Two classes of c-di-GMP-binding riboswitches have been identified30, 31
as downstream macromolecular targets in this ubiquitous second messenger signaling pathway that regulates many diverse bacterial processes1-4, 8, 10
. These two riboswitch classes have evolved different strategies for c-di-GMP recognition and consequently, differ in the structural features of c-di-GMP they require for binding and ligand specificity. Here, we have shown that the class II riboswitch utilizes fewer functional groups of both the bases and ribosyl-phosphate backbone of c-di-GMP for ligand binding and consequently is less discriminatory in second messenger recognition.
The class I riboswitch recognizes the guanine bases of c-di-GMP with greater specificity than the class II riboswitch. For example, replacing one of the guanine bases with adenine has much less of an impact on ligand binding by the class II riboswitch and adenine substitutions for both bases only abolished ligand binding by class I. In addition, we found that interactions with guanine functional groups which significantly stabilize ligand binding by the class I riboswitch are inconsequential for recognition by class II. The ability of the class II aptamer to recognize purine analogs that eliminate many of the specific contacts with the c-di-GMP bases suggests that the class II riboswitch relies largely on base stacking for ligand binding rather than specific interactions with guanine functional groups. In contrast, the class I aptamer more extensively utilizes the various structural features of guanine to sense the same ligand, resulting in increased binding specificity by this riboswitch motif.
The class I riboswitch makes specific contacts to the ribose hydroxyls of c-di-GMP whereas the class II riboswitch does not utilize this functional group for ligand binding. The specific hydrogen bonding interactions to the ribose hydroxyl groups by the class I riboswitch provide a large, stabilizing effect to the binding energy, whereas methylation of this functional group has no effect on binding by class II. However, the conformation of the ribose ring is important for ligand binding by both riboswitches. In the absence of specific recognition of the ribose hydroxyls by the class II riboswitch, 2′-deoxy substitutions still had a small effect on binding that was mitigated by 2′-fluoro substitutions. Similar effects were observed for the class I riboswitch, but the conformational penalty for 2′-deoxy sugars was much larger. Thus, both riboswitches show a preference for binding 3′-endo ribose sugars.
In accordance with these observations, contacts made to the phosphates of the ribosyl-phosphate backbone by class I are important for ligand binding whereas the class II riboswitch has not evolved a specific mechanism for c-di-GMP backbone recognition. This suggests that the c-di-GMP backbone could be modified without significant consequence to ligand affinity for the class II motif. For phosphate recognition, the class I riboswitch employs both metal coordination and hydrogen bonding interactions that allow the aptamer to differentiate between specific phosphate oxygens. These observations are again in direct contrast to what was seen for the class II riboswitch. The same phosphate backbone modifications that affected binding by the class I riboswitch had no effect on binding by class II. The greater use of the c-di-GMP ribosyl-phosphate backbone by the class I riboswitch enhances its ability relative to the class II riboswitch to discriminate between structurally similar di-nucleotide analogs.
There are a greater number of contacts made to c-di-GMP by the class I aptamer and nearly all of the interactions predicted from structural analysis42
contribute to the binding energy. While fewer specific contacts to c-di-GMP were predicted for the class II riboswitch43
, some of these interactions make little, if any, contribution towards ligand binding. It is somewhat surprising that the few contacts made between c-di-GMP and the class II aptamer do not contribute more to binding since these are the only interactions predicted to stabilize the ligand-bound complex. This further supports the prediction that this riboswitch relies heavily on base stacking for ligand binding. However, the increased recognition of c-di-GMP by the class I riboswitch likely accounts for the tighter affinities of this riboswitch class for c-di-GMP compared to the class II riboswitch. Class I ribowitches have Kd
’s for c-di-GMP as tight as 10 pM40
, whereas the affinities for class II are weaker and vary from mid-picomolar to low nanomolar31, 43
. The differential analog binding by these two c-di-GMP effectors correlates with the differences in their absolute affinities for the same second messenger ligand.
Despite the differential recognition of the c-di-GMP bases, both aptamers rely on base stacking interactions for tight ligand binding and these stacking contacts are the only conserved mechanism for guanine recognition between these two RNA effectors. The effects on binding of the 7-deaza guanine analogs were unexpectedly large for both RNAs and inconsistent with eliminating only the predicted hydrogen bonding interactions. Both of these aptamers incorporate c-di-GMP into structural elements upon binding and contain highly conserved purine nucleotides in the binding pocket that base stack with c-di-GMP42, 43
. It has been shown that duplexes containing 7-deaza guanine are less stable than the corresponding duplexes with the canonical guanine base due to decreased base pairing and stacking interactions65-67
. The considerable binding effects we observed for 7-deaza guanine substitutions may similarly be ascribed to the decreased ability of the di-nucleotide to stack with binding pocket residues. This interpretation is consistent with the structural prediction that stacking contacts are important for ligand binding and highlights the essential role of this mechanism for RNA recognition of c-di-GMP. Another possible explanation for the large effects on binding observed for 7-deaza guanine substitutions is that the additional steric bulk of protons at the C7 position results in steric clashes with nearby RNA atoms. While this possibility cannot be fully excluded, significant effects on binding by the class II riboswitch for the single 7-deaza guanine substituted analog are observed in the absence of both specific contacts to the N7 of Gα
and nearby RNA atoms that could potentially clash with a proton at this position. This suggests that the majority of binding energy lost from the 7-deaza guanine modifications is due to decreased base stacking interactions. Taken together, this implies that the similar conformation of c-di-GMP when bound to both riboswitches is functionally relevant for maintaining these high affinity base stacking interactions.
The class II riboswitch has proven to be a more promiscuous effector of c-di-GMP than the class I riboswitch, defining the challenging goal of selectively targeting the latter motif. Because the overall recognition pattern of c-di-GMP differs between the two classes, we hypothesized that analogs could be designed to preferentially bind one riboswitch class over the other. However, the identified differences in c-di-GMP recognition strategies are more easily exploited for selectively targeting the class II aptamer. In the specific context of the two c-di-GMP-binding riboswitches studied here, the N1-methyl guanine analog could potentially target the class I riboswitch over class II. We predict that the Kd
of this analog for the class I wild-type sequence (Kd
c-di-GMP, 10 pM)40
, is in the low nanomolar range and comparison of the absolute affinities of the class I and class II aptamers for this analog suggests that binding would be selective for class I. While this approach to selectively targeting the class I riboswitch is highly dependent on the affinities of c-di-GMP for both RNA aptamers in question, this demonstrates that it may be possible to differentiate between these two c-di-GMP effectors. Identifying an analog that is absolutely selective for class I RNA over class II remains desirable because it would provide a useful tool for manipulating RNA-mediated c-di-GMP signaling networks, particularly in those organisms that use both motifs for gene control. However, several tight binding second messenger analogs for both RNA aptamers were identified in this study and these are promising candidates for use in manipulating the diverse biological processes mediated by these c-di-GMP-binding riboswitches.
The larger effects on binding observed for the class I riboswitch over the class II riboswitch for the N1-methyl guanine analog indicate that the class II riboswitch is able to effectively accommodate steric bulk at positions of c-di-GMP that are in direct contact with RNA atoms. The effects on binding for rearrangement of the class II RNA to accommodate this added methyl group on the ligand were not as large as the corresponding effects from the predicted displacement of the water molecule coordinated to the N1 of Gα in the class I riboswitch. It is possible that the binding pocket nucleotides of the class I aptamer also in contact with this water molecule are strategically positioned by these hydrogen bonding interactions, suggesting that its displacement may disrupt a network of contacts that are necessary for maintaining the integrity of the c-di-GMP binding pocket. Taken together, this indicates that in comparison to the class I aptamer, the class II aptamer is a more flexible motif, which likely contributes to its greater promiscuity in ligand binding.
c-di-AMP was recently identified to be a bacterial second messenger signaling molecule68-70
and the ability of the class II riboswitch to bind this ligand hints at the possibility that c-di-AMP binding riboswitches may exist and participate in this emerging signaling pathway. Given that RNA has evolved two different strategies for binding of c-di-GMP, it is plausible that RNA has also evolved a strategy for specific, high affinity binding of c-di-AMP. The common theme for c-di-GMP recognition by these two distinct riboswitches is the use of base stacking and it is likely that the same approach would be employed by RNA for the specific recognition of c-di-AMP.
Overall, these data offer an explanation for why the class I motif is more frequently used for gene regulation by bacteria compared to the class II motif30, 31
. The ability of the class II riboswitch to recognize the biologically relevant molecule c-di-AMP, although much weaker than its cognate ligand, could have physiological and biological consequences for the cell. Diadenylate cyclase (DAC) domains, analogous to the diguanylate cyclase (DGC) domains, are broadly distributed among the bacterial kingdom suggesting that c-di-AMP may be present in many bacterial species68-70
. Initial inspection of the distribution of DAC domains (Pfam 02457) and both class I and class II c-di-GMP-binding riboswitches30, 31
across bacteria indicates that many organisms that use riboswitches for c-di-GMP signaling also have predicted DAC domains. For these organisms that potentially use both c-di-GMP and c-di-AMP signaling, the class I riboswitch would likely provide tighter genetic control than the class II riboswitch. While it has been shown that the class II riboswitch can regulate splicing in response to c-di-GMP demonstrating that this RNA can participate in complex forms of gene regulation31
, the increased binding specificity of the class I riboswitch suggests that this motif is more finely tuned to explicitly respond to c-di-GMP.
c-di-GMP analogs may be capable of discriminating between RNA and protein receptors, which would be particularly useful for differentiating between the effects of RNA-mediated and protein-mediated second messenger signaling. Recently, the crystal structures of c-di-GMP bound to several protein effectors have been reported, including the degenerate EAL domain proteins LapD29
as well as several PilZ domain proteins17, 21, 71
. When bound to both LapD and FimX, the bases of c-di-GMP are splayed apart rather than directly aligned over top of one another as they are when bound to RNA receptors, decreasing the strength of any stacking contacts formed with proteins as compared to those networks formed with RNA. This suggests that the 7-deaza guanine modified analogs would affect ligand binding by these proteins to a lesser degree than seen with riboswitch targets. In contrast, PilZ domain proteins can bind c-di-GMP as either a monomer where the guanine bases are slightly staggered overtop one another, or as an intercalated dimer. For proteins that bind the c-di-GMP dimer, interactions are not only formed between each molecule of c-di-GMP and the protein, but between the two c-di-GMP molecules as well. This suggests that ligand modifications that could potentially weaken intermolecular interactions between c-di-GMP may also affect the ability of the dinucleotide to dimerize and therefore select against PilZ domain proteins that bind the second messenger as a dimer.
These second messenger analogs may also affect the activity of the metabolic enzymes responsible for the synthesis and degradation of c-di-GMP in the cell. In particular, several of the c-di-GMP derivatives studied here may display an increased resistance to the phosphodiesterase proteins that specifically degrade this second messenger in the cell. Analogs with such properties would be especially useful for in vivo applications of riboswitch targeting, as well as for tools to further elucidate the molecular mechanisms of c-di-GMP action within the complex cellular environment.