Single Asparagine to Arginine Mutation Allows PerR to Switch from

Dec 8, 2014 - In addition to these differences in the PerR and Fur boxes, we identify in this study a residue located on the DNA binding motif of the ...
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Single Asparagine to Arginine Mutation Allows PerR to Switch from PerR Box to Fur Box Christelle Caux-Thang, Aubérie Parent, Ramakrishnan Sethu, Arhamatoulaye Maïga, Geneviève Blondin, Jean-Marc Latour,* and Victor Duarte* †

Université Grenoble Alpes, LCBM, F-38054 Grenoble, France CEA, DSV, iRTSV, LCBM, PMB, F-38054 Grenoble, France § CNRS UMR 5249, LCBM, F-38054 Grenoble, France ‡

S Supporting Information *

ABSTRACT: Fur family proteins, ubiquitous in prokaryotes, play a pivotal role in microbial survival and virulence in most pathogens. Metalloregulators, such as Fur and PerR, regulate the transcription of genes connected to iron homeostasis and response to oxidative stress, respectively. In Bacillus subtilis, Fur and PerR bind with high affinity to DNA sequences differing at only two nucleotides. In addition to these differences in the PerR and Fur boxes, we identify in this study a residue located on the DNA binding motif of the Fur protein that is critical to discrimination between the two close DNA sequences. Interestingly, when this residue is introduced into PerR, it lowers the affinity of PerR for its own DNA target but confers to the protein the ability to interact strongly with the Fur DNA binding sequence. The present data show how two closely related proteins have distinct biological properties just by changing a single residue.

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similarities, but they respond to different stimuli and regulate different sets of genes. It was originally shown in E. coli that Fur protein binds with high affinity to a 19 bp DNA sequence containing a 9−1−9 (GATAATGATAATCATTATC) palindromic motif.18 Subsequent studies by using synthetic oligonucleotides led to a different model in which Fur binds a repeated hexamer sequence (GATAAT).19 In B. subtilis, based on all identified Fur regulated genes, Helmann and co-workers came to the conclusion that Fur binds a 15 bp (TGATAATNATTATCA) consensus DNA Fur box containing a 7−1−7 palindromic motif.20,21 Interestingly, the Fur box in B. subtilis is nearly identical to the consensus PerR box (TTATAATNATTATAA).22 In addition, DNA sequence alignments indicated that Bs-Zur binds also to a similar 7−1−7 core but with a 3 bp extension on both sides (lowercase letters), aaaTCGTAATNATTACGAttt.23 The significant similarities between the DNA consensus sequences for these three regulatory proteins suggest that regulon overlap may occur. Indeed, it has been reported that mutated synthetic operator sites can be partially recognized in vitro and in vivo by two Fur paralogs.24 However, in B. subtilis, no naturally occurring sites are subject to dual regulation by Fur and PerR, thereby demonstrating that as few as two bases in each half site (positions 5 and 6) are crucial for the discrimination of the DNA binding sites by PerR, Fur, and Zur.20,25,26 This is especially the case for the PerR and Fur boxes, which differ only in position 6 of each half site of the 7−

NA binding proteins play a crucial role in many biological processes, including transcription, regulation, DNA replication, and repair. The better understanding of DNA− protein interactions has both fundamental and applied research interests. Thus, a great deal of effort has been made to figure out the principles that govern the specificity of the protein− DNA complex formation. It appears that there is no simple recognition code for a protein residue to interact with a defined nucleotide.1 Elucidation of the precise details of how the two polymers interact requires the molecular description of the process, but unfortunately this is often limited by the lack of high resolution crystal structures of protein−DNA complexes. Metalloregulatory proteins represent a subclassification of transcriptional regulators. These proteins function through a specific interaction with the promoter region immediately upstream of the regulated genes that are involved, for example, in metal uptake or detoxification.2 The Fur family of metalloregulators is found in a wide variety of Gram-negative and Gram-positive prokaryotes. It comprises four proteins implicated in metal homeostasis, Fur, Mur, Nur, and Zur, that regulate Fe, Mn, Ni, and Zn concentrations, respectively.3,4 The last member of the family, PerR, functionally equivalent to OxyR, controls the response to peroxide stress.5−7 The structural basis underlying the gene regulation by Fur proteins is well documented.8−16 These proteins generally fold into two domains consisting of an N-terminal DNA binding domain connected by a hinge region to a C-terminal dimerization domain. In spite of these studies, the structural determinants that control the DNA binding have not been elucidated. In Bacillus subtilis, three Fur paralogs, Fur, Zur, and PerR, are present.17 They share significant sequence and structure © XXXX American Chemical Society

Received: September 30, 2014 Accepted: December 8, 2014

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two single Bs-PerR mutants N61A and N61R and the reverse Bs-Fur mutant R61N. Furthermore, we designed four 33-bp DNA sequences, namely 6A, 6C, 6G, and 6T, which differ on position 6 of the 7−1−7 palindromic motif (Figure 1b). The 6G and 6T sequences represent the Fur box from the feuA promoter and the PerR box from the mrgA promoter. 6A and 6C are altered PerR boxes from the mrgA promoter. The ability of all the variants to bind a defined DNA sequence was then evaluated by using EMSA experiments to determine the dissociation constant (Kd) of the protein−DNA complex (Figure 2 and Table 1).

1−7 motif. In addition to this key position within the PerR and Fur consensus sequences, the molecular basis of this discrimination also lies in the DNA recognition helices of the two proteins. A multiple sequence alignment of selected PerR and Fur proteins allowed us to compare the DNA binding domains of the two proteins (Figure 1, see Supporting

Table 1. Affinity Constants of Fur and PerR Variants for DNA Duplexes

Figure 1. Protein and DNA sequences: (a) PerR and Fur DNA binding helices. (b) DNA palindromic sequences, 6G and 6T are Fur and PerR boxes, respectively.

protein/sequence

6A

6C

6G Fur box

6T PerR box

PerR-WT PerR-N61 R Fur-WT

1.5 (0.1) 1.0 (0.1) 1.8 (0.2)

nb 9.9 (1.5) nb

nb 0.9 (0.1) 1.0 (0.1)

0.5 (0.1) 7.6 (0.7) nb

As expected, PerR binds with high affinity to the PerR box containing the region of the mrgA operator (Figure 2aT), the estimated Kd value being 0.5 nM. In contrast to this result, no significant binding was observed for both the Fur box and the 6C DNA sequences (Figure 2aG,aC). Remarkably, the PerR protein also binds tightly to the 6A sequence (Figure 2aA); the related Kd value of 1.5 nM is equivalent to the one observed with the PerR box. To evaluate the importance of the residue in PerR position 61 for the binding of the PerR box, we prepared the N61A mutant and assessed its interaction with the four DNA duplexes. The alanine mutation abolishes PerR binding to both the PerR box and the 6A sequences (Figure 2bT,bA). Similarly, the PerR−N61A mutant did not show any affinity for the Fur box and the 6C DNA sequences (Figure 2bG,bC). To assess the possibility that the N61A mutation within the DNA recognition helix prevents the coordination of the regulatory metal to the active site and subsequently the formation of the protein DNA complex, we evaluated the Mn2+ binding capacity of PerR−N61A by EPR spectroscopy, as previously described (Supporting Information).6 A Kd value of 5.8 μM, equivalent to the one reported for PerR-WT,28 indicates that the PerR-N61A protein more than likely binds the Mn2+ ion in the regulatory site. This result strongly suggests that PerR−N61A is able to

Information Figure 1 for a more complete list). Figure 1a focuses on the potential DNA recognition H4 helices of BsPerR (Val56 to Ser69) and Bs-Fur (Leu56 to Leu69). Multiple sequence alignment shows that the TVY motif on positions 58−60 is largely conserved among PerR and Fur proteins (Supporting Information Figure 1). Indeed it has been shown in Ec-Fur that the Y60 residue is in close interaction with the corresponding DNA Fur box.27 Interestingly, the asparagine residue Asn61 (N61), which is mainly conserved in PerR, is replaced by a strictly conserved arginine (Arg, R) in Fur proteins (see Supporting Information Figure 3A,B for overall crystal structures of Bs-PerR and PaFur). Thus, we hypothesized that this residue is likely to be important for DNA binding and that it may contribute to the ability of the two proteins to discriminate between their nearly identical operator sites in Bacillus subtilis. The present results clearly demonstrate that in addition to a single base within the PerR and Fur boxes, a single residue located in the DNA binding motif of the corresponding proteins is important for the formation of the protein−DNA complexes and is critical for the discrimination of the two closely related DNA promoters. To investigate the role of both the N and R residues in the DNA binding affinity and sequence discrimination, we prepared

Figure 2. Electrophoretic mobility shift assay analyses of PerR and Fur variants to DNA sequences. Protein conc., 0.2 to 20 nM; DNA conc., 0.2 nM. B

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box and the 6A sequences. On the basis of the observation that PerR−N61R binds tightly the Fur box, we wondered if the reverse Fur mutant, namely Fur−R61N, is able to recognize a 7−1−7 PerR DNA sequence. The introduction of the R61N mutation in Fur alters its DNA binding properties and prevents its interaction with both the Fur box (Figure 2eG) and the 6A duplex (Figure 2eA). In contrast to PerR−N61R, the opposite Fur−R61N variant does not swap its high DNA binding affinity from the Fur box to the PerR box (Figure 2eT). This finding strongly suggests that other residues located on the H4 DNA binding helix are essential to the specific interaction of PerR with its DNA sequence. Indeed, several single mutants of PerR including Y60A, R64A, R67A, and E68A all failed to form a stable PerR−DNA complex (data not shown). The most striking result that emerges from this EMSA analysis of PerRWT, Fur-WT, and mutated proteins is that a single asparagine to arginine mutation allows PerR to switch from the PerR box to the Fur box. Indeed, this mutation lowers the affinity of mutated PerR for the PerR box 15-fold, while conferring for the Fur box an affinity as strong as that of Fur-WT. In addition, when Arg61 is not present in Fur or PerR variants, including PerR-WT, PerR−N61A, and Fur−R61N, the binding to the Fur box is abolished. These observations clearly suggest that in B. subtilis, where the two closely related Fur and PerR proteins are present, the strictly conserved R61 residue in the Fur protein is crucial for both the specific interaction with its DNA target and the discrimination between the Fur and PerR boxes. The crucial role of R61 in the formation of the Fur−DNA complex demonstrated here is consistent with previous computational studies on Pseudomonas aeruginosa (Pa) and Aliivibrio salmonicida (As) Fur−DNA complexes. Indeed, by using molecular dynamic simulations and binding free energy calculations, the authors pointed out that R56 in Pa-Fur and R57 in As-Fur, corresponding to R61 in Bs-Fur, are among the residues that most favorably interact with DNA.30,31 As shown in Figure 2eA, R61 is also crucial for Fur-WT to bind the 6A sequence present in H. pilori Fur-regulated promoters. In this respect, the equally high affinity of PerR-WT for this sequence may appear problematic in terms of selective promoter activation. However, so far, PerR has not been reported to be present in this organism, and conversely there is no selectivity pressure as in B. subtilis. In addition to the present study, we recently demonstrated that among the five amino acids that constitute the regulatory site of Bs-PerR, the Asp104 (D104) is a key residue that allows PerR to react with H2O2 (Supporting Information Figure 3C). In the opposite way, the corresponding Glu108 (E108) residue in Bs-Fur makes this protein poorly reactive toward H2O2.32 Remarkably, the sensitivity or insensitivity toward H2O2 of PerR and Fur seems to be entirely related to a single Asp to Glu mutation and even more precisely on the subtle changes in the carboxylate coordination mode of these residues to the regulatory metal. Indeed, a PerR−D104E mutant, equivalent to Fur-WT, shows a poor reactivity with H2O2. To the contrary, the reverse mutant Fur−E108D, analogous to PerR-WT, becomes sensitive to hydrogen peroxide.32 Together, these results and the present data prompted us to synthesize a PerR mutated protein that will harbor the properties of Fur, in terms of both reactivity toward H2O2 and DNA binding specificity, just by changing two residues. The metal binding ability of the PerR−D104E−N61R double mutant determined by EPR spectroscopy led to a Kd of 8.1 μM, consistent with the 2.7 μM value reported for Fur-WT.32 These Kd values in the

adopt the caliper like conformation which is required to bind the DNA, but the presence of the A61 residue does not favor this binding. Thus, these experiments show that the interactions of PerR with the PerR box and the 6A DNA duplex are specific. Moreover the presence of the N61 residue is required since its substitution by an alanine prevents the formation of the protein−DNA complexes. As expected, Fur-WT strongly binds to the Fur box sequence (Figure 2dG), the corresponding Kd value for the protein−DNA complex being 1 nM. In contrast to this result, no significant interactions of Fur-WT with either the PerR box from the mrgA promoter or the 6C sequence were observed (Figure 2dT,dC). Interestingly, Fur-WT binds the 6A DNA sequence with high affinity (Figure 2dA); the associated Kd value is 1.8 nM. These data suggest that the Fur protein may bind operators that contain a TAATAATTATTATTA sequence. Indeed, in Helicobacter pylori, extensive analysis of Fur regulated promoters clearly identified the latter 7−1−7 motif as the Fur binding sequence of this bacterium.29 The addition of this sequence to the promoter region of a non-Fur regulated gene appeared to be sufficient to impose Furdependent regulation in vivo. Next, we assessed the ability of the PerR−N61R variant to interact with the four designed DNA sequences (Figure 2). The corresponding DNA binding affinities are all in the nanomolar range albeit spanning an order of magnitude (Table 1, Figure 3).

Figure 3. Measurement of the PerR−N61R to DNA binding affinity by electrophoretic mobility shift assay. Autoradiograms of at least three separated experiments for each DNA sequence were quantified and plotted as percentage of DNA binding versus protein concentration and fitted as described in the Supporting Information. See Table 1 for the apparent Kd values.

The substitution of asparagine to arginine in position 61 in PerR drastically alters its DNA binding properties. First, it leads to a significantly reduced affinity for the PerR box (Figure 2cT) with a 15-fold increase of the Kd value (7.6 nM). Second, in contrast to PerR-WT, the PerR−N61R mutant shows a moderate affinity for the 6C sequence (Figure 2cC), with a corresponding Kd of 9.9 nM. Last, this mutation confers to the PerR variant affinities for the Fur box and the 6A duplex (Kd are 0.9 and 1.0 nM, respectively) similar to those obtained for FurWT (Table 1, Figure 2cG,cA and Supporting Information Figure 2). Overall, these experiments show that the N61R mutation confers to PerR a strong binding affinity for the Fur C

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Figure 4. Electrophoretic mobility shift assay analyses of PerR−D104E−N61R and Fur-WT to the Fur box and PerR-WT to the PerR box. Before (a, b, c) and after oxidative treatment (d, e, f). Protein conc., 0.2 to 20 nM; DNA conc., 0.2 nM.

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micromolar range indicate that both proteins efficiently bind the regulatory metal. The DNA binding affinity of the PerR− D104E−N61R variant for the Fur box was then compared to the affinities of Fur-WT and PerR-WT for their respective DNA targets (Figure 4a,b,c). The PerR double mutant binds tightly to the Fur box containing DNA sequence with a Kd value of 2.1 nM (Figure 4a). These data further indicate that the introduction of the D104E mutation in PerR−N61R does not significantly affect its DNA binding affinity toward the Fur box. Figure 4d,e,f shows similar EMSA experiments after the proteins were treated with stoichiometric amounts of both Fe2+ ions and H2O2 prior to gel analyses. As expected for PerR-WT (Figure 4f) and PerR− N61R (data not shown), these oxidative conditions largely affect the formation of the protein−DNA complex, whereas Fur-WT is unaffected (Figure 4e). Interestingly the interaction of PerR−D104E−N61R with the Fur box (Figure 4a) was not significantly modified by the oxidative treatment of the protein; the PerR double mutant remains bound to the Fur box sequence (Figure 4d) as is the case for the Fur-WT protein (Figure 4e). In summary our biochemical results clearly demonstrate that R61 is a key amino acid that allows Fur to recognize its DNA binding sequence and to discriminate the closely related PerR box. When introduced into PerR in place of the corresponding asparagine, this arginine residue lowers the binding affinity for the PerR box and confers to PerR a high binding affinity for the Fur box, the Kd value as low as 0.9 nM matching the one determined for the Bs-Fur-Fur box complex. We have already reported that a single mutation within the regulatory site of PerR, namely the D104E mutation, makes PerR as poorly reactive to H2O2 as Bs-Fur. Interestingly, when combining the two mutations, the PerR−D104E−N61R double mutant shows the features of the Fur-WT protein in terms of both DNA binding and reactivity toward hydrogen peroxide. This work further illustrates how subtle changes within a protein sequence, such as Asp to Glu and Asn to Arg mutations, confer to the protein completely different attributes. In the case of Bs-PerR, these two mutations modify the function of the protein and the genes that are expressed, both of these properties being part of biological events that are crucial for the microorganism. From another point of view, this work gives us insight on how nature has evolved two closely related proteins to have distinct biological properties just by changing two key residues.

ASSOCIATED CONTENT

S Supporting Information *

Supporting Information including experimental methods and supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J.-M.L. and V.D. acknowledge the financial support of IFCPAR (Project No. IFC/4109-1) and Labex ARCANE (ANR-11LABX-0003-01).

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