The limited information available from analysis of the backbone resonances of the RT RNase H domain, particularly in the region of the C-terminus, led us to investigate the behavior of the sidechain resonances. Surprisingly, the spectrum of the [13
-Ile] RNase H domain revealed a significant sensitivity of the Ile556 methyl resonance to pH ( and Supplementary Figure S2
). A fit of the titration data for Ile556 yields total titration shifts of Δ13
C = −2.34 ppm; Δ1
H = −0.146 ppm, both corresponding to a pK
= 5.77, with the resonances moving upfield as the pH is reduced. There are no titratable residues in the sequence surrounding Ile556: LVSAGI556
consistent with the observed pK
value, and the influence of residues more distant in sequence but in spatial propinquity is inconsistent with the disordered conformation of this segment.
Figure 1. pH and Mg2+ titration of the Cδ resonances of [13CδH3-Ile]RNase H domain from HIV-1 RT. The construct is identical to that used in previous studies (18), and labeling was achieved by growth on a medium containing [4-13C,3,3,-2H2]2-oxobutyrate. (more ...)
There is increasing evidence that the 13
C shift values for many amino acid sidechain resonances appear to be dominated by conformationally-dependent ‘γ-effects’ first characterized in studies of aliphatic hydrocarbons by Grant and coworkers (32
). According to this analysis, the upfield shift observed for Ile556 δ1 methyl is typical of Ile residues with χ2 in the g− conformation (19
). Using the relation developed by Hansen et al.
), the Ile556 χ2 distribution changes from ~45% g− conformer at neutral pH, to ~84% g− conformer at low pH, so that its pH dependence may be qualitatively described by the relation:
represents a random coil mixture. Since the g−
conformation represents a less energetically favorable rotamer than the predominant t
conformation, the high g−
probability that characterizes the low pH behavior of Ile556 is inconsistent with a random coil state. Thus, this behavior indicates that at low pH, Ile556 experiences significant inter-residue stabilizing interactions that compensate for the higher intrinsic energy of the g−
Crystallographic studies of RT provide limited information on the behavior of the RNase H C-terminus; however, recent studies of RNase H–inhibitor complexes show the positions of both bound Mg2+
ions as well as most of the C-terminal residues (34–37
). Interestingly, although none of the structures of apo RT show an Ile556 residue with a χ2 = g−
conformation, all of the RNase H–inhibitor complexes show this conformation to be present (). In each of these structures, the sidechain of Ile556 packs against the central β-sheet and continues the amphiphilic motif of Val548 and Val552. These structures reveal the role of the bound Mg2+
ions, and particularly MgA
, in stabilizing the orientation of helix E by bridging Asp443, located on the central β-sheet, and Asp549, located on helix E (a). It is reasonable to hypothesize that the observed pH dependence of the Ile556 shift results from formation of a hydrogen bonding interaction between the same two aspartyl residues, such that the repulsive interaction at higher pH is replaced by a positive interaction as the hydrogen bond forms at lower pH. The pK
value of 5.77 obtained from the Ile556 shifts is well above that expected for an isolated aspartyl sidechain (38
), but is consistent with a shared hydrogen bond.
Figure 2. Crystal structure of the active site of the HIV RT RNase H domain. (a) Isolated RT RNase H domain–inhibitor complex [pdb code: 3K2P (34)].The inhibitor, which binds to both Mg ions, is not shown. The four acidic residues that complex the two Mg (more ...)
We also considered the alternative hypothesis that the pH-dependent shifts of Ile556 and Ile542 might result from a conformational transition induced by titration of His539. This hypothesis is based on the proximity of His539 to Glu546 and Asp549 in some of the structures of RNaseH–inhibitor complexes, as well as the similarity of the pK
values obtained above to typical histidine pK
values. In order to evaluate the role of such interactions, we analyzed the pH behavior of the isoleucine residues in an RNase H (H539S) mutant. The titration behavior of Ile556 in the mutant is essentially unaffected by this mutation, while there is a somewhat larger perturbation of the Ile542 titration, consistent with its proximity to the mutated residue (Supplementary Figure S3
). Thus, titration of His539 has no effect on the behavior of Ile556, consistent with the above interpretation that the pH-dependent effect on Helix E results from an effect on the electrostatic repulsion of the active site aspartyl residues.
It is likely that both the integrity of the helix, as well as its position is also destabilized by loss of the interactions with the β-sheet. However, the exchange broadening of the amide resonances as well as the Ile556 13Cδ shift behavior discussed above indicate that the structure does not unwind and produce a random coil, but equilibrates between the packed conformation observed in the inhibitor complex and a partially disordered conformation. In summary, comparison of the titration data with the structures of RNase H–inhibitor complexes strongly suggests that lowering the sample pH is exerting a conformational effect that is in some respects analogous to the conformation observed for the Mg2+–inhibitor complexes.
Effects of divalent metal ions
Based on the above analysis, we also titrated the RNase H sample with MgCl2
. The binding of Mg2+
influences the Ile556 Cδ shift similarly to a pH reduction (b). Interestingly, the Ile556 and (smaller) Ile542 shift responses to both H+
are qualitatively similar, while the responses of the remaining methyl groups are very different (a and b). This result is consistent with the conclusion that Ile542 and Ile556 are sensitive to the changing position/stability of helix E, while for the remaining isoleucine resonances, H+
exert different conformational effects. Analysis of the Mg2+
titration study yielded an apparent
of 6.3 mM (Supplementary Figure S4
), falling between the values of 3.2 and ~35 mM previously obtained for Mg2+
binding to the apo RNase H domain (18
). This intermediate apparent
value probably results from partial occupancy of both the A and B divalent ion sites, since A-site binding would be expected to have a much more significant stabilizing effect on helix E.
Crystal structures of the isolated HIV-1 RT RNase H domain (39
) and the Escherichia coli
RNase HI (40
) in the presence of Mn2+
indicate that both divalent ion positions are occupied. In order to achieve an analogous occupation of both divalent ion sites without the associated paramagnetic effects on resonance linewidth, we utilized another transition metal ion, Zn2+
. The ionic radii of Zn2+
are nearly identical, and it has been reported that optimal RT Rnase H activity is achieved at ~25 µM Zn2+
, much lower than the concentration of Mg2+
required for optimal activity (although the maximum rate obtained with Zn2+
is substantially below that determined in the presence of Mg2+
). Addition of Zn2+
at a 1:1 molar ratio with RNase H results in small shift changes for several isoleucine methyl resonances, particularly Ile482 and Ile505 but produces a minimal perturbation of the Ile556 resonance (). This result is consistent with the conclusion that the initially bound Zn2+
ion exhibits sufficient differential affinity for the A and B sites such that only the higher affinity B site is significantly occupied, and selective occupation of site B by a divalent ion is insufficient to significantly influence the stability of helix E (a). Further addition of Zn2+
to produce a 2:1 complex results in a large, upfield shift of Ile556, with the δ13
C = 9.8 ppm (b), lower than the values observed at low pH or at 64 mM Mg2+
(~10.3 ppm). These results clearly indicate that at a 2:1 ratio, both divalent ion sites are largely occupied, with the occupation of site A resulting in significant helix E stabilization. The more readily resolved Ile505 resonance also shows clearly the effects of the two separate Zn2+
complexes that are formed. Formation of the 1:1 complex produces a shift to a slightly lower δ13
C ~ 11.9 ppm value. Subsequent formation of the 2:1 complex results in a distinct resonance with a larger δ13
C ~ 12.4 ppm (b). Further increases in the Zn2+
concentration resulted in only very small additional shifts of the Ile556 resonance, although the resonance becomes more intense, consistent with a reduction in the exchange broadening (Supplementary Figure S5
Figure 3. Effect of Zn2+ on RT RNase H Ile methyl resonances. (a) Overlay of the 1H–13C HMQC spectra of the 15 µM [13CδH3-Ile]RNase H domain (black), 20 µM RNase H plus 20 µM Zn2+ (red). (b) Overlayed spectra of 20 µM (more ...)
C HMQC spectrum of the [13
complex was also obtained; however, the paramagnetic broadening produced by the Mn2+
ion prevents observation of the isoleucine methyl resonances arising from the closer Ile482, Ile542 and Ile556 residues (Supplementary Figure S6
Effect of a Mg2+–inhibitor complex and evaluation of a helix probability factor
As is apparent from b, even at 64 mM Mg2+
, the Ile556 resonance exhibits substantial exchange broadening, indicating that even very high Mg2+
concentrations are insufficient to strongly stabilize the completely folded state of the RNase H domain. Similarly, the backbone amide resonances of helix E remain difficult to observe even in the presence of high Mg2+
). In order to evaluate the behavior of a more completely stabilized ternary complex, we obtained a representative dual Mg2+
-binding ligand, 2-hydroxyisoquinoline-1,3(2H,4H)-dione, the synthesis of which has been described by Billamboz et al.
The 2-hydroxyisoquinolone and structurally related ligands interact with both MgA
, forming a stable RNaseH•Mg2+
•isoquinolone complex. Addition of 4 mM Mg2+
+ 0.5 mM isoquinolone ligand resulted in further spectral changes, such that the Ile556 13
Cδ is shifted to a more extreme position (δ13
C = 8.6 ppm), and the exchange broadening is dramatically reduced (a). Thus, we assign this Ile556 shift to Ile556 locked in the χ2 = g− conformation characteristic of the fully folded state (21
Figure 4. Effect of Mg2+ and 2-Hydroxyisoquinoline-1,3(2H,4H)-dione ligands on RNase H Ile resonances. (a) Overlay of the 1H–13C HMQC spectra of the isolated [13CδH3-Ile]RNase H domain (black), plus 4 mM Mg2+ (red), after subsequent addition of (more ...)
Since Ile556 is not located directly in the active site, the shift behavior of the Ile556 Cδ resonance can be used to provide an estimate for the formation of helix E as a function of different experimental conditions that is largely independent of the details of active site structure and ligands. Defining the probability of a well-formed helix to be 1 in the presence of both Mg2+
and the isoquinolone inhibitor, and estimating that in the absence of a helix, the shift parameters for this Ile556 should be similar to those of a random coil (42
), we obtain:
Using this expression, the helix E orientational probability varies from ~14% at pH 7.1 up to ~63% at pH 4.5 (). These values are generally consistent with the observed exchange broadening that makes the backbone amide resonances difficult to observe. Even a pH of 4.5 or a Mg2+
concentration of 64 mM is insufficient to stabilize the helix by >~65%. We note that the calculation of Equation (4)
provides an estimate of the helix E orientational probability in RNase H based on the behavior of the Ile556 resonance, and is distinct from the g−
estimate that can be made using the relation given by Hansen et al.
), which is based on an analysis of the relationship between δ(Cδ1
) and 3
)values for the residues in a set of six proteins.
Figure 5. Dependence of helix E probability on pH and Mg2+. The helix probability factor, calculated from the Ile556 δ1-methyl 13C shift using Equation (4) and fit to the relations described in ‘Materials and Methods’ section, is shown as (more ...)
Behavior of the RNase H domain in full RT
In order to evaluate whether a similar transition can be observed for the full RT molecule, we prepared [13CδH3-Ile]66RT, containing isoleucine Cδ methyl labels in the p66 subunit. In the spectrum of RT obtained in the absence of Mg2+, the resonance for Ile556 was not readily apparent, possibly due to overlap with other resonances or to more severe exchange broadening. Surprisingly, addition of 4 mM Mg2+ was found to produce a much more extreme shift for Ile556 in RT than in the isolated RNase H domain (9.7 versus 11.4 ppm) (b). In the full RT molecule, the N-terminus of helix E including Ile542 interacts with the p51 subunit. The interface includes a hydrophobic-binding pocket on p51 that interacts with Ile542 and, in some structures, additional hydrogen bonding interactions between Arg28451 and residues Gln547 and/or Glu546 on the RNase H domain (). We thus conclude that the interaction of the RNase H domain on the p66 subunit with the thumb subdomain on the p51 subunit, helps to orient helix E, leading to a more completely preformed MgA-binding site with higher Mg2+ affinity, so that addition of a fixed concentration of Mg2+ leads to a greater shift of 9.2 ppm for Ile556. Based on the observed Ile556 13C shift of 9.2 ppm, we obtain pHelixE = 86% for the RH domain of RT, compared with 35% for the isolated RNase H domain, both evaluated in the presence of 4 mM MgCl2. Addition of the isoquinolone inhibitor leads to a more extreme shift of the Ile556 methyl resonance and eliminates the exchange broadening, consistent with a well-defined helix orientation. In contrast with the result obtained in the presence of Mg2+, the Ile556 resonance shifts observed for the ternary complexes of both RT and the isolated RNase H domain with Mg2+-isoquinolone are similar. In general, the relative rarity of the χ2 = g− shift conformation makes it easy to identify the corresponding resonances even in enzymes such as RT that contain many isoleucine residues.
Figure 6. Intersubunit interactions contribute to RNase H domain helix E stability. The N-terminus of the RNase H domain helix E (green) is stabilized by hydrophobic interactions involving Ile542 and residues of the p51 subunit (cyan) and by hydrogen bonding interactions (more ...)
An analogous difference of pH sensitivity was also observed between the isolated RH domain and intact RT. At pH 6.1, Ile556 δ13C = 11.7 ppm for the isolated RH domain, and 9.6 ppm in RT (data not shown). It thus appears likely that the additional structural features present in RT that help to preform the MgA-binding site also lead to elevation of the pK value that characterizes the Ile556 shift. This result is consistent with the presence of a bridging interaction in which Asp443 and Asp549 share a proton.
Structural and catalytic role of Arg557
The data presented above support the conclusion that the conformations of the C-terminal residues observed in the recently determined inhibitor-complex structures, which are not apparent in structures of the apo enzyme, can be significantly populated at low pH or in the presence of divalent ions. In addition to the role of Ile556 in stabilizing the orientation of helix E, this structural analysis also supports a functional role for Arg557, in orienting the active site Asp549 residue, stabilizing the helix E structure, and presumably interacting with the RNA strand of the substrate.
In order to better characterize the behavior of Arg557, we obtained 1
N HSQC spectra of the region containing the arginine NHε resonances at pH 5.5, at which the slower exchange of Hε facilitates the observations (). The well-resolved resonance is assigned to Arg463 on the basis of structures showing a side-bonded guanidino group of this residue with the carboxylate of Glu438 [e.g. pdb 3K2P, (34
)]. This salt bridge plays an important role in limiting the accessibility of the Phe440–Tyr441 protease cleavage site in the RNase H domain (43
). Two other Arg NHε resonances are observed, with an intensity ratio of 2:1. The Arg557 resonance was assigned based on the spectrum of an RNase H(R557S) mutant as indicated in , and the remaining resonance, with approximately double the intensity of each resolved peak, corresponds to Arg448 and Arg461. The resolution of the Arg557 NHε resonance, despite the fact that Hε is not involved in a hydrogen bond, is consistent with the positional constraint of the Arg557 guanidino group. In contrast, the NHε resonances for Arg448 and Arg461 are degenerate. A small upfield shift for the Hε resonance of Arg463 is also observed for the RNase H (R557S) mutant. Presumably, this results from a longer range influence of the helix E structure on the adjacent β-sheet, which includes Arg463.
Figure 7. Arginine NHε spectra of RT RNase H domain. A region of the 1H–15N HSQC spectra of U-[15N]RNase H containing the Arginine NHε resonances. (a) apo RNase H (black) overlayed with RNase H (R557S) (red); (b) RNase H + 200 µM (more ...)
The limiting shift of Ile556 observed in the presence of both Mg2+
and the isoquinolone inhibitor is also somewhat smaller for the R557S mutant (δ13
C = 9.8 ppm; Supplementary Figures S7
). We interpret this difference as a consequence of the backbone restraint imposed by positioning of the Arg557 sidechain. In the absence of this conformational restraint, the backbone of the adjacent Ile556 is also less constrained and this will also influence the sidechain conformation as it packs against the β-sheet and the Lys454 sidechain.
Identification of a new RNase H ternary complex
Coupling of the 13C shift of Ile556 Cδ with the position of helix E and assembly of the active site makes this resonance well suited as a sensitive probe for the discovery of new RNase H active site complexes. The location of the probe >10 Å from the active site itself suggests that the shift should be sensitive to how well positioned the helix is, without being strongly dependent on the detailed nature of the ligand complex. After surveying a series of divalent cations and available ligands, we found that Zn2+-ATP was also capable of producing a shift of the Ile556 Cδ resonance that was similar to that produced by the Mg2+–isoquinolone inhibitor complex (c and b).
We also investigated the effect of this ternary complex on the behavior of the 1H–15N HSQC spectrum containing the arginine NHε resonances. Addition of 200 µM Zn2+ to the sample at pH 5.5 resulted in small decreases in intensity of several resonances. In contrast, addition of 200 µM Zn2+ + 100 µM ATP decreased the intensity of the Arg557 resonance by ~50% (c). The most probable interpretation of this result is that the bound Zn-ATP helps to restrain the position of the Arg557 sidechain, resulting in a decreased T2 value, reduced polarization transfer and hence reduced resonance intensity, although several alternative interpretations are also possible. Regardless of the basis for the intensity perturbation, this observation is consistent with the suggestion above that the Arg557 guanidinium sidechain helps to stabilize the interaction of the negatively charged Asp549 sidechain and a negatively charged ligand—either the substrate or in this case, ATP.