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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Mol Biol. Author manuscript; available in PMC Apr 3, 2009.
Published in final edited form as:
PMCID: PMC2665032
NIHMSID: NIHMS97250

Structural Basis of Transcriptional Regulation of the Proline Utilization Regulon by Multifunctional PutA

Summary

The multifunctional Escherichia coli PutA flavoprotein functions as both a membrane-associated proline catabolic enzyme and transcriptional repressor of the proline utilization genes putA and putP. To better understand the mechanism of transcriptional regulation by PutA, we have mapped the put regulatory region, determined a crystal structure of the PutA ribbon-helix-helix domain (PutA52) complexed with DNA and examined the thermodynamics of DNA binding to PutA52. Five operator sites, each containing the sequence motif 5′-GTTGCA-3′, were identified using gel-shift analysis. Three of the sites are shown to be critical for repression of putA, whereas the two other sites are important for repression of putP. The 2.25 Å resolution crystal structure of PutA52 bound to one of the operators (operator 2, 21-bp) shows that the protein contacts a 9-bp fragment, corresponding to the GTTGCA consensus motif plus three flanking base pairs. Since the operator sequences differ in flanking bases, the structure implies that PutA may have different affinities for the five operators. This hypothesis was explored using isothermal titration calorimetry. The binding of PutA52 to operator 2 is exothermic with an enthalpy of −1.8 kcal/mol and a dissociation constant of 210 nM. Substitution of the flanking bases of operator 4 into operator 2 results in an unfavorable enthalpy of 0.2 kcal/mol and 15-fold lower affinity, which shows that base pairs outside of the consensus motif impact binding. The structural and thermodynamic data suggest that hydrogen bonds between Lys9 and bases adjacent to the GTTGCA motif contribute to transcriptional regulation by fine-tuning the affinity of PutA for put control operators.

Keywords: proline utilization A, X-ray crystallography, isothermal titration calorimetry, ribbon-helix-helix, proline catabolism

Introduction

Proline is used as a source of carbon, nitrogen and energy through two oxidative steps catalyzed by proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH).17 In enteric bacteria such as Escherichia coli, proline utilization requires the two genes, putP and putA. The former encodes the PutP high-affinity Na+-proline transporter and the latter encodes the multifunctional flavoprotein PutA (Proline utilization A).8,9 PutA is unique in that it functions as both a transcriptional repressor of the put genes and a membrane-associated bifunctional proline catabolic enzyme.2,1012 The enzymatic and transport functions of the putA and putP genes, respectively, are conserved among different Gram-negative bacteria, whereas the genetic organization and regulatory mechanisms that control the expression of these genes are highly divergent.7,1018 The focus of this work is to provide a molecular and structural understanding of the regulation of put genes in E. coli by PutA.

PutA from E. coli combines PRODH, P5CDH, and transcriptional regulatory activities into a single polypeptide of 1320 amino acids.2,19 Insights into the organization of the functional domains in PutA have been gained from molecular dissection and characterization of truncated PutA proteins. The PRODH and P5CDH active sites are located within residues 261-612 and 650-1130, respectively, with the PRODH active site utilizing an FAD cofactor and P5CDH activity requiring NAD+. Structural studies have shown that the PRODH domain forms a unique (βα)8 barrel,20,21 and that reduction by dithionite causes dramatic conformational changes in the FAD ribityl chain.22 Molecular dissection studies showed that the DNA-binding domain is contained in residues 1-47.23 Subsequently, the crystal structure of a polypeptide corresponding to E. coli PutA residues 1-52 (PutA52) was solved, which showed that PutA is a member of the ribbon-helix-helix (RHH) family of transcriptional regulators.23,24

While knowledge of PutA structure and function continues to build, a considerable gap remains in our understanding of critical PutA-DNA interactions in the put control DNA region. To further understand the regulation of proline metabolism in E. coli, we have identified the PutA binding sites in the put regulatory region, elucidated the roles of these operators in repressing expression of putA and putP, determined the crystal structure of PutA52 bound to one of the identified operators, and investigated the thermodynamics of DNA binding to PutA52 using isothermal titration calorimetry.

Results

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 (Fig. 1a, 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 (Fig. 1a, 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 ...

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 (Fig. 1b), and it appears five times in the 183 - 412 bp region of the put control DNA (Fig. 1b). Thus, five potential operator sites, denoted O1 - O5, were proposed, as shown in Fig. 1c.

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 (Fig. 2a, 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 Fig. 2b, 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 ...

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) (Fig. 3a, 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 Fig. 1c), 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 (Fig. 3a). 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 ...

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 Fig. 3b. PutA repressed lacZ reporter gene expression by about 47 % relative to control cells (Fig. 3b, WT). Apparently PutA represses putP promoter activity less than the putA promoter, consistent with previous results suggesting PutA is a stronger regulator of putA than putP.26,27 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) (Fig. 3b) 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 (Fig. 3b). Thus, PutA binding to O1 and O2 is responsible for repressing the putP promoter.

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 (Fig. 4).

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 ...

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 (Fig. 4).

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 (Fig. 4).

The β-sheet of PutA52 inserts into the DNA major groove (Fig. 4). 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 (Fig. 1b), 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 Fig. 5a. 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 Fig. 4). Note that this fragment contains the GTTGCA motif. Interactions with the 9-bp fragment are summarized schematically in Fig. 5b. and shown in detail in Fig. 6.

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 ...
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 ...

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 (Fig. 5b). 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 (Fig. 6), 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 Fig. 7a. 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 ...

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 (Fig. 7a), while the backbone carbonyl accepts a hydrogen bond from C12 of strand 2 (Fig. 7b). 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 (Fig. 7b) 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 (Fig. 7b). In chain B, Gly7 forms van der Waals interactions with the C5 methyl of T9 (Fig. 7c). Note also the close contacts between DNA bases and Thr5(A) in this region of the structure (Fig. 7c). 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 (Fig. 6). 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 (Fig. 7B). 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 (Fig. 6), 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 (Fig. 7a). 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 Fig. 7a). Finally, the Cδ atom of Pro29 forms close contacts (3.4 Å) with oxygen atoms of the phosphate backbone (Fig. 7a).

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 (Fig. 8a), whereas, in phosphate buffer at the same pH, the reaction was weakly exothermic (Fig. 8b). 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 ...

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 (Fig. 8c). This analysis shows that the binding of O2 to PutA52 is intrinsically exothermic, with ΔH = −1.8 kcal/mol (Table 2), and K = 4.8 × 106 M−1, which corresponds to Kd = 210 nM (Table 2). 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.

Table 2
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 (Fig. 8d), but in phosphate buffer the reaction is nearly isenthalpic (Fig. 8e).

Global analysis of the data from the two O2fb4 titrations (Fig. 8f) shows that binding of this oligonucleotide to PutA52 is marginally endothermic, with intrinsic enthalpy change of only 0.18 kcal/mol (Table 2). 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

Discussion

Transcriptional regulation of the put regulon

Based on the arrangement of the five PutA-DNA binding sites, PutA most likely represses the put genes by hindering the σ70-dependent binding of E. coli RNA polymerase to the putA and putP promoter regions.32 We did not find additional PutA consensus binding sites in the coding regions of putA and putP, indicating that PutA binds only to the put intergenic region. Previous reports suggested that proline, via PutA, regulates expression of putA more tightly than putP.26,27 Here we have shown that PutA is a stronger repressor of the putA promoter than the putP promoter. Therefore, putP expression appears to be regulated relatively weakly by PutA, which would allow proline uptake under a variety of environmental conditions leading to subsequent activation of putA expression. In addition to proline-specific regulation by PutA, the put genes are also responsive to global regulators. The cAMP receptor protein has been proposed to function as an activator by increasing putA and putP promoter activity in nutrient-poor environments.26

We evaluated put control DNA sequences from other bacteria in which PutA contains the conserved RHH domain and is predicted to function as an autogenous transcriptional repressor. The GTTGCA sequence was found in every putA promoter region of the 39 genome sequences analyzed.

We also examined put control DNA sequences in bacteria that share the same genetic organization of putA and putP as found in E. coli. Fig. S1 (supplemental material) shows an alignment of put control DNA sequences from E. coli, Shigella boydii, Salmonella typhimurium, Klebsiella aerogenes, Yersinia pestis and Pseudomonas putida. The length of the intergenic region ranges from 361 base pairs in P. putida to 577 base pairs in Y. pestis. Each sequence has at least three exact repeats of the GTTGCA motif. Operators O1, O3 and O4 are present in all six sequences. O2 is present in all six organisms, except P. putida. O5 is the least conserved site. Y. pestis and P. putida have additional exact repeats of the motif that do not align with the E. coli sites. These results suggest that the GTTGCA motif is the fundamental transcriptional control element of the PutA autogenous repression system. Therefore, the biochemical and structural results reported here for E. coli are likely applicable to other organisms in which PutA serves as a transcriptional repressor.

Importance of the GTTGCA motif for PutA - operator binding

Mutation of the consensus GTTGCA motif to GTcatA was found to severely impact binding to PutA, based on gel shift analysis. In fact, mutation of all five operators eliminated binding. Moreover, this mutation was found to affect gene transcription as monitored by cell-based reporter assays. These results show that recognition of the middle of the consensus motif is essential for PutA binding and proper transcriptional control.

The basis for these results is evident from the crystal structure. The mutated triplet corresponds to base pairs T9:A13, G10:C12, and C11:G11 of the structure. As shown in Fig. 5B, the protein directly contacts T9, C12, C11 and G11. Mutation of T9 to C eliminates van der Waals interactions between the thymine C5 methyl and Gly7(B) (Fig. 7c). Replacement of C12 with T eliminates the hydrogen bond with the backbone carbonyl of Thr5(A) (Fig. 7b). In fact, this mutation would position two hydrogen bond acceptors - carbonyl of Thr5(A) and O4 carbonyl of thymine - close to each other, which is unfavorable. Moreover, the C5 methyl of the thymine would clash with Thr4(A). Interestingly, changing C11:G11 to T:A is predicted to have little effect on the protein-DNA interaction surface. Mutation of G11 to A would preserve the hydrogen bond donated by Gly7(A) (Fig. 7b), since both adenine and guanine have the hydrogen bond donor, N7. Likewise, one can imagine the thymine O4 carbonyl engaging Thr5(B) in a hydrogen bond analogous to the one formed with the C11 in Fig. 7B. Thus, the observed deleterious effect on binding due to the GTcatA triple mutation appears to be primarily due to the change of TG to CA.

Whereas E. coli has five exact repeats of the GTTGCA motif, Y. pestis has single nucleotide substitutions in O1 (GTTaCA) and O2 (GTTGtA) (Fig. S1). As described in the preceding paragraph, the G-to-A variation in O1 is predicted to disrupt the protein-DNA interface. On the other hand, the GTTGtA variation is likely to be accommodated by the protein without significant structural penalty. Hence, the Y. pestis O1 site may have a limited role in transcriptional control. We note that Y. pestis has an additional GTTGCA sequence motif upstream of O1, and that this site could substitute for a nonfunctional O1.

Importance of bases flanking the GTTGCA motif

Given the conservation of the GTTGCA motif in put intergenic regions, it is not surprising that interactions with these bases are essential for binding. Interestingly, the structure reveals that the footprint of PutA52 extends beyond the GTTGCA motif, indicating that bases flanking the consensus sequence may be important for operator recognition.

The structure shows that PutA52 contacts a 9-bp fragment, which we denote XGTTGCAYZ. Lys9 interacts with XG and the complementary bases of Y and Z. Based on the structure, it appears that G is preferred for X and CC is preferred for YZ, because this sequence maximizes the number of hydrogen bonds formed by Lys9 (eight total, four with each DNA strand).

Operators 1–3 of E. coli have G for base X while operators 4 and 5 have A (Fig. 1b). Substitution of A in place of G at position X can be simulated by imagining adenine in place of G6 in Fig. 7a. This change would eliminate one of the hydrogen bonds formed by Lys9. All five operators have C for position Y except O4, which has A. The effect of this variation can be seen by changing G7 in Fig. 7A to thymine. Elimination of one hydrogen bond is again predicted. Operators 1 and 3 have T and A, respectively, at position Z, which requires Lys9 to interact with A and T, respectively. Both variations would eliminate one hydrogen bond with Lys9, relative to optimal case of O2. This simple hydrogen bond inventory analysis implies that PutA exhibits different affinities for the five operator sites, with O2 predicted to have the highest affinity (eight hydrogen bonds to Lys9) and O4 having the lowest affinity (six hydrogen bonds).

This hypothesis was tested with ITC experiments that compared the binding of PutA52 to two ligands differing only in the bases flanking the consensus motif: O2 and O2fb4. The former ligand is the one used in crystal structure determination. The latter is identical to O2 except that it has the flanking bases of O4 (X,Y = A). These two ligands thus mimic the two extremes of the predicted affinity spectrum of the PutA operator sites.

The ITC analysis showed that PutA52 binds to O2 with fifteen times higher affinity than O2fb4 (Table 2), in agreement with our structure-based predictions. The binding enthalpy accounts almost entirely for the difference in affinity (Table 2); ΔH for O2 is more exothermic than that of O2fb4 by about 2 kcal/mol. That the difference in affinity is enthalpic in origin is consistent with our prediction that Lys9 forms more hydrogen bonds with the flanking bases of O2 than O4. These results suggest that the five operator sites are nonequivalent in terms of binding affinity, which is potentially significant because differential binding could be important for proper transcriptional regulation.

In light of these results, it is interesting that PutA is a weaker repressor of the putP promoter than the putA promoter, yet the highest affinity operator (O2) is involved in repression of putP whereas the lowest affinity operator (O4) is involved in repression of putA. We suggest that PutA-DNA binding affinity is only one of several factors to consider when assessing the potential impact of the operators on transcriptional repression. Other key factors include the number of transcription start sites and the location of the operators relative to the promoter regions. The putP gene was previously shown to have multiple transcription start sites and three functional promoters.26 The three putP promoters are positioned 14, 26, and 58 base pairs upstream of the O1 site. Thus, PutA binding at O1 and O2 is not predicted to directly interfere with RNA polymerase at each of the putP promoters resulting in weaker repression of the putP gene by PutA. On the other hand, the putA gene has only one promoter and a single transcriptional start site with O3 and O4 located within the putA promoter region. Thus, these sites are positioned optimally for PutA to interfere with RNA polymerase, which could explain why PutA is a stronger repressor of putA expression.

Parenthetically, this ITC analysis underscores the value of examining binding reactions in buffers having distinct ionization enthalpies. Besides revealing the involvement of protonation in protein-ligand associations, inclusion of the buffer ionization enthalpy can, in select cases, significantly improve the quality of the titration data. For example, the intrinsic binding enthalpy for the interaction between PutA52 and the O2fb4 oligo (0.18 kcal/mol) is effectively zero. Absent the contribution of phosphate or Tris ionization, this binding reaction would be invisible by ITC. Although the intrinsic enthalpy for the PutA52-O2 association is somewhat larger (−1.79 kcal/mol), the interaction would nonetheless be difficult to characterize in phosphate buffer alone because the heat of buffer ionization reduces the observed enthalpy to just −0.8 kcal/mol. In striking contrast, the highly endothermic Tris ionization event renders the reaction much more amenable to analysis and facilitates, via a global fitting strategy, treatment of the data collected in phosphate buffer.

Possible roles of PutA-specific residues

PutA is a unique member of the RHH family of transcription factors. With a polypeptide chain in excess of 1300 residues, PutA is the largest protein known to contain an RHH domain. Furthermore, to our knowledge, PutA is the only protein to have a flavin redox regulatory domain coupled to an RHH domain.

PutA is also distinguished from other RHH proteins at the primary sequence level.24 In particular, Gly7 and Pro29 are absolutely conserved among PutAs, yet rarely found in other RHH domains.

We suggest three possible roles for Pro29. First, proline may facilitate initiation of αB and thus help position Thr28 and His30 for interaction with the DNA backbone. Second, Pro29 may provide a steric “backstop” for the DNA backbone and thereby contribute to recognition of a 9-nucleotide fragment of B-form DNA. A third possibility is that Pro29 may be donating C-H…O hydrogen bonds to the DNA backbone. This suggestion is based on the observation Pro29 Cδ forms close contacts with oxygen atoms of the phosphate backbone (Fig. 7a).

There is precedent for proline Cδ donating hydrogen bonds. For example, when proline is located in the middle or C-terminus of an α-helix, Cδ donates hydrogen bonds to the backbone carbonyl 3-5 residues preceding the proline.33 These unconventional hydrogen bonds enable proline to appear in α-helices despite lacking a free N-H group for classic i to i+4 hydrogen bonding. We note that the CH…O distance of 3.4 Å observed in the PutA52/O2 complex is identical to the average distance for proline intrahelix C-H…O hydrogen bonds.33

The unique location of Pro29 at the beginning of αB, juxtaposed to the DNA backbone, also supports a hydrogen bonding role. Analysis of RHH/DNA structures shows that there are two conserved hydrogen bonds donated by the backbone of residues at the N-terminus of αB to the DNA backbone.30 We observe only one of these conserved hydrogen bonds, and it involves the backbone N-H of His30 (Fig. 7a). Since proline does not have a free N-H group, the second conserved hydrogen bond is missing. We suggest that the unconventional C-H…O hydrogen bonds substitute for the missing conserved hydrogen bond.

Gly7 represents an interesting sequence variation for the RHH family. Typically, this position of the β-sheet is occupied by a polar residue, such as Thr or Asn, which forms hydrogen bonds with DNA bases. The PutA52/O2 structure shows that Gly7 participates in base recognition, despite lacking a side chain. It donates a hydrogen bond to a guanine base and forms van der Waals contacts with the C5 methyl group of thymine.

Gly7 may underlie a more global structural aspect of DNA recognition by PutA: deep penetration into the major groove. Absence of a side chain at this position allows the β-sheet to penetrate further into the major groove, compared to other RHH proteins. As a measure of depth of penetration, we calculated the distance of closest approach between the DNA axis and each of the Cα atoms of the three canonical β-sheet residues responsible for base recognition (Thr5, Gly7, Lys9 in PutA). The values for PutA52/O2 are 5.0 Å, 5.1 Å, and 8.2 Å for chain A and 7.3 Å, 6.0 Å, and 7.4 Å for chain B. The corresponding values for Arc,34 MetJ,35 CopG,36 NikR,37 omega38 and FitA39 bound to DNA span the ranges 8.0 – 11.9 Å, 5.8 – 8.1 Å, and 8.2 – 11.7 Å, respectively, for these three residue positions. Thus, PutA52 penetrates deeper into the major groove than the other RHH proteins. The role of this close encounter in transcriptional regulation of the put regulon is unknown, but the universal conservation of Gly7 in PutAs, and its absence in the rest of the RHH family, suggests functional significance.

Materials and Methods

Materials

Chemicals and buffers were purchased from Fisher Scientific and Sigma-Aldrich, Inc. unless otherwise stated. Restriction endonucleases and T4 DNA ligase were purchased from Fermentas and Invitrogen, respectively. BCA reagents used for protein quantitation were obtained from Pierce. Goat anti-rabbit secondary antibody was purchased from Amersham Inc. E. coli strains XL-blue and BL21 DE3 pLysS were purchased from Stratagene. E. coli strain JT31 putA lacZ was a generous gift from J. Wood (University of Guelph, Guelph, ON, Canada). Synthetic oligonucleotides for site-directed mutagenesis, cloning, DNA-binding assays and co-crystallization were purchased from Integrated DNA Technologies. All experiments used Nano-pure water. LB medium and Terrific broth were used for general culture growth and protein production, respectively, while M9 minimal medium was used for cell-based transcription assays.

Gel-shift assays

Full-length PutA and PutA52 were expressed as C-terminally His-tagged proteins from vector pET23b (Novagen) and purified as described previously.23,24,40 The C-terminal His tags were retained after purification. Purified full-length PutA was dialyzed into 50 mM Tris (pH 7.5) containing 10 % glycerol and stored at −70 °C. PutA52 was dialyzed into 50 mM phosphate buffer (pH 7.3) containing 200 mM NaCl and stored at −70 °C. The concentrations of the PutA proteins were determined using the BCA method (Pierce) with bovine serum albumin as the standard and spectrophotometrically using molar extinction coefficients of 12,700 M−1 cm−1 at 451 nm for PutA and 6970 M−1 cm−1 at 280 nm for PutA52.24,41

Nondenaturing gel electrophoretic mobility shift assays were used to test the binding of the PutA proteins to the put control intergenic DNA as previously described.23,41 Different regions of the put control DNA (putC) were PCR amplified (non-labeled) using synthetic primers and purified. The purified products were incubated with full-length PutA (0, 0.6, and 1.5 μM) for 20 min at 20 °C in 50 mM Tris buffer (pH 7.5, 100 mM NaCl). The protein-DNA complexes were separated using a native polyacrylamide gel (4 %) at 4 °C. The gel was then stained with ethidium bromide and visualized by Bio-Rad Quantity One. Binding assays with synthetic oligonucleotides corresponding to base pairs 183-210 (O1), 342-365 (O3/4), and 388-412 (O5) of putC were performed similarly. The concentration of oligonucleotide for these assays was 100 nM. Duplex DNA of each oligonucleotide was prepared by annealing the complementary oligonucleotides in buffer (10 mM Tris, pH 8.0, 50 mM NaCl, 1 mM EDTA) by first heating at 95 °C for 5 min and then gradually cooling down the sample to room temperature.

Gel-shift assays utilizing fluorescently labeled put intergenic DNA were also performed. The synthetic oligonucleotide (M13 forward primer) was 5′ end-labeled with IRdye-700 (LI-COR, Inc.) and used as one of the primers in a PCR reaction to amplify wild-type put intergenic DNA or mutant put intergenic DNA containing different combinations of PutA binding site mutations. The resulting IRdye-700 labeled put intergenic DNA was purified and quantitated by measuring the nucleic acid concentration at 260 nm and the absorbance of the IRdye-700 at 685 nm using an extinction coefficient of 170 mM−1 cm−1 according to the recommendations of the manufacturer. PutA (0–900 nM) or PutA52 (0–200 nM) was incubated with 2 nM put intergenic DNA in a total volume of 25 μl in 50 mM Tris, 50-250 mM NaCl, pH 7.5, containing 10 % glycerol for 20 min (20 °C) before electrophoresis. Calf thymus competitor DNA (100 μg/ml) was also added to the binding mixtures to prevent nonspecific protein-DNA interactions. The PutA-DNA and PutA52-DNA complexes were separated using a native polyacrylamide gel electrophoresis at 4 °C. The gels were visualized using a LI-COR Odyssey Imager.

LacZ reporter assays

E. coli strain JT31 putA lacZ was cotransformed with PutA-pUC18 and the reporter construct PputA:lacZ-pACYC184 or PputP:lacZ-pACYC184. Details about the cloning procedures and the primers used for generating the above constructs are provided in the supplemental material and Table S1. To test which PutA binding sites are critical for repressing put gene expression, E. coli strain JT31 putA lacZ containing different combinations of the PutA-pUC18 construct and the PputA:lacZ or PputP:lacZ reporter constructs were grown at 37 °C in M9 minimal medium supplemented with ampicillin (50 μg/ml), kanamycin (40 μg/ml) and chloramphenicol (34 μg/ml) to OD600 ~ 1.0. PutA expression from the lac promoter on pUC18 was not induced as no isopropyl-β-D-thiogalactopyranoside was added to the culture medium. Cells from the various cultures were pelleted, resuspended in Tris-HCl buffer (20 mM, pH 7.5) and broken using the B-PER II bacterial protein extraction reagent from Pierce (20 mM Tris-HCl, pH 7.5).23 β-galactosidase activity assays were performed in a 1-ml volume of 100 mM sodium phosphate, pH 7.3, containing 1 mM MgCl2, 50 mM β-mercaptoethanol, and 2 mM o-nitrophenyl-β-D-galactopyranoside. The initial velocity was determined by measuring the increase in absorbance at 420 nm. The reported β-galactosidase activities are averaged values from four independent experiments.

Expression of PutA was confirmed by Western blot analysis using an antibody directed against a polypeptide containing PutA residues 1-47 (PutA47). PutA47 was purified without a 6xHis tag as described previously.23 Antiserum directed against purified PutA47 was prepared by Proteintech Inc. For Western blot analysis, cell pellets from 5 ml culture grown in minimal medium (OD ~ 1.0) were resuspended in 100 μL of SDS sample buffer and boiled for 10 min. After SDS denaturing electrophoresis the protein bands were transferred onto a sequi-blot polyvinylidene difluoride (PVDF) membrane (Bio-Rad, 0.2 μm pore size) using an EBU-4000 semi-dry electrophoretic blotting system. Immunoreactive bands were detected using Enhanced chemiluminescence Western blotting reagents (Amersham Inc.).

Crystallization

Exhaustive crystallization experiments were conducted using two different RHH domain constructs paired with several different DNA fragments, as described in Supplemental Material. Successful co-crystallization required the use of an RHH domain construct consisting of E. coli PutA residues 1-52 (PutA52) fused to a cleavable N-terminal histidine tag. Expression and purification protocols for this protein have been described.24 The N-terminal tag was removed by proteolysis prior to crystallization trials as described.24 The purified, tag-free protein was concentrated using an Amicon Ultra centrifugal filtration device (MWCO 5,000) to 10.8 mg/mL in a buffer of 20 mM Tris pH 8.0, 500 mM NaCl, 20 mM imidazole. The protein concentration was estimated with the BCA method.

The oligonucleotide used for co-crystallization with PutA52 corresponds to nucleotides 211-231 of the put control region, which contains the second of five operator sites for PutA (denoted O2 in Fig. 1):

5′-TTTGCGGTTGCACCTTTCAAA-3′(strand 1)

3′-AAACGCCAACGTGGAAAGTTT-5′(strand 2)

Each DNA strand was dissolved in 10 mM Tris, 50 mM NaCl, 1 mM EDTA, pH 8.0 to a concentration of 6 mM. The two strands were annealed as follows. Equal volumes of the oligonucleotide solutions were combined, and the mixture was placed in a water bath at room temperature. The temperature of the bath was then set to 94 °C, and once the target temperature was reached, the power to the bath was turned off to slowly cool the sample back to room temperature.

The PutA52 and dsDNA stock solutions were mixed so that the molar ratio of PutA52 dimer to dsDNA was approximately 1:3. The mixture was injected onto a Sephacryl S-100 HiPrep 16/60 gel filtration column equilibrated with 10 mM Tris, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT, pH 8.0. Fractions were pooled and concentrated to 11.6 mg/mL (BCA assay) using an Amicon Ultra centrifugal filtration device (MWCO 10,000).

The PutA52/DNA mixture was input to several crystal screens to identify initial crystallization conditions. The crystals used for data collection were obtained directly from Index Screen reagent 54, which consists of 30 % PEG-MME 550, 50 mM CaCl2, and 100 mM Bis-Tris pH 6.5.

The crystals were prepared for low temperature data collection by soaking them in a solution of 32 % PEG-MME 550, 50 mM CaCl2, 100 mM Bis-Tris pH 6.5, 15 % PEG 200. The crystals were then picked up with Hampton loops and plunged into liquid nitrogen.

X-ray diffraction data collection, phasing and refinement

Several crystals were analyzed at Advanced Light Source beamline 4.2.2 using a NOIR-1 CCD detector. The data were integrated with MOSFLM42 and scaled with SCALA.43 The crystals have space group C2 with unit cell parameters of a = 90.9 Å, b = 44.1 Å, c = 55.2 Å, and β = 101.5°. The asymmetric unit contains one PutA52 dimer and one DNA duplex, which corresponds to 50 % solvent and VM = 2.3 Å3/Da.44,45 The best data set had a high resolution limit of 2.25 Å, and consisted of 180 frames collected with oscillation angle of 1°/frame, exposure time of 7 s/frame and detector distance of 120 mm. Data collection and processing statistics are listed in Table 1.

Table 1
Data Collection and Refinement Statisticsa

The structure was solved using molecular replacement with a PutA52 dimer serving as the search model. CNS was used for molecular replacement calculations.46 The fast-direct method was used for cross-rotation function calculations. Prior to translation function calculations, the orientations from the cross-rotation function calculation were optimized with Patterson correlation refinement using two groups corresponding to the two protein chains of the dimer. The top solution from the translation function calculation had correlation coefficient of 0.257. Rigid body refinement resulted in a model with Rcryst = 0.488 and Rfree = 0.493 for data to 3.0 Å resolution. Simulated annealing refinement in CNS lowered the R-factors to Rcryst = 0.450 and Rfree = 0.467 for all reflections.

The model from simulated annealing was used as the starting point for iterative cycles of model building in COOT47 and refinement with TLS in REFMAC5.48 After a few cycles, the R-factors for a protein-only model were Rcryst = 0.416 and Rfree = 0.427. At this point, electron density representing DNA base pairs was evident. The DNA part of the model was gradually built up over about two dozen cycles of model building and refinement. Solvent was added during the latter stages of model building.

The final model consists of one PutA52 dimer, one dsDNA molecule and 27 water molecules. The modeled protein chains include residues 3 - 46 for chain A and 4 - 48 for chain B. The DNA strands include nucleotides 4 - 21 for strand 1 and 1 - 19 for strand 2. Electron density near the end of the DNA duplex containing nucleotides 1-3 of strand 1 and 19 - 21 for strand 2 was rather weak, and indicated fraying of the base pairs and possibly more than one conformation. But, the density was not of sufficient quality to allow reliable modeling of this end of the DNA ligand. We note that this end of the oligonucleotide is far from the protein-DNA interface. Refinement statistics are listed in Table 1.

Structure analysis

Structures were analyzed graphically using COOT and PyMOL.49 CNS was used to calculate buried surface area.46 DNA conformation was analyzed with 3DNA.28 The depth of penetration of the β-sheet into the DNA major groove was analyzed for RHH proteins. An operational definition of penetration depth was adopted for this purpose as the shortest distance between selected Cα atoms of the β-sheet and the DNA helical axis. DNA helical axes were calculated using 3DNA.28 The 9 - 10 base pairs corresponding to the region contacted by the protein were used for the axis calculation. Distances between Cα atoms and DNA axes were calculated using a program written by Damian Coventry, which implements theory by Paul Bourke.

Isothermal titration calorimetry (ITC)

ITC experiments were conducted at 25 °C in a VP-ITC calorimeter (MicroCal, LLC). Prior to analysis, the protein and oligonucleotide were dialyzed extensively against the appropriate reaction buffer, which was either 50 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 8.0, or 50 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA, pH 8.0. The dimeric quaternary structure of PutA52 was confirmed by equilibrium analytical ultracentrifugation under conditions similar to those used in ITC experiments (20 μM PutA52 in 50 mM Tris, 50 mM NaCl, pH 8.0). The data could be fit very well to a single-species model with apparent molecular weight corresponding to that of the homodimer (~13.5 kDa). No evidence of a monomeric species was present at 20 μM. Sample and titrant were degassed under vacuum immediately before being loaded into the sample cell and buret, respectively. Following thermal equilibration, aliquots (7 or 10 μL) of titrant were added to the 1.41 mL sample at 240-second intervals. A 2 μL pre-injection was included at the start of each titration. The heat associated with this addition – invariably inaccurate due to diffusion of titrant from the buret during the equilibration period – was neglected during the fitting process.

Samples of PutA52 were titrated with two oligonucleotides, designated O2 and O2fb4 (O2 with flanking bases of O4). O2 is the oligonucleotide used for co-crystallization. O2fb4 is identical to O2 except that the base pairs flanking the consensus motif are those of operator 4 (Fig. 1B):

5′-TTTGCAGTTGCAACTTTCAAA-3′(strand 1)

3′-AAACGTCAACGTTGAAAGTTT-5′(strand 2)

Experiments were conducted in both phosphate and Tris, two buffers with distinct ionization enthalpies. The raw data were integrated with software supplied with the instrument. Blank titrations, injection of titrant into buffer, were performed for each oligonucleotide-buffer combination. The average injection heats associated with these experiments were used to correct the corresponding protein titrations for the nonspecific heat of mixing/dilution.

The apparent protein-DNA binding enthalpies differed profoundly in Tris and phosphate, indicating that the PutA52-DNA interaction is accompanied by protonation. Accordingly, the data from the two buffer systems were subjected to simultaneous least-squares analysis, employing a model that explicitly includes the heat of buffer ionization. The following equation describes the cumulative heat after the ith titrant addition:

Qi=V[M]t(ΔH+nΔHbuf)[Kap[DNA]1+Kap[DNA]]
(1)

where V is the sample cell volume, [M]t is the total protein concentration, ΔH is the intrinsic binding enthalpy, ΔHbuf is the heat of buffer ionization, n is the number of protons taken up by the protein-DNA complex during the binding reaction, Kap is the apparent association constant, and [DNA] is the concentration of free DNA.

Because protein-DNA binding is linked to protonation, the apparent free energy change for the reaction (ΔGap) includes a contribution from buffer ionization (ΔGbuf):

ΔGap=ΔG+ΔGbuf
(2)

Thus,

RTlnKap=RTlnKnRTlnKbuf
(3)

or

Kap=K(Kbuf)n
(4)

where K is the intrinsic binding constant for the protein-DNA association, and Kbuf is equal to

Kb=[B][BH+]u=exp[2.303(pHpKa)]
(5)

In equation 5, [BH+] and [B] represent the concentrations of the conjugate acid and base forms of the reaction buffer, and pKa is the appropriate value for the particular buffer under consideration. K, ΔH, and n were global fitting parameters; pH was a fixed global parameter; and ΔHbuf and pKa were fixed titration-specific parameters obtained from the literature.50

The ith injection heat (qi) was modeled as the difference in the cumulative heats associated with the ith and (i+1)th additions:

qi=(QiQi1)+dViV(Qi+Qi12)
(6)

The second term in equation 6 is a correction for the heat associated with the volume of solution displaced from the sample cell by the ith titrant addition, where dVi is the volume of the ith injection. Fitting was performed in Origin (v. 7.5, OriginLab), employing a LabTalk script generated in-house.

PDB accession code

Atomic coordinates and structure factor amplitudes have been deposited in the PDB51 as entry 2RBF.

Supplementary Material

supplementary

Acknowledgments

This research was supported by NIH grants GM065546 (JJT) and GM061068 (DFB), and NSF grant MCB0091664 (DFB). CAB was supported by a postdoctoral fellowship from the National Library of Medicine (2-T15-LM07089-14). We thank Damian Coventry and Paul Bourke for providing computer code that was used in penetration depth calculations. Part of this research was performed at the Advanced Light Source, which is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division, of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory. This work is a contribution of the University of Nebraska Agricultural Research Division, supported in part by funds provided through the Hatch Act. This publication was also made possible by NIH Grant Number P20 RR-017675-02 from the National Center for Research Resources. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

Abbreviations used

PRODH
proline dehydrogenase
P5CDH
Δ1-pyrroline-5-carboxylate dehydrogenase
PutA
Proline utilization A
PutA52
polypeptide corresponding to residues 1-52 of E. coli PutA
RHH
ribbon-helix-helix
PutA47
polypeptide corresponding to residues 1-47 of E. coli PutA
bp
base pair
ITC
isothermal titration calorimetry

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