The physiologic state of a protein can dictate its biological function. For RRs, phosphorylation plays an essential role in the signal relay of the TCSTS. In this work, we examined the S. mutans RR ComE, focusing on its phosphorylation state and its capacity to form oligomers. We provide evidence that phosphorylation fails to strongly affect both the specificity and affinity of binding of ComE to its cognate binding site. In contrast, phosphorylation appears to affect the oligomeric state of the protein, perhaps being critical for downstream functions after binding.
A variety of mechanisms have been identified by which phosphorylation activates RRs. Here we examined if the positional negative charge was necessary and sufficient to replace phosphorylation and the active site aspartate. A change in the conserved aspartic acid to glutamic acid can mimic the phosphorylated form of some RRs, and the consequences of this change differs from one species to another (3
). To examine the effect of this mutation on ComE, the putative active site aspartic acid was altered to glutamate or alanine. From binding affinity experiments, we found that ComE:DE has a similar equilibrium dissociation constant as ComE (). However, ComE:DA has a higher Kd
than either ComE or ComE:DE, which suggests that the replacement of aspartic acid with alanine, in either negative charge or configuration, has a marginal effect on ComE binding, demonstrating that phosphorylation is not an intrinsic quality of protein-DNA interactions.
We further tested these two mutant ComE proteins in DNase I footprinting assays with two comC
substrates. We found the identical protection pattern for both wild-type ComE and ComE:DE (). Interestingly, the footprint of ComE:DA showed a difference compared to ComE and ComE:DE ( and ). Similar protection of the comC
substrate was observed; however, the protection was modestly weaker and no hypersensitive sites were found on the ComE:DA footprint (although there was a hypersensitive site on the comC
Δ substrate) (). Hypersensitive spots from footprinting analysis usually indicate the bending of the DNA by the protein of interest. As described previously by Hoover et al., integration host factor (IHF) facilitated the activation of nitrogen fixation nif
operon by bending the regulatory region of the DNA to promote the interaction of transcription factor NifA and RNA polymerase (9
). Although the role of DNA bending by ComE has not been elucidated, the lack of hypersensitive spots by ComE:DA could mean that the aspartic acid plays a significant role in the biological function of ComE. It is plausible that the change from aspartic acid to alanine results in a less active form of ComE, which is consistent with the finding that ComE:DA has a slightly higher equilibrium dissociation constant and a lack of hypersensitive sites as judged by DNase I footprinting. The similarity in binding pattern and affinity for ComE:DE and ComE is not unusual and has been seen with Caulobacter crescentus
CtrA D51E, which binds to DNA with similar affinity as the wild-type unphosphorylated CtrA (33
As previously indicated, phosphorylation of an RR plays an essential part in activation and often increases its affinity for target DNA. In order to test the effects on binding activity of phosphorylated ComE, we directly phosphorylated ComE with small molecule phosphate donors and purified ComE from an E. coli pta-ackA double mutant strain, which eliminated the production of acetyl-P and reduced the likelihood of an endogenously phosphorylated ComE. ComE612 showed identical protection and hypersensitive site cleavages of the comC substrate as the wild-type ComE (). This indicates that the gross binding and structural imposition of each protein on the DNA are indistinguishable. However, we did note a 2-fold difference in binding affinity of the ComE612 protein to DNA but in the absence of any additional data cannot determine if this difference is intrinsic to the protein itself or to the preparations of protein purified; therefore, the binding affinity difference remains an open question. We do, however, distinguish the DNA binding abilities of ComE:DA and ComE612 despite similar DNA binding affinities; the DNase I footprint of ComE612 appears identical to ComE while the ComE:DA footprint does not. Finally, we tried to phosphorylate ComE612 with acetyl-P but observed no improvement in the DNA binding activity in EMSA experiments (data not shown).
One possibility is that ComE cannot be phosphorylated by acetyl-P. In fact, not all RRs are phosphorylated when pretreated with acetyl-P (23
). However, other phosphodonors, such as carbamyl phosphate and PA, have been used to phosphorylate RRs in vitro
). We used PA as an alternative donor to phosphorylate ComE. Treatment with PA reduced ComE binding activity to comC
substrate in EMSA. There are at least three possibilities for why we may not have seen an enhancement of binding affinity when ComE was phosphorylated by a phosphate donor. First, unphosphorylated ComE may be the active form of ComE as has been observed with B. subtilis
). Another possibility is that ComE is efficiently phosphorylated but has a fast and spontaneous autodephosphorylation rate as reported recently by Thomas et al. (37
). However, since ComE lacks some of the important amino acids identified in the Thomas study, which were indicative of high autodephosphorylation rates, we were unable to predict with confidence at what relative rate ComE is predisposed to autodephosphorylate. An empirical experiment would have to be performed to determine the autodephosphorylation rate of ComE. A third possibility is that while phosphorylation might be important to activate RRs for gene expression, it may not play a role in ComE binding, as has been observed for S
. Typhimurium NtrC (43
). Currently, we do not know whether phosphorylation is required for ComE to regulate gene expression, but our analysis of ComE binding strongly favors that phosphorylation does not play a role in DNA binding affinity.
In addition to an increase in binding affinity and activation of gene regulation by phosphorylation, some RRs oligomerize upon phosphorylation. NtrC of enteric bacteria oligomerizes when phosphorylated, and this oligomer catalyzes the isomerization of closed complexes between σ54
holoenzyme and the promoter to open complexes, which activate transcription (40
). On the other hand, simple DNA recognition by the RR can promote oligomerization (25
). In E. coli
, the RR involved in osmotic regulation, OmpR, was shown to dimerize upon phosphorylation or by DNA binding (25
). To address if ComE forms an oligomer when phosphorylated, we used a homobifunctional cross-linker, DMS, to examine the effect of phosphorylation on ComE oligomerization. We showed that when ComE is phosphorylated by PA, a shift to a dimer state becomes favored. However, we observed dimerization only in a small fraction of ComE. There may be a kinetic barrier preventing dimer formation, for example, this phosphorylation-induced dimer could be very unstable or the conformation necessary for dimerization has too short a half-life. Alternatively, it is possible that the DMS reacts nonproductively so that dimers are not prone to form. A similar phenomenon was observed for OmpR in which only a portion of OmpR became dimerized in the presence of PA and the cross-linking reagent (25
). However, in contrast to OmpR (25
), addition of DNA did not increase the dimer formation in the cross-linking reaction (data not shown). Interestingly, when ComE:DE and ComE:DA were incubated with PA and DMS, we did not observe oligomerization of either protein. This result is consistent with D60 of ComE being the active-site aspartic acid for phosphorylation and phosphorylation facilitating oligomerization.
The significance of the phosphorylation-induced ComE dimer remains unknown, but we considered two possibilities. One model is that unphosphorylated ComE binds to one repeat with high affinity as a monomer, and this binding induces a cooperative interaction to the second direct repeat. Once two ComE protomers occupy each direct repeat, phosphorylation of the ComE monomers would regulate gene expression. This model seems unlikely since we were able to show that phosphorylation of ComE by PA also induces cooperative binding to approximately the same degree as unphosphorylated ComE (data not shown). A second possibility is that phosphorylation induces the dimer formation of ComE before binding to the direct repeats and then this dimer binds to the target site to regulate gene expression. In addition, the orientation of the ComE dimers with respect to the RNA polymerase might serve as a mechanism to distinguish between activation and repression of genes. Studies have shown that NtrC activates transcription by contacting RNA polymerase by means of a DNA loop, which allows the polymerase to gain access to the template DNA strand in a productive way (30
). It is possible that ComE binds in a head-to-tail fashion, and the formation of the oligomer (greater than dimers) extends ComE monomers to accomplish a similar contact with RNA polymerase, e.g., when the head of ComE contacts the RNA polymerase, the gene is turned off, and when the tail contacts the RNA polymerase, gene expression is activated or vice versa (). Previously, we have published that ComE acts bifunctionally where it both activates mutacin (nlmC
) production and represses CSP biosynthesis through the same intergenic space (13
). This model accounts for how ComE could regulate these two genes differently while utilizing the same intergenic sites.
Fig 7 Proposed mechanism on how ComE activates nlmC and represses comC. ComE (open arrow bar) binds to a consensus binding site in a head-to-tail fashion and forms oligomers to extend and contact with RNA polymerase (RNAP; diamond shape). When the head contacts (more ...)
In summary, we have characterized conditions that lead to the oligomeric state of ComE. We determined that phosphorylation can facilitate dimer formation of ComE and is likely responsible for downstream activities, possibly through interactions with RNA polymerase.