 2+ .Ca 2+ binds to the same site as Mg 2+ and amino acids located at the active site also showed the largest chemical shift changes in response to Ca 2+ .Our NMR titration data demonstrated small and localized chemical shift perturbation patterns in HIV-RH in response to the divalent metal ions, Mg 2+ and Ca 2+ , when the buffer pH and ionic strength were carefully maintained.It is clear that Mg 2+ and Ca 2+ bind specifically to the active site of the RNase H domain, but do not significantly change the global conformation and dynamics of the protein.However, the C-terminus of our construct is still mostly disordered even at high concentrations of divalent metal ions at the testing buffer conditions.Most of the C-terminal residues of HIV-RH were not stable enough to produce measurable signals in the HSQC spectra in the presence of 80 mM Mg 2+ or 15 mM Ca 2+ .

Sequential Binding of Two Mg 2+ Ions to the Active Site
In the Mg 2+ titration experiments, a few peaks, including G444, Q500, W535ω, and V536, became broader and broader as the Mg 2+ concentration increased.More interestingly, they split into two sets of peaks at 80 mM Mg 2+ (Figure 2(a)).The splitting of these signals indicated the existence of two slowly-exchanging protein conformations, and the occupancy of the second metal binding site at high Mg 2+ concentrations (80 mM or higher).At 80 mM Mg 2+ , the second metal-binding site was approximately half-occupied by Mg 2+ , whereas the first metal-binding site was almost fully occupied.Interestingly, the affinity of the second Mg 2+ ion for HIV-RH was reported to be ~35 mM [11].fit simultaneously into the active site.Although we saw evidence of the sequential binding of two Mg 2+ ions in our experiments, we were not able to obtain enough data points to determine the binding constant of the second Mg 2+ ion through curve fitting.

Interaction of Metal-Ions to Metal-Chelating Compounds
Divalent metal ions, especially Mg 2+ , are directly involved in the catalytic activities of HIV-RH, and two Mg 2+ ions bind to one molecule of HIV-RH at the active site.To target the active site, it is a common strategy to design metal-chelating compounds binding to two divalent metal ions at the active site [2,16].The effect of compound/metal binding property on enzyme inhibition is always of interest in the lead generation and optimization of metal-chelating compounds.In this study, we utilized 1D 1 H-NMR experiments to identify the metalbinding sites of our lead compounds through chemical shift perturbations induced by metal ions.Again, similar to the protein/metal interaction, protons that are near the metal-binding site generally experience larger perturbations.Based on Equation (2), we determined the binding affinities of the lead compounds to metal ions through chemical shift changes.The sample preparation, data acquisition and data processing were all automated for a rapid measurement of K d .Derivatives of unsaturated and saturated tricyclic N-hydroxypyridones, shown in Figures 3(a) and (b) respectively, were designed and synthesized in-house for targeting HIV-RH.The unsaturated tricyclic N-hydroxypyridone derivatives contain a flat and rigid three-ring core and two hypothetical metal-binding motifs (Figure 3(a)).In the Mg 2+ titration experiments, all protons on the tricyclic N-hydroxypyridone core showed significant and incremental chemical shift changes as the Mg 2+ concentration increased; the example of compound U-1 is shown in Figure 3(c).Meanwhile, only slight chemical shift changes were observed with the other protons, which were located in the R1 and R2 substitute groups.H-13 gave the largest chemical shift changes (up to 130 Hz) when the Mg 2+ concentration changed.Therefore, H-13 is the proton nearest to the Mg 2+ ion (Figure 3(a)).Three more unsaturated tricyclic N-hydroxypyridone compounds were tested and H-13 showed the largest chemical shift change in all cases.The chemical shift changes of the core protons at the compound/Mg 2+ ratio of 1:20 when the titration curves reached the plateaus were listed in Table 1.The chemical perturbations of the other three core protons H-5, H-8 and H-12 showed only half of the change of H-13.This indicates that Mg 2+ preferably binds unsaturated tricyclic N-hydroxy-pyri-done derivatives at site I by interacting with the two oxygen atoms of the O 14 ∸N 1 ∸C 2 ∸O 15 motif (Figure 3(a)).The K d values of the two unsaturated compounds (U-1 and U-4) were determined through chemical shift changes, and equivalent with each other within the measurement deviation range.The chelator-Mg 2+ complexes of the other two compounds (U-2 and U-3) had poor solubility.As the Mg 2+ concentration was approximately equivalent to the compound concentration, we saw precipitates in the NMR tubes and the proton signals became too weak to measure.Consequently, we were not able to calculate the K d due to the lack of points.Interestingly, when [Mg 2+ ] was higher than 20 times of the compound concentration, the compound proton signals became stronger and therefore measurable.The similar phenomena were also observed with compound U-1 and U-4.As shown in Figure 3(c), proton signals of U-1 became broader and broader and then sharper and sharper as the Mg 2+ concentration increased gradually from 0 to 80 mM.In addition, when [Mg 2+ ] was over 40 times of the compound concentration, the compound proton signals shifted to an opposite direction.We believe this was related to the second metal-ion binding and the ternary complexes (compound/(Mg 2+ ) 2 ) had better solubility than the binary complexes (compound/Mg 2+ ).The chemical shift change "turn-over" occured all at a Mg 2+ concentration of 16 -20 mM, suggesting that with ~20 mM Mg 2+ , the second metal-binding site is approximately half-occupied by Mg 2+ , whereas the first metal-binding site was almost fully occupied and therefore the K d of the second Mg 2+ to this series compound was estimated as ~10mM.
In our NMR study, we could not obtain sufficient data to determine K d of the second Mg 2+ binding using the double binding curve fit due to the maximum concentration of MgCl 2 in stock solution of 0.5 M.
The observations were different when the tricyclic N-hydroxypyridone core was saturated at the carbon-carbon bond between C-12 and C-13 (Figure 3(b)).Saturation of the C-C bond makes the six-member-ring more flexible and no longer flat.Figure 3(d) shows the shift of the proton signals of 0.20 mM compound S-5 in the Mg 2+ titration measurements.Table 1 lists the chemical shift perturbations of the core protons of four saturated tricyclic N-hydroxypyridones at a concentration ratio of compound: Mg 2+ of 1:100 when the ∆ vs. [Mg 2+ ] titration curves reached the plateaus.All four saturated derivatives showed the same interaction mapping, which obviously was different from the mapping of the unsaturated analogs.Instead of H-13 in the unsaturated cases, proton H-5 of saturated compounds presented the largest chemical shift change, indicating Mg 2+ preferably boundat site II and interacts with the nitrogen and oxygen at-oms of the N 4 ∸C 3 ∸C 2 ∸O 15 motif when the C12-C13 bond was saturated.In the NMR spectra, only one set of triplet peaks was observed for either of the two H-12 or two H-13 (Figure 3(d)), indicating the equivalence of the

Ternary Complex of HIV-RH, Mg 2+ , and
The use of metal chelation as an anchor to the active site -thujaplicinol to the first Mg 2+ ion, as determined from und s-7), which has a weak bindin tw xypyridone ring is relatively flexible and exchanges between chair and boat conformations.Four saturated Nhydroxypyridones had very similar K d values, in a tight range of 2 -5 mM (Table 1) which was 10 to 20 times larger than those of the unsaturated analogs.The overall chemical shift changes and the differences between the largest and the other perturbations of the core protons of the saturated compounds were both smaller than those observed with the unsaturated ones.We also observed the sharp-broad-sharp line-shape changes as the Mg 2+ concentration increased, but didn't see the second binding of Mg 2+ as well, probably because the Mg 2+ concentration was not high enough.

Metal-Chelating Compounds
is a common strategy in designing inhibitors to target HIV-RH and the highly-related HIV integrase enzymes [2,16,32].The final product of the design is a ternary complex (compound/ metal-ion/protein).Therefore, the correlation between compound/metal or protein/metal binding affinity and enzyme inhibition is always of high interest.Sometimes, metal ions are critical for inhibition by the designed compound.-thujaplicinol, a selective inhibitor of HIV RT, is a typical metal chelator [33].Our study demonstrated that it binds to two Mg 2+ ions sequentially.Its proton signals incrementally shifted downfield and became broader and broader as the concentration of Mg 2+ increased.When the concentration of Mg 2+ reached 8 mM, the signals started to shift in the opposite direction.As the Mg 2+ concentration increased further, the signals shifted upfield and became narrower.The K d of the proton chemical shift changes, is 0.64  0.10 mM.Interestingly, -thujaplicinol introduced no chemical shift perturbations in the HSQC signals of the HIV-RH domain when metal ions were absent.On the contrary, in the presence of 8 mM Mg 2+ , significant chemical shift perturbations were observed in the HSQC spectrum of the HIV-RH protein attributable to 500 M -thujaplicinol.The experiments indicated that the presence of a metal ion is necessary for -thujaplicinol to interact with the HIV-RH domain, in agreement with the crystallographic results [21].
On the other hand, 500 M saturated tricyclic N-hydroxypyridone (compo g affinity (K d 4.4 mM) to Mg 2+ , induced less than 25 Hz of chemical shift changes in the signals of 14 residues of HIV-RH in the presence of 8 mM Mg 2+ .Three detectable chelating residuals of the DEDD motif, i.e., D443, E478 and D498, were among them.The chemical shift changes were 14, 10 and 13 Hz, respectively.We also observed line-shape-broadening of the E478 signal.In comparison with Figure 1(c), Figure 4 indicates that the compound interacted with the active site of HIV-RH through Mg 2+ chelation in the buffer containing 8 mM Mg 2+ .Interestingly, in the absence of divalent metal ions, this compound was found to bind HIV-RH at an alternative binding site through our NMR and crystallographic studies (unpublished data).Although compound s-7 induced small chemical shift perturbations in the HIV-RH domain, it showed strong inhibition against HIV-RH with an IC 50 of 0.038 M.One reason could be that the experimental conditions used for the IC 50 measurement were very different from those used in the NMR experiments.In our biochemical assay, we used substrate oligonucleo la much less active in RNA hydrolysis than intact HIV-RT [22,23,28,30].In addition, the interactions between RT and oligonucleotide duplexes may affect the conformation of the protein, thereby altering the affinity of RNase H for metal ions.
As shown in Table 1, we did not observe a correlation between the compou ti-HIV-1 activity of the inhibitors.The interactions of Mg 2+ with either HIV-RH or the tricyclic N-hydroxypyridones are weak, with K d values in the mM range.However, the HIV-RH IC 50 of the tricyclic N-hydroxypyridone compounds range from 2.2 μM to 0.038 μM.We believe that the metal-binding ability of this category of compounds plays an important role in anchoring the compound to the active site of HIV-RH specifically.Since the binding affinity of metal/compound and metal/ protein are weak, the inhibitory potency of the compound is not driven by metal chelation, but by direct interactions between the compound and the protein instead.Therefore, the goal of chemical design is to improve the inhibitory potency rather than to increase the metal chelation affinity.In fact, the strategy of focusing on protein/compound interaction is practically more efficient.Firstly, the protein/compound direct interaction is also the key to improve the selectivity of designed compounds.Secondly, very strong metal chelation property (for instance, in the low nM range) is not preferable because it may cause toxicity by chelating metal ions in the blood or in tissues.

Conclusions
properties and enzy metal ions with HIV-RH and two series of HIV-RH inhibitors using NMR chemical shift perturbations.We noticed that the chemical shift perturbation maps of the HIV-RH protein and the compounds are sensitive to the pH, ionic strength and concentration of divalent metal ions.A neutral pH of 6.5 was chosen and 150 mM salt was used to maintain the ionic strength in the metal ion binding studies.When the buffer pH and ionic strength were carefully maintained, the HSQC titration data demonstrated that Mg 2+ and Ca 2+ ions introduce small and localized chemical shift perturbation patterns in HIV-RH.Both Mg 2+ and Ca 2+ bind specifically to the active site of the HIV-RH domain, with binding constants of 13 and 3 mM, respectively.We also observed that two Mg 2+ ions bind sequentially to the RNase H domain, whereas only a single binding event was observed for Ca 2+ ions.
The metal-chelating affinities of the saturated or unsaturated tricyclic N-hydroxypyridone inhibitors were also measured and c e same tricyclic core showed similar binding affinity to metal ions at the same primary chelating site.Metal ions interact primarily at site I with the unsaturated tricyclic N-hydroxypyridones, but at site II with the saturated analogs (Figures 3(a) and (b)).The flexibility of the N-hydroxypyridone ring gained from the saturation reduced the metal-binding affinity from 0.2 to 4.0 mM.With 8 mM Mg 2+ present, our NMR mapping data demonstrated that the tricyclic N-hydroxypyridone compounds bind to the active site of HIV-RH.Interesting gardless of the weak interaction of Mg 2+ with either HIV-RH target or the tricyclic N-hydroxypyridones (the K d 's are in the mM range), the HIV-RH IC 50 of those compounds were much higher, from 2.2 μM to 0.038 μM.The poor correlation between the compound/metal chelating affinity and the anti-HIV-1 activities of the inhibitors reveals the direct protein/compound interactions of the series of inhibitor.The metal-binding property is necessary for the metal chelating inhibitors to bind specifically to the active site through a metal ion bridge, but the design should focus on the direct interactions of the compound with the protein for inhibitory potency and selectivity.

Figure 2 (Figure 2 .
Figure 2. (a) Regions of the HSQC spectra of HIV-RH showing peak shifting with increasing Mg 2+ concentrations, at 0, 2.5, 5.0, 10, 20, 40, and 80 mM, in the direction indicated by the arrows.The dashed circles show peak splitting for V536 and G444 and broadening for E478 and G453 at 80 mM Mg 2+ .(b) Mapping of splitting HSQC signals on the HIV-RH structure.Red: AA with splitting HSQC signals, Yellow: not observed or assigned.(c) Mapping of the shift perturbations of HIV-RH by .0 mM Mg 2+ on the HIV-RH structure as shown in Figure 2(c) in a different orientation.5

Figure 4 .
Figure 4. Mapping of the shift perturbations on the HIV-RH structure by compound s-7 in the presence of 8 mM