To identify residues that contribute to formation of the E:S complex, we created eight mutants of residues within the crystallographically determined RNA−protein interface (
): D40A, K42A, Y47F, H49A, K110A, K111A, K113A, and D143A. The anionic residues form direct and solvent-mediated substrate contacts. D40 forms an outer sphere coordination to a potassium ion located in the tetraloop of the SRL. D143 is the only other anionic residue that interacts with the substrate in any of the available structures. Cationic residues K42, K110, K111, and K113 form contacts to the SRL. K110 and K111 form salt bridges to phosphate oxygen atoms of the SRL RNA (>3.4 Å). Given its location, K42 is also expected to form a strong electrostatic interaction with the backbone (3.7 Å). In contrast, K113 is expected to form longer-range electrostatic interactions (~6.6 Å). K110, K111, and K113 form contacts to the bulged G motif, with K113 forming sequence-specific contacts to the bulged G. Y47 and H49 are in the active site. H49 is expected to carry a partial positive charge because its measured pKa
of 7.7 in the presence of a dinucleotide substrate (21
) is similar to the pH of 7.5 that was used in the assays. In contrast, Y47 is not charged and serves as a control.
To test if the targeted residues affect formation of the E:S complex, we determined the change in salt dependence for SRL cleavage by mutants at these positions [Δn
(Table and Figure )]. Cleavage of a 32
P-labeled SRL oligonucleotide was performed at 37 °C under k2
conditions (E0 K1/2
, and S0
) in 10 mM Tris (pH 7.5) containing 0.05% Triton X-100 and 30−100 mM KCl. Other chloride salts show equivalent salt dependence for formation of the E:S complex (NH4
Cl and LiCl; supplementary Figure 2A,B in ref (4
)). The salt dependence of each mutant, n
, is the slope of a plot of log k2
versus log[KCl]; the value of n
was then subtracted from that of the wild-type protein to yield the change in salt dependence, Δn
, for each mutant. Mutation of a cationic residue that contributes to binding is expected to produce a smaller salt dependence (Δn
> 0) because fewer cationic residues are available to interact with the anionic RNA substrate and therefore displace fewer anions from the RNA surface upon complex formation. Conversely, mutation of an anionic residue that contributes to binding is expected to produce a greater salt dependence (Δn
< 0) because the net increase in positive charge strengthens the interactions with RNA and requires displacement of a greater number of ions from the RNA surface upon binding. Removal of a single protein charge from RNA−protein interfaces produced Δn
values of ~0.5 (22
). Mutation of residues that do not contribute to electrostatic binding is expected to result in little or no change in salt dependence (Δn
KCl Dependence of SRL Cleavage by Restrictocina
Figure 1 Salt dependence of restrictocin mutants. (A) Cleavage of the SRL by D40A at varying salt concentrations ranging from 12 to 100 mM KCl. Reactions were conducted under multiple-turnover conditions at 37 °C in 10 mM Tris (pH 7.4) and 0.05% Triton (more ...)
Three lines of evidence suggest that the altered salt dependence upon mutation results from disruption of electrostatic interactions between the RNA and the endonuclease rather than salt-induced structural changes to the endonuclease or RNA in the complex. First, similar salt dependence profiles are observed for both a minimal specific substrate (the SRL) and nonspecific substrates (single-stranded substrates) (4
), indicating that the observed salt dependence is independent of the RNA structure. Second, as demonstrated herein, the neutral mutation Y47F does not change the salt dependence (Δn
= 0), despite an ~280-fold decrease in k2
relative to that of the wild-type enzyme. Only mutations of charged residues lead to changes in the salt dependence (see below), suggesting that the salt inhibition reflects disruption of electrostatic interactions rather than structural rearrangements upon formation of the E:S complex. Third, comparison of crystal structures of restrictocin alone or in complex with substrate analogues reveals negligible structural changes in the protein.
It is unlikely that the changes in salt dependence arise from structural changes due to amino acid substitution. As alanine substitutions do not remove backbone atoms, changes to the protein structure are not expected (24
). Consistent with this notion, replacement of three active site residues with glutamine (H49Q/E95Q/H136Q) was structurally isomorphous (M. J. Plantinga and C. C. Correll, unpublished observations).
Alanine mutants of cationic residues within the RNA−protein interface exhibit a shallower slope in their salt dependence plots (Table and Figure ). For three of the mutants, Δn
approaches unity: K110A has a Δn
of 0.8, K111A a Δn
of 0.7, and K113A a Δn
of 0.9. These residues (designated the lysine triad) cluster to form a highly positive patch in loop 4 (L4, residues 98−118) at the edge of the predicted RNA−protein interface (Figure D). In accord with the contribution of these three lysine residues to formation of the electrostatic E:S complex, they form a patch with the highest positive potential (Figure A). The other mutants, K42A and H49A, have a smaller effect on salt dependence, with Δn
values of 0.4 and 0.3, respectively. K42 is found in loop 2 (L2, residues 36−48), near the lysine triad. H49 is located in the active site; the observed moderate decrease in the salt dependence for the H49A mutant supports the previous finding that this residue carries a partial charge in the ground-state complex (26
Figure 2 Isopotential contours mapped onto the surface of the substrate and enzyme. (A and B) Isopotential contours mapped onto the active site face (A) and back face (B) of restrictocin, using the molecular orientations from Figure D. Electrostatic (more ...)
For the alanine mutants of anionic residues, D40A shows a steeper slope in the salt dependence plot (Δn = −0.5) whereas D143A has the same salt dependence as wild-type restrictocin (Δn = −0.1). D40 is located in L2 near K42 and the lysine triad in L4 (Figure D). The change in salt dependence for the D40A mutant is consistent with removal of an anionic residue increasing the net cationic character of the protein and thus strengthening electrostatic interactions with the anionic RNA substrate. In contrast, the lack of a change in the salt dependence for D143A demonstrates that this residue does not contribute electrostatically to formation of the E:S complex, although D143 is located near the active site.
Our analysis of the salt dependence data for interface and noninterface mutants indicates that only mutations of charged residues located on the active site face exhibit significant changes in salt dependence (Table and Figures and ). Changes in salt dependence can result from disruption of direct and/or long-range electrostatic interactions. Mutation of residues outside the E:S interface does not alter the salt dependence, indicating that these residues form neither direct nor long-range electrostatic interactions with the substrate. In contrast, residues on the active site face alter the salt dependence when mutated, consistent with direct electrostatic interactions with the RNA substrate. These findings strongly support a model in which restrictocin uses its active site face to bind to its RNA substrate in the E:S complex, thereby facilitating subsequent specific recognition and cleavage.
To test whether formation of the E:S complex can be described by the nonlinear Poisson−Boltzmann (NLPB) model, we calculated salt dependencies for eight mutants (Figure and
). Kinetic studies indicate that the ground-state E:S complex is not a single structure but rather an ensemble that is partially represented by two restrictocin−substrate analogue cocrystal structures (4
). Thus, both structures were used for these calculations. The ncalc
values correlate well with experimental n
values for complexes of point mutants (Figure A), and the Δncalc
values are independent of the structure used (Figure B). Importantly, the rank order of the change in salt dependence is the same for experimental and theoretical values. These results support the validity of these calculations and provide further evidence that the structures used provide a reasonable representation of the E:S complexes. In contrast, calculations do not agree with the experimental results for the noninterface R21D/K28D/K63D triple mutant; the Δncalc
is 3.0 for both structures, but the experimental Δn
is 0.2 (4
). Half of the Δncalc
for the triple mutant arises from removal of the three positive charges via alanine substitutions (data not shown); the remaining half arises from addition of negative charges at these positions. Perhaps the large change in net charge for this mutant (−6) alters the RNA−protein interactions in the ensemble of E:S complexes enough to negate the relevance of the crystal structures used for the calculations.
Figure 3 Comparison of experimental data with electrostatic calculations. (A) Correlation between experimental and calculated salt dependencies. The R2 values shown are for linear regression fits to the data. (B) Comparison of theoretical and experimental salt (more ...)