The DNA-binding mechanism of the TCS response regulator ArlR from Staphylococcus aureus

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The DNA-binding mechanism of the TCS response regulator ArlR from Staphylococcus aureus ⁎

⁎

Hui Yan, Qing Wang, Maikun Teng , Xu Li

Hefei National Laboratory for Physical Sciences at Microscale, National Synchrotron Radiation Laboratory, School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, China

A R T I C LE I N FO

A B S T R A C T

Keywords: TCS ArlR DNA binding property Crystal structure Phosphorylation

ArlRS is an essential two-component system in Staphylococcus aureus that regulates the transcription of virulence factors and participate in numerous pathogenic and symbiotic processes. In this work, we identified different DNA binding properties and oligomerization states among the DNA-binding domain of ArlR (ArlRDBD) and the phosphorylated and unphosphorylated full-length ArlR. Based on a 2.5-Å resolution crystal structure of ArlRDBD and subsequent mutagenesis experiments, we confirmed the DNA-binding site of ArlR and the preferred binding sequences in the agr promoter that enables the DNA recognition process. Finally, we propose a putative transcription regulation mechanism for ArlR. This work will facilitate our understanding of the DNA binding affinity regulatory mechanism between the phosphorylated and unphosphorylated response regulator in the two-component system.

1. Introduction The two-component system (TCS) is a family of signal transduction proteins that is typically comprised of a receptor histidine kinase (HK) and a cognate response regulator (RR) (Hoch, 2000). TCS is a significant system for bacterial, lower eukaryotes, and plants to sense and respond to environmental changes by modifying gene expression or modifying the biochemical activities of target proteins (Capra and Laub, 2012). In bacteria, TCS has been implicated in the adaptation to a variety of stress conditions, pathogenic and symbiotic interactions with eukaryotic hosts, and essential cellular pathways (Groisman, 2016). Upon receipt of a stimulus, the HK autophosphorylates a conserved histidine residue and relays the phosphoryl group to a conserved aspartate on RR (Stock et al., 2000). The RR consists of a well-conserved receiver domain (RD) and a DNA-binding domain (DBD) (Galperin, 2006). Regarding regulation, the receiver domain is phosphorylated, and DNA binding affinity changes and leads to transcription alteration (West and Stock, 2001). Given their characteristics, TSCs have been regarded as a promising target for therapeutic intervention of diseases caused by pathogenic microorganisms (Stephenson and Hoch, 2002). Staphylococcus aureus is an opportunistic pathogen that may invoke a wide range of infections, including life-threatening sepsis, endocarditis, and pneumonia (Lowy, 1998; Projan, 1997). The diversity

and severity of these diseases depend on the expression of virulence factors (e.g., a serine protease (Ssp) and protein A (Spa)) in S. aureus (Hartleib et al., 2000; McGavin et al., 1997). The coordinated temporal expression of these virulence factors is tightly regulated by many regulatory elements, including global regulators (e.g., SigB, SarA and SarA homologs), regulatory RNA molecules (e.g., RNAIII) and two-component regulatory systems (e.g., agr, saeRS and arlRS) (Fan et al., 2015). S. aureus has 16 TCSs to adapt to the diverse microenvironments encountered during its life cycle, including host tissues and implanted medical devices (Burgui et al., 2018). It was recently reported that ArlRS is a necessary TCS for establishing invasive S. aureus disease and is encoded by the arl (autolysis-related locus) locus (Fournier and Hooper, 2000); its mutation augmented the synthesis of RNA II and RNA III (the gene belongs to the agr operon, which influences the expression of 138 genes in S. aureus). In the ArlRS TCS, ArlS is the membrane-anchored sensor protein, and ArlR is the intracellular response regulator, which belongs to the OmpR/PhoB family whose structure contains an N-terminal receiver domain and a C-terminal DNA binding domain. While the extracellular domain of ArlS receives some signals (i.e., the manganese starvation signal (Radin et al., 2016)) from environmental changes, the conserved residues on the intracellular domain of ArlS are autophosphorylated and relayed the signal to ArlR (Fournier and Hooper, 2000).

Abbreviations: TCS, two-component system; HK, histidine kinase; RR, response regulator; RD, receiver domain; DBD, DNA binding domain; EMSA, electrophoretic mobility shift assay; CD, circular dichroism; FPA, fluorescence polarization assay ⁎ Corresponding authors. E-mail addresses: [email protected] (M. Teng), [email protected] (X. Li). https://doi.org/10.1016/j.jsb.2019.09.005 Received 2 July 2019; Received in revised form 5 September 2019; Accepted 10 September 2019 1047-8477/ © 2019 Elsevier Inc. All rights reserved.

Please cite this article as: Hui Yan, et al., Journal of Structural Biology, https://doi.org/10.1016/j.jsb.2019.09.005

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In addition to regulating other transcriptional regulators, ArlRS is also involved in many essential aspects of the pathogenesis of S. aureus. For example, arlRS inactivation leads to a > 70% reduction in endothelial damage, which is regarded as a critical step in the pathogenesis of S. aureus endovascular infections (Seidl et al., 2018). ArlRS helps S. aureus overcome nutritional immunity when the host imposed manganese and zinc starvation on invading pathogens (Radin et al., 2016), negatively modulates the expression of autolysin and prevents cell autolysis (Memmi et al., 2012). ArlRS also contributes to the regulation of antibiotic-resistance processes by repressing biofilm development (which help bacteria to adhere to its host and resist to antibiotics), and the expression of multidrug-resistant transporter NorA (which protected S. aureus from a number of lipophilic and monocationic compounds) (Fournier et al., 2000; Toledo-Arana et al., 2005). The deletion of arlRS decreases the effect of DNA supercoiling modulators on protein A expression, demonstrating the relationship between Arl proteins and topological alteration of DNA (Fournier and Klier, 2004). However, to date, very little information is available on the mechanism of the above physiological functions of ArlRS. In this research, we report different binding properties of ArlR, the DNA-binding domain of ArlR (referred to ArlRDBD) and the D52E mutant of ArlR (which mimic the phosphorylated ArlR, referred to ArlRD52E). Various assemblies of these molecules in solution were also observed using size-exclusion chromatography and dynamic light scattering assays. Based on the 2.5-Å resolution crystal structure of ArlRDBD and subsequent mutagenesis experiments, we confirmed the DNA-binding site of ArlR and the preferred binding sequences on the agr promoter in this DNA recognition process. Finally, we propose a putative transcription regulation mechanism for ArlR. This study will facilitate our understanding of the DNA binding affinity regulatory mechanism between phosphorylated and unphosphorylated RR in TCSs and provide insight into the physiological function mechanism of ArlRS.

Table 1 Data-collection and refinement statistics for ArlRDBD. PDB ID

6IJU

Data-collection statistics Space group a, b, c (Å) α, β, γ (°) Wavelength (Å) Resolution limits (Å)# No. of unique reflections Completeness (%) Redundancy Rmerge† (%) Mean I/σ(I)

P6422 67.235, 67.235, 153.228 90, 90, 120 0.9791 50.00–2.40 (2.44–2.40) 8660 88.6 (64.7) 35.9 (33.4) 9.9 (95.9) 47.6 (5.0)

Refinement statistics Resolution limits (Å) Rworkǂ/Rfree§ (%) R.m.s.d., bond lengths (Å) R.m.s.d., angles (°) B factor (Å2) Protein H2 O No. of non-H protein atoms No. of water O atoms Ramachandran plot (%) Most favoured regions Additional allowed regions Generously allowed regions

32.00–2.40 21.15/24.07 0.008 0.878 42.593 41.311 813 32 95.79 4.21 0.00

† Rmerge = ∑hkl∑i|Ii(hkl) − I(hkl)|/∑hkl∑iIi(hkl), where Ii(hkl) is the ith observation of reflection hkl and (I(hkl)) is the weighted average intensity for all i observations of reflection hkl. ǂ Rwork = ∑hkl||Fobs| − |Fcalc||/∑hkl|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors for reflection hkl, respectively. § Rfree was calculated in the same way as Rwork but using a randomly selected 5% of the reflections which were omitted from refinement. # Values in parentheses are for the highest resolution shell.

(Wang et al., 2018). ArlRDBD was crystallized in space group P6422 with cell unit parameters a = b = 67.235 Å, c = 153.228 Å, α = β = 90°, and γ = 120°. The data were processed and scaled using the HKL-2000 package (Otwinowski and Minor, 1997). Molecular replacement was performed using Phaser-MR in Phenix (Adams et al., 2010), and the structure of the DNA-binding domain of PhoB from E. coli was used as a search model (sequence identity: 41%). Further refinement was achieved using Refmac5 (Murshudov et al., 2011) in CCP4 suite and manual rebuilding in COOT (Emsley and Cowtan, 2004). The final crystallographic parameters are listed in Table 1.

2. Materials and methods 2.1. Protein expression and purification The full-length ArlR (residues 1–219) and ArlRDBD (residues 122–219) genes were amplified by PCR from genomic S. aureus NCTC 8325 DNA and cloned into pET-28a (+) to yield an N-terminally His6tagged fusion protein. Overexpression of the recombinant proteins was induced in E. coli BL21 (DE3) cells (Novagen) utilizing 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) when the cell density achieved an OD600 of 0.6–0.8. After growth at 289 K for 20 h, cells were harvested, resuspended and lysed in buffer (500 mM NaCl, 25 mM Tris-HCl pH 7.5, 5% glycerol). The supernatant was applied into a Ni-NTA column (Qiagen) and recombinant protein was eluted with buffer supplemented with 200 mM imidazole. The proteins were further purified by using HiTrap QFF (5 ml) and size exclusion chromatography with a Superdex 200 column (GE Healthcare). ArlRDBD protein was concentrated to 10 mg ml−1 for crystallization. All of the ArlRDBD and ArlR mutants were obtained from pET-28aArlRDBD and pET-28a-ArlR by site-directed mutagenesis using Primer STAR DNA polymerase (Takara). Subsequently, the overexpression of all mutants was achieved based on the procedures described above.

2.3. Electrophoretic mobility shift assay The promoter of the agr operon was PCR amplified from genomic S. aureus NCTC 8325 DNA with a pair of 5′ FAM labelled primers agr-F ( 5′-CAACTATTTTCCATCACATCTCTGT-3′) and agr-R (5′-TTTTACACCA CTCTCCTCACTGTCA-3′). Briefly, 50 nM DNA probes were incubated with increasing concentrations of ArlR, ArlRDBD, and ArlRD52E protein on ice, and all the mutants were added in the 6-μl reaction mixture composed of 25 mM Tris-HCl pH 7.5, 50 mM NaCl, 0.5 μM unlabelled competitor and 1 μM non-specific competitor. After 30-minute incubation on ice, all the samples were subjected to electrophoresis in a 6% native polyacrylamide gel in 0.5 × TBE for 1 h. Protein-DNA binding was imaged using fluorescence block in ImageQuant LAS4000 (GE Healthcare).

2.2. Crystallization, data collection and structure determination Crystals were grown using the hanging-drop vapour diffusion method at 289 K. Microcrystals were obtained when protein was mixed in a 1:1 ratio with well solution containing 0.1 M sodium acetate trihydrate pH 4.5 and 2.0 M ammonium sulfate after 3 days. Crystals were soaked in cryoprotectant consisting of 20% (v/v) glycerol and flashcooled in liquid nitrogen. X-ray data were collected from a single crystal on beamline 17U at the Shanghai Synchrotron Radiation Facility (SSRF)

2.4. DNaseⅠfootprinting assay The 5′ FAM-labelled agr was obtained using the same method as described above. DNaseⅠfootprinting assays were performed using a modified method based on previous studies (Xue et al., 2009). Briefly, 20 μl of 1 pM DNA was incubated with 20 μl of reaction buffer (25 mM 2

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Fig. 1. Binding of ArlRDBD, ArlR, and ArlRD52E to the agr promoter. (A) Gel shift of purified ArlRDBD with the 5′ FAM-labelled agr promoter (nt 1451–1685). Lane 1 represents the labeled DNA probe alone. Increasing amounts of ArlRDBD at 50, 60, 70, and 80 µmol, respectively, were added to the probe (lanes 2–5). Unlabelled 235bp agr promoter (lane 6) in a 10-molar ratio excess was added to a lane containing 80 µmol of the purified ArlRDBD protein as a specific competitor, while a 235-bp AlaX gene fragment with a 20-molar ratio excess was also added to 80 µmol of ArlRDBD (lane 7). (B) Gel shift of purified ArlR with the 5′ FAM-labelled agr promoter. A procedure similar to that described for ArlRDBD was employed, but different protein concentration gradients at 40, 80, 120 and 160 µmol were used. (C) Gel shift of purified ArlRD52E with the 5′ FAM-labelled agr promoter. The procedure is similar to that described for ArlRDBD, but different protein concentrations gradients were employed: 2.5, 5, 10, and 15 µmol in.

Tris-HCl pH 7.5, 50 mM NaCl), 20 μl of 150 μM ArlRDBD protein and 20 μl of 10 μM ArlRD52E for 20 min at room temperature. Then, 50 μl containing 0.6 U DNase I was added to digest the sample for one minute followed by addition of 90 μl stop solution (200 mM NaCl, 30 mM EDTA, 1% SDS) to terminate the reaction. DNA was extracted by ethanol precipitation. Sangon Biotech (Shanghai) Co., Ltd. performed the sequencing, and the results were analysed using Peak Scanner software.

2.8. Dynamic light scattering assay Dynamic light scattering assay was performed using a Zetasizer μV from Malvern Instruments (Malvern Worcs, UK). Protein samples diluted to 1 mg ml−1 were assessed in buffer containing 50 mM NaCl and 25 mM Tris-HCl pH 7.5. 2.9. Circular dichroism spectroscopy ArlRDBD protein and its mutants were transferred into 50 mM sodium phosphate buffer pH 7.5. All the proteins were diluted to 0.2 mg ml−1 and loaded into a quartz cuvette, and CD spectra were detected from 190 nm to 260 nm on a Chirascan qCD (Applied Photophysics). Sodium phosphate buffer was recorded as a reference, and all the samples were repeatedly measured thrice. The spectra were processed using Chirascan software, and the molar ellipticities (θ) were plotted versus wavelength.

2.5. Fluorescence polarization assay Briefly, 5′ FAM-labelled primers and their complementary strands were synthesized by General Biosystems Co., Ltd. After 50 μM of the two primers annealed (the calculated TM for agr40-50 is 44.2 °C, 40.1 °C for agr51-60, 41.2 °C for agr61-70, 58.1 °C for agr40-55 ,48.6 °C for agr56-70, 35.2 °C for agr100-110, 35.4 °C for agr111-120 67.7 °C for agr40-70, 55.2 °C for agr100-120 and 73.5 °C for agr40-120), protein samples in increasing concentrations were incubated with 40 nM 5′ FAM-labelled DNA in a final volume of 40 μl on ice for 20 min. All samples were detected at room temperature using the SpectraMax M5 microplate-reader system. Fluorescence polarization P was calculated as follows: P= , where P is the

int +

max (kd 2×R×n

3. Results 3.1. The DNA-binding properties of ArlR, ArlRDBD and ArlRD52E The ArlRS is involved in autolysis, cell division, growth, and pathogenesis (Liang et al., 2005). As a transcriptional regulator, ArlR functions through directly or indirectly binding to the promoter of the target genes. Nevertheless, whether ArlR modulates the expression of agr by directly binding to the promoter or by indirectly forming a regulatory cascade through a third factor, such as regulating the large surface protein Ebh (Crosby et al., 2016), remains unknown. To investigate the DNA-binding properties between ArlR and the agr promoter, a series of EMSA experiments were performed. As shown in Fig. 1A, while the protein concentration reached to 60 µM, the DNA binding domain of ArlR (ArlRDBD) displayed obvious protein-DNA interactions with the agr promoter. These interactions could be competed with by the free unlabelled probe but not a nonspecific competitor, which suggested that sequence specificity or preference exists during ArlRDBD binding to the agr promoter. However, similar interactions should be observed in ArlR when the concentration reached to 160 µM (Fig. 1B), indicating that the unphosphorylated receiver domain of ArlR would hinder the interaction between ArlRDBD and its target genes. To assess this assumption, we expressed and purified the ArlR variant by replacing the conserved aspartic acid phosphorylation site with glutamic acid (ArlRD52E), a phospho-mimic variant previously shown to be constitutively active in other systems (Arribas-Bosacoma et al., 2007). As shown in Fig. 1C-D, the strong interaction was observed between ArlRD52E and agr promoter when the protein concentration was greater than 5 µM. This result supported our assumption that the unphosphorylated receiver domain acted as an obstacle for ArlR to bind its target genes and was consistent with the previous finding that PhoBDBD had a higher affinity for specific DNA binding than unphosphorylated

+H+n

× R − −4 × n × H × R + (−kd − H − n × R)2 ) observed polarization, int is the polarization of DNA without protein, H is the total protein concentration, R is labelled DNA concentration, n is binding stoichiometry, and kd is equilibrium dissociation constant. Curve fitting was accomplished with Origin 8.0 software.

2.6. Crosslinking assay Chemical crosslinking assays were performed utilizing BS3 (bissulfosuccinimidyl suberate) as a crosslinker. Briefly, 0.8 μg purified ArlRDBD, ArlR and ArlRD52E protein in crosslinking buffer (20 mM NaCl, 10 mM KCl, 2 mM DTT, 20 mM Hepes pH 7.5) was incubated with BS3 for different time intervals. The reactions were terminated by adding SDS-PAGE loading buffer, and all the samples were analyzed by SDSPAGE.

2.7. Size-exclusion chromatography assay Size-exclusion chromatography was performed with a Superdex 200 column attached to an ÄKTA pure system (GE Healthcare). Protein samples and the molecular-mass standards were applied on to the column at 0.3 ml min−1 and eluted with 200 mM NaCl and 25 mM TrisHCl pH 7.5. Protein was detected by the absorbance measured at 280 nm. 3

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Fig. 2. The specific binding regions in the agr promoter for ArlRDBD and ArlRD52E. (A) DNaseⅠfootprint of the agr promoter with ArlRDBD. The regions protected from DNaseⅠ digestion are marked by red (nt 40–70) and green (nt 100–120) boxes. The corresponding sequence on agr are listed below the box, A and T base are highlighted in red and green in each sequence separately. (B) DNaseⅠfootprint of the agr promoter with ArlRD52E. The regions protected from DNaseⅠ digestion are marked by red (nt 45–70) and green (nt 100–120) boxes. The corresponding sequence on agr are listed below the box, A and T base are highlighted in red and green in each sequence separately. (C) Fluorescence polarization of ArlR shows no binding affinity to three agr promoter fragments (nt 40–70, nt 100–120 and nt 40–120) or the full-length agr promoter. (D) Fluorescence polarization of ArlRDBD shows obvious binding affinity to three agr promoter fragments (nt 40–70, nt 100–120 and nt 40–120) and the full-length agr promoter. (E) Fluorescence polarization of ArlRD52E shows the strong binding affinity between ArlRD52E and all of the agr promoter probes. The above fluorescence polarization data are representative of three independent experiments. Curve fitting results using a 1:1 binding model. The dissociation constants are summarized in the table (F). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

influence RNA III synthesis (Fournier et al., 2001). The second mutation is the 100–120 region of agr promoter (agr100-120), which is located approximately 50 bp upstream of −35 in the agr P2 promoter. No obvious convergence was observed in these two sequences, but both of them have high AT content (24/31 and 18/21). Similar DNase I footprinting assay results of ArlRD52E also revealed that two regions (agr45-70 and agr100-120) were protected by ArlRD52E at a lower concentration of 10 μM, which is consistent with the higher affinity between ArlRD52E and agr promoter than that of ArlRDBD (Fig. 2B). The second specific binding region was same as that of ArlRDBD, but the first one was shorter than that of ArlRDBD. These results indicate that the DNA binding interface of ArlRD52E differs from that of ArlRDBD. To verify the specificity of these two binding sites, two 5′-FAM-

PhoB (Ellison and McCleary, 2000). 3.2. The specific binding regions in agr To characterize the ArlR-agr interactions and map the possible core sequence responsible for ArlRDBD and ArlRD52E recognition, DNase I footprinting assays with the entire 235-bp agr promoter DNA were performed. Two regions, which yield prominent readout in the presence of ArlR, appear to be protected when 150 μM ArlRDBD was added (Fig. 2A). The first segment encompasses the 40–70 region of the agr promoter (agr40-70), which is located between the −10 element and −35 element in the agr P3 promoter (Novick et al., 1995) that encodes the actual effector of the agr response RNA III. This finding was also consistent with a previous report that the ArlR mutation would 4

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Fig. 3. Oligomerization states of ArlR, ArlRDBD, and ArlRD52E in solution. (A) Size-exclusion chromatography of ArlR, ArlRDBD, and ArlRD52E shows that ArlR and ArlRDBD are monomers in solution and ArlRD52E exhibits more complicated oligomerization states. (B) SDS-PAGE profile of ArlR cross-linking. Briefly, 0.8 µg protein is displayed on a denaturing acrylamide gel with 2 mM BS3 as cross-linker after 15, 30, 45-min incubation at room temperature. Only a monomer band could be observed. (C) SDS-PAGE profile of ArlRDBD cross-linking. Briefly, 0.8 µg protein is displayed on denaturing acrylamide gel with 2 mM BS3 as cross-linker after 15, 30, 45-min incubation at room temperature. The appearance of only one band at approximately 26 kDa indicates the dimerization of ArlRDBD. (D) SDS-PAGE profile of ArlRD52E cross-linking. Briefly, 0.8 µg protein is displayed on denaturing acrylamide gel with 2 mM BS3 as crosslinker after 15, 30, 45-min incubation at room temperature. The appearance of only one band at approximately 42 kDa indicates the dimerization of the ArlRD52E. (E) Dynamic light scattering was performed on ArlRDBD, and the diameter of main particle of ArlR was 4.2 nm. (F) Dynamic light scattering was performed on ArlR, and the diameter of main particle of ArlR appeared was 6.5 nm. (G) Dynamic light scattering was performed on ArlRD52E, and the diameter of main particle of ArlRD52E shifted to 13.5 nm, indicating changes in oligomerization.

agr40-120 was 9.77 ± 2.27 μM. ArlR still had no obvious interaction with this probe. Given that the binding affinity to agr40-120 was similar to that of agr40-70 or agr100-120, the two specific binding sites did not show a synergistic effect when ArlR binds to the agr promoter.

labelled fragments representing agr40-70 and agr100-120 were synthesized and used for fluorescence polarization assay studies with purified ArlRDBD, ArlR, and ArlRD52E, respectively. As shown in Fig. 2C-F, the binding affinities of ArlRDBD to agr40-70 and agr100-120 were 182.83 ± 36.15 μM and 170.09 ± 42.20 μM, respectively, ArlR had no obvious interaction with these two probes. The binding affinities of ArlRD52E to agr40-70 and agr100-120 were 4.27 ± 1.39 μM and 8.82 ± 2.51 μM, respectively, and these values are 20-fold stronger than that of ArlRDBD. The above data support our DNase I footprinting assay results and confirmed that ArlR phosphorylation liberates its binding ability to its target genes again. Based on the above findings, we wanted to assess whether a synergistic effect existed between the two specific binding sites described above during the ArlR binding process. Thus, a 5′-FAM-labelled 80-bp fragment that encompassed 40–120 bp of the agr promoter (agr40-120) was synthesized. As shown in Fig. 2C-F, the binding affinity of ArlRDBD to agr40-120 was 183.45 ± 35.8 μM; the binding affinity of ArlRD52E to

3.3. Assembly of ArlR and ArlRD52E in solution In many RRs, such as PhoB and KdpE, the α4-β5-α5 surface of RDs in the inactive conformation sequesters the DBD to occlude interaction with DNA. After phosphorylation, RRs assemble as a dimer by the α4β5-α5 surface; thus, the inhibition is relieved (Bachhawat et al., 2005; Narayanan et al., 2014). To verify whether phosphorylation influences the oligomerization state of ArlR, size-exclusion chromatography was performed for ArlRDBD, ArlR, and ArlRD52E (Fig. 3A). The outcomes demonstrated that ArlRDBD and ArlR mostly existed as a monomer; the phospho-mimic variant ArlRD52E is inclined to form dimers and even higher polymers. Furthermore, a dynamic light scattering assay 5

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Fig. 4. The structural comparison between ArlRDBD and its homologues. (A) The overall structure of ArlRDBD. (B) The structural superposition among ArlRDBD (in yellow), PhoB (in magenta, PDB entry: 1GXQ) and KdpE (in cyan, PDB entry: 3ZQ7) shows that main structural variations are located in N-terminal regions. (C) The overall structure of the ArlRDBD dimer. (D) Surface view of the electrostatic potentials of the ArlRDBD dimer. (E) The sequence alignment among the DNA-binding domains of ArlR, PhoB and KdpE. The secondary structural elements of ArlRDBD are shown at the top of the sequence. Strictly conserved residues and similar residues are depicted in red and white, respectively. Residues labeled with a blue pentagram were mutated in this study. (F) The structural comparison between ArlRDBD (in yellow) and PhoBDBD-DNA complex (in magenta, PDB entry: 1GXP). (G) The structural comparison between ArlRDBD (in yellow) and KdpEDBD-DNA complex (in cyan, PDB entry: 4KFC). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

indicated that the major particle size of ArlRDBD appeared at 4.2 nm, which is similar to that of ArlR at 6.5 nm. The particle size peak shifted to approximately 13.5 nm for ArlRD52E, demonstrating that the diameter of ArlR molecule increased two-fold after phosphorylation (Fig. 3E-G). A series of crossing-linking assays also showed more directly that ArlRD52E yields dimer molecular weight bands (Fig. 3D), but ArlR does not (Fig. 3B). The above phenomena were similar to that reported for PhoB and KdpE and implied that phosphorylation would

induce ArlR dimerization, which is quite distinct compared with unphosphorylated state. However, the crossing-linking assay for ArlRDBD revealed that ArlRDBD could also form a dimer in solution (Fig. 3C), and this result was distinct compared with size-exclusion chromatography and dynamic light scattering assay results for ArlRDBD. However, the dimer band for ArlRDBD did not appear in the first fifteen minutes, which hinted us that it would have great possibility to be the nonspecific dimer band due to the longer incubation time. 6

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Fig. 5. The interactions between four mutants of ArlRDBD and agr. (A, B, C, D) Gel shift of four purified ArlRDBD mutants (R193A, K198A, K200A, R212A) with the 5′ FAM-labelled agr promoter (nt 1451–1685). Lane 1 represents the labeled DNA probe alone. Increasing amounts of four ArlRDBD mutants (40, 80, 120, and 160 µM) were added to the probe (lanes 2–5, respectively). 160 µM ArlRDBD was added to probe in the final lane to function as a positive control. (E) Circular dichroism of recombinant ArlRDBD and its four mutants (R193A, K198A, K200A, and R212A).

3.4. The overall structure of ArlRDBD

indicating that ArlRDBD could form a dimer in solution. Combined with the size-exclusion chromatography and dynamic light scattering assay results of ArlRDBD, we conclude that ArlRDBD possesses dimerization capability; however, the contact between the two monomers is not sufficiently strong. Thus, in the solution, the monomer is the dominant oligomerization state of ArlRDBD.

To illuminate the DNA-binding affinity regulatory mechanism of ArlR, we attempted to crystallize ArlRDBD, ArlR, ArlRD52E, and corresponding protein-DNA complexes. Unfortunately, only the structure of ArlRDBD was solved at 2.5-Å resolution (Table 1). The crystal of ArlRDBD belongs to space group P6422, and each asymmetric unit contains one molecule. The structure includes four N-terminal β-strands (β1-β4) followed by four α-helix bundles (α1-α4) and an antiparallel β-sheet (β5-β6) at the C terminal (Fig. 4A). Similar to the structural composition of homologs, the α2 and α3 helices comprise a classical helix-turnhelix motif, and the C-terminal antiparallel β-sheet (β5-β6) forms a βhairpin structure that comprises the winged motif. In homologs, the loop between these two helices is called a transactivation loop given its ability to interact with RNA polymerase subunits to activate transcription (Makino et al., 1993). In addition, the α3 helix and the Cterminal winged motif play a vital role in dsDNA recognition and are responsible for contact with the major and minor grooves of dsDNA, respectively, in many OmpR/PhoB family members (Martínez-Hackert and Stock, 1997). A structural comparison between our ArlRDBD and the DNA-binding domain of PhoB (PDB entry 1GXQ) and KdpE (PDB entry 3ZQ7) revealed minimal structural variation between their main chains (Fig. 4B) with a root-mean-square deviation (r.m.s.d) of 0.922 Å2 for 74 Cα atoms between ArlRDBD and PhoBDBD and 0.789 Å2 for 63 Cα atoms between ArlRDBD and KdpEDBD. The main structural differences between these 3 DNA-binding domains lie in the N terminal regions (with an r.m.s.d of 6.162 Å2 and 6.192 Å2 for 26 Cα atoms of PhoBDBD and KdpEDBD, respectively), while the C-terminal regions resembled each other (with an r.m.s.d of 0.810 Å2 for 63 Cα atoms of PhoBDBD and 0.615 Å2 for 49 Cα atoms of KdpEDBD), implying a similar DNA binding pattern. Furthermore, based on a symmetry assessment of the ArlRDBD crystal structure, we observe domain swapping by the N-terminal domains (β1-β2 sheet) between two adjacent ArlRDBD monomers. This domain swapping contributes a 1180.1 Å2 buried surface area (which covers 17.8% of total accessible surface area) between these two adjacent ArlRDBD monomers and indicates that ArlRDBD could assemble into a dimer (Fig. 4C). The major contacts on the dimer interface include hydrophobic interactions and hydrogen bonds. Six hydrophobic residues (I130, T131, I132, A136, F137, and V139) on the interface potentially contribute to hydrophobic interactions, whereas an additional six residues (N128, T131, D133, Y153, T140 and R202) are involved in stabilizing the dimeric interface through eight hydrogen bonds. This finding was consistent with the cross-linking assay results

3.5. The putative DNA binding site of ArlR To reveal more detailed interaction information between ArlRDBD and dsDNA, sequence alignment (Fig. 4E) and structure comparison (Fig. 4F-G) of ArlRDBD, PhoBDBD-DNA complex (PDB entry 1GXP) and KdpEDBD-DNA complex (PDB entry 4KFC{Narayanan, 2014 #97}) were performed. The above comparisons indicated that a positive charge enrichment area composed of the C-terminal recognition helix and the winged motif region represent the probable DNA binding site of ArlRDBD. Based on the sequence alignment and the structure comparison of above three DNA-binding domains, two conservative residues (Arg193, Arg212) and two nonconservative residues (Lys198, Lys200) distributed among positively charged enrichment area of ArlRDBD were predicted to primarily contribute to interact with dsDNA. To assess the contribution of these four residues in the ArlRDBD-agr interaction process, four ArlRDBD mutants (R193A, K198A, K200A, and R212A) were constructed and analyzed by EMSA experiments. As shown in Fig. 5A-D, the four mutations abolished the binding of the agr promoter, separately. The corresponding circular-dichroism (CD) spectroscopy results (Fig. 5E) indicated that all the mutants were folded. The above results proved that this positively charged area of ArlRDBD is the DNA binding site of ArlR, and these four residues play a significant role in this DNA binding process. Because the dimerization pattern has been not observed in the homologs of ArlRDBD, we sought to reveal how DNA-binding domain dimerization influences the DNA-binding process. In this dimerization pattern, two-winged motifs in the dimer adopt different orientations and are maintained far away from each other, thus preventing two ArlRDBD subunits from binding to the major groove of dsDNA at the same time (Fig. 4C). Meanwhile, the positively charged area of the above two-winged motifs are separated by a huge negative charged gap region, which also hinders the dsDNA from simultaneously binding to these two ArlRDBD subunits (Fig. 4D). Based on the above structural analysis and the low occupancy of dimer for ArlRDBD, we suggest that the dimerization of ArlRDBD has no obvious influence on its dsDNA binding process.

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Fig. 6. The dsDNA recognition preference of ArlR. (A, B, C) Surface view of the electrostatic potentials of ArlRDBD, PhoBDBD, and KdpEDBD (with no DNA bound structure). The white dash lines display the distinct length of the consecutive positively charged areas of these three DNA binding domains. (D, E) Fluorescence polarization assay of ArlRDBD binding to 10-bp and 15-bp long fragments. Briefly, 50 nmol 5′ FAM DNA probes were incubated with indicated concentrations of ArlRDBD to measure fluorescence polarization, demonstrating that ArlRDBD binds 15-bp fragments but not 10-bp fragments. (F, G) Fluorescence polarization assay of ArlRD52E binding to 10-bp and 15-bp long fragments. Briefly, 50 nmol 5′ FAM DNA probes were incubated with indicated concentrations of ArlRD52E to measure fluorescence polarization, showing that ArlRD52E could not bind 10-bp or 15-bp fragments. (H) The dissociation constants of aforementioned fluorescence polarization assay are summarized in this table. (I) Partial view of the structural superposition of ArlRDBD (in yellow) and PhoBDBD-DNA complex (in magenta, PDB entry: 1GXP). The distance between two non-conservative residues (K198 and K200 in ArlRDBD) and DNA (from PhoBDBD-DNA complex) was measured. (J) Fluorescence polarization assay of ArlRD52E+Q69A binding to the agr promoter. Briefly, 50 nmol 5′ FAM DNA probes were incubated with indicated concentrations of ArlRD52E+Q69A to measure fluorescence polarization, showing that ArlRD52E+Q69A still possessed partial binding affinity for the agr promoter. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

recognition preferences. PhoBDBD binds two 7-base pair direct repeats separated by an A/T-rich region of 4 bp (Blanco et al., 2002), while KdpEDBD displays affinity to two 6-base pair sequences separated by 5bp sequences (Narayanan et al., 2014). Given that the positively charged area of ArlRDBD was smaller and shorter than that of PhoBDBD but similar to that of KdpEDBD, we hypothesized that ArlRDBD might bind to a shorter specific sequence than that of PhoBDBD. To verify this assumption, we equally divided agr40-70 into three 10bp long fragments (agr40-50, agr51-60 and agr61-70) and two 15-bp long fragments (agr40-55 and agr56-70). In addition, agr100-120 was also averagely separated into two 10-bp long fragments (agr100-110 and agr111120 ). All the fragments were utilized as substrates for fluorescence polarization assays. The results showed that the ArlRD52E dimer could not obviously bind to these 10- or 15-bp fragments (Fig. 6F-G); the monomer ArlRDBD could obviously bind to both 15-bp fragments of

3.6. The dsDNA recognition preference of ArlR Despite using a similar DNA binding site to recognize dsDNA, the electrostatic potential of each DNA binding site interface regions of the ArlRDBD, PhoBDBD, and KdpEDBD (with no DNA bound) displayed obvious differences. Given the variation in amino acid constitution, the positively charged area of PhoBDBD was centralized and larger than that of the other two DNA-binding domains (with a length of 25.0 Å from the side chain of R201 to the side chain of R219, Fig. 6B). The positively charged areas of ArlRDBD and KdpEDBD were distributed on the DNA binding interfaces, and the lengths of consecutive positively charged areas were 19.2 Å (from the side chain of R193 to the side chain of R212) and 19.3 Å (from the side chain of R193 to the side chain of R211), respectively (Fig. 6A, 6C). The difference in the positive-charge distribution pattern of PhoBDBD and KdpEDBD results in different dsDNA 8

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agr40-55 and agr56-70 but not any 10-bp fragments (Fig. 6D-E). The above results indicated that, ArlRDBD could bind to the approximately 15-bp long dsDNA via fitting with the shorter positively charged DNA-binding site. However, given the dimerization of ArlRD52E, the DNA-binding site of ArlR expanded and requires a longer DNA substrate than that of the ArlRDBD monomer.

of China, for assistance during data collection. This work was supported by the Chinese National Natural Science Foundation [grants U1732114, 31971124, 31770788, U1932120 and 31770895]; The National Key Research and Development Program of China [grant 2017YFA0503600]; the Fundamental Research Funds for the Central Universities.

4. Discussion

Author contributions

The two-component system ArlRS represses the transcription of the agr operon in S. aureus via agr promoter interaction with ArlR; however, the binding characteristics and regulatory mechanism remain unclear. In this study, we confirmed the direct interaction between ArlR and the agr promoter and identified two specific binding regions on agr. Combining the distinct oligomerization states of ArlRDBD, ArlR, and ArlRD52E and their different dsDNA-binding properties, we revealed the DNA-binding affinity regulation process of ArlR. ArlRDBD possesses some DNA binding capability. However, when ArlR monomer is unphosphorylated, its receiver domain interacts with the DNA binding domain and significantly decreases the DNA binding affinity of ArlR. When the receiver domain of ArlR receives upstream signals and is phosphorylated (Parkinson, 2010), ArlR assembles as a dimer, and the blocked DNA binding domains are released to constitute a new DNAbinding site with approximately 10-fold increased dsDNA binding affinity compared with ArlRDBD monomer. Then, the phosphorylated ArlR dimer executes its transcription regulation task with high efficiency. Although the regulation process of ArlR is very similar to that of its homologs, the DNA recognition characteristics and the regulatory interaction process between the receiver domain and the DNA binding domain also display some differences with that of homologs. Given the similarity to homologs, such as PhoB and KdpE, DNA recognition by ArlR was mediated by the helix-turn-helix motif and the winged motif. However, compared to the structures of PhoB-DNA and KdpE-DNA complexes, the electrostatic potential distribution pattern of ArlRDBD was different from that of PhoBDBD and KdpEDBD. On the other hand, two non-conservative residues (K198 and K200) participate in the ArlRDBD-DNA interaction, but the corresponding residues in PhoBDBD are located far away from the dsDNA (with 4.6 Å for K200 and 7.0 Å for K198, respectively, Fig. 6I). The above findings indicated that the recognition characteristic between ArlR and its dsDNA substrate might be somewhat different from that of homologs. Further structure analyses of the ArlRD52E-DNA complex are necessary to illuminate above differences. We proposed a putative working model for ArlR; however, the structural basis of the phosphorylation-triggered relief of DNA-binding inhibition remains unknown. In the KdpE-DNA complex, the interactions between RD and DBD are stabilized by hydrogen bonds between water and the carbonyl oxygen of Q69 from the RD and the side chain of N165 from the DBD (Narayanan et al., 2014). We mutated the conservative glutamine69 to alanine in ArlRD52E; however, the DNAbinding affinity of ArlRD52E+Q69A was only slightly decreased to 25.91 ± 3.43 μM, which was different from the obvious effect of the Q69A mutation in KdpE (Fig. 6J). This result implies that the regulatory interaction between the receiver domain and the DNA binding domain of ArlR employs different mechanisms.

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Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments We would like to thank the staff of BL17U1/BL19U1 beamlines at Shanghai Synchrotron Radiation Facility, Shanghai, People's Republic 9

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