Identification of PutA binding sites
Initial localization of PutA binding sites in the put
control DNA region was performed by gel mobility shift assays using different fragments of the 419-bp put
control DNA. Systematic evaluation of different regions of the put
control DNA indicated that PutA does not bind to the 1–170 bp region immediately downstream of putP
(, lanes 3–4). However, PutA was observed by gel mobility shift assays to bind to regions 183-231 and 342-412 of the put
control DNA (data not shown). Additional assays indicated that PutA binds to oligonucleotides 183-210, 342-365 and 388-412 (, lanes 5–10). Previously, we showed by gel mobility shift assays that PutA also binds to oligonucleotide 211-231, with apparent binding stoichiometry of one DNA duplex per PutA dimer.25
Figure 1 Localization of PutA binding sites in the put control DNA region. (a) Gel mobility shift assays of PutA with different regions of put control DNA. Separate binding mixtures of PutA (0–1.5 μM) with full-length put control DNA 1–419 (more ...)
Sequence alignment of the four oligonucleotides that bound to PutA (183-210, 211-231, 342-365, 388-412) revealed a GTTGCA consensus sequence. This motif is present in each of the four oligonucleotides (), and it appears five times in the 183 - 412 bp region of the put control DNA (). Thus, five potential operator sites, denoted O1 - O5, were proposed, as shown in .
The proposed binding sites were further examined by changing each one from GTTGCA to GTCATA by site-directed mutagenesis of the put control DNA. Gel mobility shift assays show that simultaneously mutating all five sites disrupts PutA binding to the put control DNA (, Δ12345) confirming that PutA specifically recognizes only the five binding sites in the put control region. Gel mobility shift assays were then used to test PutA binding to the five sites incrementally using PutA52 to resolve the different complexes. As shown in , decreasing the number of binding sites in the put control DNA reduces the observed mobility shift of the protein-DNA complex. This further confirms that the put control DNA contains five PutA binding sites, and suggests that PutA52 is able to bind all five sites simultaneously
Figure 2 Gel mobility shift assays of full-length PutA and PutA52 with wild-type put control DNA and put control DNA with an increasing number of mutated binding sites. (a) Separate binding mixtures of full-length PutA (0–0.25 μM) with wild-type (more ...)
Autorepression of putA
Cell-based reporter gene assays were performed to test the role of each PutA binding site in repressing expression of putA
. For these assays, E. coli
strain JT31 putA− lacZ−
was cotransformed with PutA-pUC18 and PputA:lacZ
-pACYC184 constructs (wild-type and single or multiple operator site mutations in the put
control DNA). Western analysis confirmed expression of PutA. Consistent with previous results, PutA repressed expression of the lacZ
reporter gene by over 75 % relative to control cells (pUC18 alone and wild-type PputA:lacZ
construct) (, WT).22
Mutations of O1 (Δ1) and O2 (ΔO2) singly or in combination (ΔO1-2) did not increase β-galactosidase activity. Because PutA repression of the lacZ
reporter gene (~ 73 %) was not diminished by mutating operator sites O1 and O2, PutA binding to these sites is not necessary for repressing transcription of putA
. Mutating O3 (ΔO3) greatly reduced lacZ
reporter gene expression in the control cells (data not shown) to ~ 10 % of wild-type put
control DNA. Because of the intrinsically low reporter gene expression of the ΔO3 mutant construct, we were not able to directly assess the impact of site O3 on PutA repression of putA
. O3 is located in the −35 region of the putA
promoter (see ), thus, the mutation at site O3 most likely decreases the binding of the σ subunit of E. coli
RNA polymerase to the −35 element. We thus consider O3 to be an important operator for autorepression of putA
despite the fact that we could not test its role using the reporter gene assay. Mutating sites O4 (ΔO4) or O5 (ΔO5) increased β-galactosidase activity and lowered repression of the lacZ
reporter gene to about 50 % relative to the control cells (). Simultaneously mutating O4 and O5 (ΔO4-5) generated an additive effect with a 3-fold increase in β-galactosidase activity relative to WT resulting in only 20 % repression of the lacZ
reporter gene. Thus O3, O4 and O5 are the most critical sites for PutA autorepression of putA
Figure 3 β-galactosidase activity from lacZ reporter constructs containing various mutations in the put control DNA. (a) Relative percent β-galactosidase activity in E. coli strain JT31 containing PutA-pUC18 and wild-type (WT) PputA:lacZ and various (more ...)
Regulation of putP by PutA
The binding sites critical for regulating putP
were also identified. In these assays, E. coli
strain JT31 putA− lacZ−
was cotransformed with the PutA-pUC18 construct and the PputP:lacZ
-pACYC184 construct (wild-type and single or multiple operator site mutations in the put
control DNA). These results are shown in . PutA repressed lacZ
reporter gene expression by about 47 % relative to control cells (, WT). Apparently PutA represses putP
promoter activity less than the putA
promoter, consistent with previous results suggesting PutA is a stronger regulator of putA
Mutation of O1 (ΔO1) increased β-galactosidase activity thereby decreasing the repression of the lacZ
reporter gene to about 30 % relative to control cells. Mutating O2 singly (ΔO2) or in combination with O1 (ΔO1-2) resulted in about 20 % repression relative to control cells. In contrast to the putA
promoter, mutation of O3, O4, and O5 individually (ΔO3, ΔO4, ΔO5) () or in combination (ΔO3-5) (data not shown) did not significantly increase β-galactosidase activity or alter the repression of the lacZ
reporter gene. Mutating all five binding sites (ΔO1-5) resulted in the same repression of lacZ
expression (20 %) as ΔO1-2 put
control DNA (). Thus, PutA binding to O1 and O2 is responsible for repressing the putP
Overall structure of PutA52 bound to O2
The crystal structure of PutA52 bound to O2 was solved in order to understand the three-dimensional structural basis of DNA recognition by PutA. This structure is the first one of a PutA RHH domain bound to DNA, and it is currently the highest resolution structure of a RHH/DNA complex. The asymmetric unit contains one PutA52 dimer bound to one O2 duplex ().
Figure 4 Overall structure of PutA52 bound to O2. PutA52 chains A and B are colored green and magenta, respectively. DNA is represented as sticks, with strand 1 colored yellow and strand 2 colored white. The electron density map is a 2Fo-Fc map with Fc and phases (more ...)
Each PutA52 chain adopts the RHH fold, which consists of a β-strand (β1) followed by two α-helices (αA, αB). The two protein chains assemble into a dimer featuring an intermolecular two-stranded antiparallel β-sheet ().
The bound DNA ligand adopts the B conformation, based on analysis of projected phosphorus positions (zP
) using 3DNA.28
Values of zP
< 0.5 are diagnostic of B-form DNA, whereas zP
> 1.5 Å indicate A-form DNA.28,29
All but three of the 17 base pair steps of O2 have zP
< 0.5 Å. The three exceptions have zP
= 0.52 – 0.58 Å. Thus, binding of PutA52 to O2 does not cause significant distortion of the DNA from the expected B conformation. Also, the double helix displays no discernable curvature ().
The β-sheet of PutA52 inserts into the DNA major groove (). Residues of the sheet contact DNA bases, while residues near the N-terminus of αB interact with the DNA backbone. This general mode of binding is typical for RHH proteins.30
Although the five operators that we identified each contain the 6-bp consensus sequence of GTTGCA (), the structure shows that PutA52 contacts a larger fragment of DNA. A plot of the surface area buried by nucleotides in the protein-DNA interface is shown in . The bimodal shape of the plot reflects the two-fold symmetries of the protein dimer and the DNA double helix. The surface area calculations, along with detailed inspection of the protein-DNA interface, show that the footprint of PutA52 encompasses the 9-bp fragment from G6:C16 to C14:G8 (see boxed base pairs in ). Note that this fragment contains the GTTGCA motif. Interactions with the 9-bp fragment are summarized schematically in . and shown in detail in .
Figure 5 Footprint of PutA52 on O2 derived from the crystal structure. (a) Surface area contributed by DNA nucleotides to the protein-DNA interface. (b) Schematic diagram of protein-DNA interactions. Dotted lines indicate electrostatic interactions. Thick, solid (more ...)
Figure 6 Detailed stereographic view of the protein-DNA interface. PutA52 chains A and B are colored green and magenta, respectively. DNA strands 1 and 2 are colored yellow and white, respectively. Cyan spheres represent water molecules. Dotted lined indicate (more ...)
Interactions with DNA bases
Structures of RHH domains bound to DNA show that, typically, two polar residues and one Arg/Lys from each β-strand form hydrogen bonds to DNA bases. In PutA, this critical triad corresponds to Thr5, Gly7 and Lys9, and all three residues interact with DNA bases. We note that these residues are identically conserved among PutAs.24
Lys9 binds to the pair of guanine bases located at the 5′ ends of each strand (). Lys9 of chain A interacts with the guanine bases of strand 2, while Lys9 of the B chain interacts with the guanine bases of strand 1. The two sets of interactions are nearly identical (), which is expected since they involve the palindromic ends of the DNA fragment. Each Lys9 forms four hydrogen bonds, two with each base of the guanine pair. These interactions are shown for Lys9(B) in . The hydrogen bond distances are 2.5 – 3.1 Å for the inner base (G7 of strand1, G9 of strand 2) and 3.2 – 3.5 Å for the outer base (G6 of strand 1, G8 of strand 2). We note that only guanine has two appropriately placed hydrogen bond acceptors for interaction with Lys9, so these interactions appear to enforce a preference for binding a 9-bp fragment containing GG at the 5′ ends of both strands.
Figure 7 Close-up views of selected protein-DNA interactions. In all three panels, PutA52 chains A and B are colored green and magenta, respectively, DNA strands 1 and 2 are colored yellow and white, respectively, and black dotted lines indicate electrostatic (more ...)
Thr5 forms hydrogen bonds with three different base pairs and both DNA strands. In chain A, the hydroxyl of Thr5 donates a hydrogen bond to T8 of strand 1 (), while the backbone carbonyl accepts a hydrogen bond from C12 of strand 2 (). Since the hydrogen bond with T8 involves the palindromic GGT end of the DNA, one might expect Thr5(B) to form an analogous interaction with T10 of strand 2. Interestingly, Thr5(B) accepts a hydrogen bond from C11 of strand 1 () rather than hydrogen bonding with T10. The expected two-fold symmetry is broken by a conformational change of Thr5(B). The χ1 angle of Thr5(B) is +60°, whereas this angle is −60° for Thr5(A). We note that Thr5 has χ1 = −60° in all chains of ligand-free PutA52 structures (PDB codes 2AY0, 2GPE). Thus, binding to DNA induced a conformational change in Thr5(B), which introduces asymmetry in PutA52.
Gly7 helps confer sequence specificity despite lacking a side chain. In chain A, Gly7 donates a hydrogen bond (2.9 Å) to the N7 atom of G11 (). In chain B, Gly7 forms van der Waals interactions with the C5 methyl of T9 (). Note also the close contacts between DNA bases and Thr5(A) in this region of the structure (). The tight packing of the T9:A13 base pair against Gly7 and Thr5 could contribute to sequence specificity.
Finally, there are no water molecules bridging the protein with DNA bases. There is, however, one water molecule (Wat6) strategically located in the protein-DNA interface on the pseudo two-fold axis that relates the two chains (). It is equidistant from the two Gly7 residues of the β-sheet, and forms hydrogen bonds with G10 of DNA strand 1 and G11 of strand 2 (). Wat6 appears to fill the void created by the lack of a side chain at residue 7. Indeed, mutation of Gly7 in silico to any other residue causes steric clash with this water molecule as well as with DNA bases.
Interactions with the DNA backbone
Thr28, Pro29 and His30 bind the DNA backbone. Thr28 is the Ncap of αB, while Pro29 and His30 are the first two residues of αB. The interactions display nearly perfect two-fold symmetry (), so just one set of interactions will be described. The side chains of Thr28 and His30 form electrostatic interactions with the phosphate group connecting the two G nucleotides at the 5′ end of the 9-bp fragment (). In addition, the backbone of His30 donates a hydrogen bond to the phosphate group of the T nucleotide at the 5′ end of the 9-bp fragment (T8 of strand 1, T10 of strand 2, see ). Finally, the Cδ atom of Pro29 forms close contacts (3.4 Å) with oxygen atoms of the phosphate backbone ().
Isothermal titration calorimetry
The binding of O2 to PutA52 at pH 8.0 was studied using ITC to gain insights into the thermodynamic basis of DNA recognition. In Tris buffer, the association reaction was evidently endothermic (), whereas, in phosphate buffer at the same pH, the reaction was weakly exothermic (). Since the enthalpy of ionization of Tris (11 kcal/mol) differs substantially from that of dihydrogen phosphate (1 kcal/mol), these results suggest that the DNA-binding event is coupled to the ionization reaction of the buffer at pH 8.0. Moreover, the fact that the titration in Tris yielded the more endothermic result implies proton uptake by the protein-DNA complex during association.
Figure 8 ITC-based analysis of the interaction between PutA52 and the O2 and O2bf4 duplexes. (a) Raw data for the titration of 12 μM PutA52 with 0.15 mM O2 (10 μL additions) in NaCl, Tris, pH 8.0. (b) Raw data for the titration of 21 μM (more ...)
The data from the four titrations with O2 were fit simultaneously as described in Materials and Methods to estimate the intrinsic binding enthalpy, association equilibrium constant and number of protons transferred (). This analysis shows that the binding of O2 to PutA52 is intrinsically exothermic, with ΔH
= −1.8 kcal/mol (), and K
= 4.8 × 106
, which corresponds to Kd
= 210 nM (). The latter value agrees favorably with the estimate from gel-shift analysis of Kd
< 200 nM for O2 binding to full-length PutA.25
The estimated number of protons transferred to the protein/DNA complex is 0.7.
Isothermal titration calorimetry data for DNA binding to PutA52 at 298K
A second set of titrations was performed using oligonucleotide O2fb4, which is identical to O2 except that the bases flanking the GTTGCA motif are those of O4. These measurements were performed to assess the impact of bases outside of the consensus motif on affinity. As with O2, the apparent enthalpy of binding of O2fb4 to PutA52 at pH 8.0 is dependent on buffer choice. In Tris buffer, the association appears to be strongly endothermic (), but in phosphate buffer the reaction is nearly isenthalpic ().
Global analysis of the data from the two O2fb4 titrations () shows that binding of this oligonucleotide to PutA52 is marginally endothermic, with intrinsic enthalpy change of only 0.18 kcal/mol (). The association constant from global fitting is K = 3.2 × 105 M−1, which corresponds to Kd = 3100 nM. As with the O2 titrations, there is an uptake of 0.7 protons during binding, which suggests that the binding mechanisms of the two ligands are qualitatively similar. Notice, however, that the association constant for O2 is fifteen times higher than that of O2fb4. These results show that bases outside of the consensus motif impact the affinity of PutA52, and presumably PutA, for put control sites.
The binding of PutA52 is entropy-driven for both ligands. This result, combined with the observation that the protein-DNA interface is nearly devoid of bound water molecules, suggests that desolvation of macromolecular surfaces is important for DNA binding. We note that the free energy of the RHH protein MetJ binding to a metbox operator also includes a substantial favorable entropic component at 25 °C, particularly in the absence of the corepressor S-adenosylmethionine.31