Oxygen and Bis(3′,5′)-cyclic Dimeric Guanosine Monophosphate

Article pubs.acs.org/biochemistry

Oxygen and Bis(3′,5′)-cyclic Dimeric Guanosine Monophosphate Binding Control Oligomerization State Equilibria of Diguanylate Cyclase-Containing Globin Coupled Sensors Justin L. Burns,† Shannon Rivera,† D. Douglas Deer, Shawnna C. Joynt, David Dvorak, and Emily E. Weinert* Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, Georgia 30307, United States S Supporting Information *

ABSTRACT: Bacteria sense their environment to alter phenotypes, including biofilm formation, to survive changing conditions. Heme proteins play important roles in sensing the bacterial gaseous environment and controlling the switch between motile and sessile (biofilm) states. Globin coupled sensors (GCS), a family of heme proteins consisting of a globin domain linked by a central domain to an output domain, are often found with diguanylate cyclase output domains that synthesize c-di-GMP, a major regulator of biofilm formation. Characterization of diguanylate cyclase-containing GCS proteins from Bordetella pertussis and Pectobacterium carotovorum demonstrated that cyclase activity is controlled by ligand binding to the heme within the globin domain. Both O2 binding to the heme within the globin domain and c-di-GMP binding to a product-binding inhibitory site (I-site) within the cyclase domain control oligomerization states of the enzymes. Changes in oligomerization state caused by c-di-GMP binding to the I-site also affect O2 kinetics within the globin domain, suggesting that shifting the oligomer equilibrium leads to broad rearrangements throughout the protein. In addition, mutations within the I-site that eliminate product inhibition result in changes to the accessible oligomerization states and decreased catalytic activity. These studies provide insight into the mechanism by which ligand binding to the heme and I-site controls activity of GCS proteins and suggests a role for oligomerization-dependent activity in vivo.

B

Within the GCS family, a number of putative proteins are predicted to contain diguanylate cyclase (DGC) output domains, which are the enzymes responsible for synthesizing c-di-GMP, a bacterial second messenger that is a major regulator of biofilm formation.20,21 Recently, DGC-containing GCS proteins from Shewanella putrefaciens17 and Bordetella pertussis9 have been found to alter intracellular c-di-GMP levels and biofilm formation in an O2-dependent manner, suggesting potentially important role(s) for GCS signaling in controlling bacterial phenotypes in response to changing O2 levels. Most bacteria have multiple DGC proteins encoded within their genomes and sensing or regulatory domains differentially regulate the activity of these diguanylate cyclases.20,21 These enzymes function as dimers, with each monomer binding one molecule of GTP and cyclization to c-di-GMP occurring across the dimer interface.22−26 In addition, DGC domains often contain a productbinding inhibitory site (I-site) that consists of an Arg-X-X-Asp (RxxD) motif. Binding of c-di-GMP to the I-site, typically as an intercalated c-di-GMP dimer, results in product inhibition and decreased cyclase activity.22−25,27,28 Detailed biochemical and structural characterization of DGC proteins, such as PleD from

acteria are constantly sensing changes in their environment to alter intracellular pathways that maximize their ability to survive. Among the numerous environmental signals, oxygen (O2) concentration is an important variable for nearly all organisms due to its role in respiration.1,2 In addition to modulating growth and metabolism, bacteria have been found to alter phenotypes, such as virulence, in response to altered O2 levels, suggesting broad importance of O2 as a signal.1,3,4 While a number of previously characterized proteins have been shown to serve as intracellular redox sensors,1,5 direct O2 sensors (proteins that reversibly bind diatomic oxygen) recently have been shown to play key roles in controlling bacterial phenotypes. A family of heme proteins, termed globin coupled sensors (GCSs), has been identified and found to consist of an N-terminal sensor globin domain linked through a middle domain to a variety of output domains, such as methyl accepting chemotaxis proteins (MCP), diguanylate cyclases, phosphodiesterases, STAS domains, and kinases.3,4,6−14 GCS proteins are widely distributed throughout bacteria, as well as in a number of archaea, and are represented within the genomes of environmental bacteria, plant pathogens, and human pathogens, suggesting widespread importance in controlling O2-dependent bacterial phenotypes.10,15 Ligand binding to the heme within the sensor globin domain of GCSs has been shown to alter activity of the output domain, leading to phenotypes such as aerotaxis and biofilm formation.6,7,9,16−19 © 2016 American Chemical Society

Received: May 24, 2016 Revised: November 13, 2016 Published: November 14, 2016 6642

DOI: 10.1021/acs.biochem.6b00526 Biochemistry 2016, 55, 6642−6651

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Biochemistry

Figure 1. Sequence alignment of representative diguanylate cyclase containing globin coupled sensors EcDosC (P0AA89.1),7,29,34 PccGCS (YP_003018185.1),6 BpeGReg (NP_882025),6,9 SpDosD (Sputcn32_3244),17 and HemDGC (YP_063937).16 Key resides are highlighted by arrows ((a) distal tyrosine; (b) proximal histidine; (c) interdomain interaction residue identified in tDGC crystal structure;24 (d) RxxD I-site; (e) GG(D/E)EF active site).

translated with EcDosP, the other member of the direct oxygen sensing (DOS) operon in E. coli. Furthermore, EcDosC and EcDosP form hetero-oligomeric complexes in vitro and in vivo.7,29,33 Therefore, interactions with EcDosP may be a factor in the differential activation of EcDosC in vitro. The current understanding of GCS activation has been hampered by the lack of full-length crystal structures in active and inactive ligation states. Isolated sensor globin domains have been crystallized, and globin domains were found to exist as dimers in the crystallographic unit.19,34−36 In addition, structures of the three individual domains from EcDocC recently were crystallized, and all of the domains formed dimers within the unit cells.34 The majority of sensor globins that have been crystallized were in either the FeII or FeIII−CN states to yield insight into the inactive and “active-like” conformations. These studies have suggested that ligand binding leads to subtle rearrangements of the heme pocket and rotation around the globin dimer interface, although to a smaller extent in EcDosC, that may be transmitted to the output domains. Resonance Raman studies on the GCS from Bacillus subtilis (HemAT-Bs, MCP output domain) also identified multiple hydrogen bonding patterns within the heme pocket and effects of the output domain on heme pocket conformation.37 However, without full-length structural information, the broader rearrangements that occur upon ligand binding have remained elusive. Both structural information and the DGC enzymatic mechanism suggest that homo-oligmerization is likely an important structural element of GCS proteins. DGC proteins require formation of a dimer for activity,26 with each monomer

Caulabacter crescentus and WspR from Pseudomonas f luorescens, has shown that activation of DGC proteins leads to conformational changes that result in formation of dimeric assemblies that exhibit high catalytic activity. In contrast, product binding leads to inhibition by shifting the equilibrium away from dimeric assemblies, to monomeric and tetrameric/extended dimeric assemblies for PleD and WspR, respectively.22,23 Characterization of DGC-containing GCS proteins has been reported for proteins from Escherichia coli (EcDosC),7,29 Desulfotalea psychrophila (HemDGC),16 B. pertussis (BpeGReg),6,9 Pectobacterium carotovorum (PccGCS),6 and Shewanella putrefaciens (SpDosD).17 In general, GCS proteins show modest to poor sequence similarity, with absolute conservation only of the proximal histidine that ligates the heme (Figure 1).10−12,15,30−32 In addition to the conserved proximal histidine, all of the characterized DGC-containing GCS proteins also contain a distal tyrosine that provides hydrogen bonding to bound diatomic ligands, and the conserved DGC active site (GG(D/E)EF) and I-site (RxxD) motifs. However, HemDGC differs from the other GCS proteins due to its shorter middle domain (∼40 amino acids vs ∼135 amino acids, respectively).16 To date, activity of DGC-containing GCS proteins has been demonstrated to be regulated by ligation or oxidation state of the heme within the sensor globin domain.6,9,16,17,29 In all cases characterized so far, GCS with FeII heme resulted in basal activity and O2 binding resulted in greater cyclase activity. In contrast to the other characterized proteins, FeIII EcDosC exhibits even greater cyclase activity than FeII−O2 EcDosC.7 However, EcDosC also differs in that it is known to be cotranscribed and 6643

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Single oligomeric states (≥85% purity) were isolated by gel filtration as previously described.6 Samples of BpeGReg and PccGCS bound to c-di-GMP were made by incubating the proteins with GTP in the absence of EcDosP for 60−90 min at room temperature. Based on previous work6 and preliminary tests, the enzymes had become product inhibited at this point and behaved similarly to proteins incubated with c-di-GMP. Phosphodiesterase EcDosP39 was purified in an analogous manner to the GCSs, with the exception that it was expressed from pET-28a with pGro7 (Takara Bio), as previously described,39 so cells were grown in the presence of kanamycin (30 μg mL−1) and chloramphenicol. Electronic Absorption Spectroscopy. All spectra were recorded on an Agilent Cary 100 with Peltier accessory. Preparation of complexes was carried out as previously described except that the proteins were prepared in buffer C.38,40,41 Analytical Gel Filtration. GCS oligomers were detected via size exclusion chromatography using an Agilent 1200 infinity system with a Sepax SEC-300 (7.8 mm × 300 mm, 300 Å) and diode array detector (simultaneous detection at 214, 416, and 431 nm), as previously described.6 Proteins were reduced in an anaerobic chamber and then allowed to bind O2 following mixing with aerobic buffer before injection onto SEC-300 column. The mobile phase for all experiments consisted of 150 mM sodium phosphate, pH 7.0 (AGF buffer), with 1 mM DTT. Spectra were collected for each peak during the SEC run to confirm that the heme remained in the FeII−O2 ligation state. Globular proteins (Sigma-Aldrich) consisting of thyroglobulin (669 kDa), ferritin (443 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), and conalbumin (29 kDa) were used as molecular weight standards for calibration curves. Anaerobic Analytical Gel Filtration. AGF buffer was sparged with helium and then equilibrated in an anaerobic chamber (Coy) for at least 24 h to ensure all O2 had been removed. Sodium dithionite (1 mM) was added to one bottle of AGF buffer (AGF buffer B). Once the AGF buffers were removed from the glovebag and installed on the HPLC, both buffers were kept under postitive N2 pressure to minimize any diffusion of O2 into the buffers. The HPLC and AGF column were deoxygenated by washing the instrument in 98% AGF buffer and 2% AGF buffer B (final dithionite concentration = 500 μM). The injection needle and sample loop were deoxygenated by injecting a sample of 500 μM dithionite in AGF buffer. GCS samples were reduced in the anaerobic chamber6,38 and sealed in HPLC sample vials, which were removed from the glovebag immediately prior to injection. Spectra (200−600 nm) were obtained for all protein peaks to ensure that the GCS proteins remained FeII (and did not bind O2 or oxidize) during HPLC analysis. Calibration curves were obtained using molecular weight standards as described above. Oligomer Percentage Peak Fitting. Oligomer percentages were calculated by peak fitting the 416 nm AGF traces (corresponding to FeII−O2 absorbance maximum) or the 430 nm AGF traces (corresponding to FeII absorbance maximum) using Igor Pro (Wavemetrics) Multipeak Fitting 2 analysis package (Figure S1). AGF traces were obtained for each protein from multiple protein preps on multiple days to ensure that the observed results were not due to an instrument or protein anomaly. These traces were analyzed using the Igor Multipeak Fitting analysis package by providing initial peak estimates for the program (including peak width, height, and location) and allowing the program to minimize the residuals to provide a final

binding one GTP and the active site spanning the dimer interface, suggesting that DGC-containing GCS proteins also must exist as dimers, at least during catalysis. Indeed, EcDosC, HemDGC, PccGCS, and BpeGReg have been shown to form oligomers in solution;6,16,34 however the oligomeric states accessed by each of these proteins differs. HemDGC was found to exist as a tetramer by native PAGE,16 while EcDosC was found to exist primarily as a dimer, with small percentages of higher molecular weight species, when analyzed by analytical ultracentrifugation and gel filtration.34 BpeGReg and PccGCS exist as mixtures of oligomers, with BpeGReg purifying as a monomer−dimer−tetramer mixture and PccGCS as a dimer− tetramer−octamer mixture.6 These studies suggest that the factors controlling oligomerization of GCS proteins are not yet understood. The existence of multiple slowly re-equilibrating oligomeric species for BpeGReg and PccGCS allowed for comparison between oligomeric states, identifying tetrameric assemblies of BpeGReg and PccGCS as being 4−5-fold more active than equal per-monomer concentrations of dimeric assemblies.6 Therefore, shifting the equilibrium between dimer and tetramer potentially could alter GCS cyclase activity and lead to changes in O2dependent signaling in vivo. This work demonstrates the role of ligand binding to the heme in triggering a shift between oligomeric states, as well as the effects of product binding to the Isite on the cyclase domains. Together, these studies highlight the interplay between ligand sensing within the globin and cyclase domains on oligomerization, heme pocket conformation, and cyclase activity.

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MATERIALS AND METHODS Materials and General Methods. Unless otherwise noted, all reagents were purchased in the highest available purity and used as received. Protein Expression and Purification. GCS proteins were expressed and purified as previously described, with minor modifications.6 Plasmids with codon optimized GCS genes (pET20-BpeGReg; pET20-PccGCS) were transformed into Escherichia coli Tuner (DE3) pLysS cells (Novagen) via electroporation, and positive transformants were selected on LB medium containing ampicillin (100 μg mL−1) and chloramphenicol (30 μg mL−1). Expression strains were grown in yeast extract medium38 to late exponential phase (OD600 = 0.8) at 37 °C. The temperature then was lowered to 25 °C, and cells were then induced with IPTG (100 μM final concentration). Cells were allowed to express WT proteins for 6 h, while I-site variants were induced for 16−20 h. Cells were harvested by centrifugation (3500g, 4 °C, 20 min), and resulting cell pellets were either stored at −80 °C or resuspended in buffer A (50 mM Tris, 50 mM NaCl, 1 mM DTT, 20 mM imidazole, pH 7.4) for immediate purification. Cell pellets were homogenized using a homogenizer (Avestin, Inc.), and resulting lysates were centrifuged at 130 000g in a Beckman Optima L-90X ultracentrifuge at 4 °C for 1 h. All subsequent purification steps were performed at 4 °C. Supernatants were applied to a pre-equilibrated HisPur Ni-column (Fisher Scientific), and proteins were eluted with buffer B (buffer A with 250 mM imidazole, pH 7.4). Purified proteins were desalted using a S200 gel filtration column (GE Healthcare) into buffer C (50 mM Tris, 50 mM NaCl, 1 mM DTT, 5% glycerol (v/v), pH 7.0). Proteins were collected and concentrated via ultrafiltration (YM-10, 10 kDa MWCO filter, Millipore), aliquoted, flash frozen, and stored at −80 °C until use. 6644

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Figure 2. Ligand-dependent changes in oligomeric states. (A, B, E, F, G, H) Qualitative comparisons of representative AGF traces. (C, D) Stacked bar graphs of oligomer percentages quantified from AGF traces. (A) Solid line, PccGCS FeII−O2; dashed line, PccGCS FeII. (B) Solid line, PccGCS FeII−O2; dashed line, PccGCS FeII−O2 + c-di-GMP. (C) Comparison of PccGCS FeII−O2, FeII−O2 + c-di-GMP, FeII, and FeII + c-di-GMP. (D) Comparison of PccGCS WT FeII−O2, BpeGReg WT FeII−O2, BpeGReg R364A FeII−O2, and BpeGReg R364A FeII−O2 + c-di-GMP. (C, D) Vertical stripe, monomer; white bar, dimer; gray bar, tetramer; horizontal stripe, octamer/HMW. Measurement percentages and errors can be found in Table S1. (E) Solid line, BpeGReg FeII−O2; dashed line, BpeGReg FeII. (F) Solid line, BpeGReg FeII−O2; dashed line, BpeGReg FeII−O2 + c-di-GMP. (G) Solid line, BpeGReg WT FeII−O2; dashed line, BpeGReg R364A FeII−O2. (H) Solid line, PccGCS WT FeII−O2; dashed line, PccGCS R377A FeII−O2.

fit. The entire fitting process (starting from an unfitted trace) was performed at least three times on each AGF trace, varying initial peak parameters and peak numbers, to ensure that that the peak fits were reproducible and provided the best fits of the raw data.

Fits were considered optimized when consistent peak areas were obtained with residuals of each fit 20-fold faster than reequilibration of isolated FeII−O2 oligomers.6 Quantification of AGF traces yielded a ∼1.5-fold increase in the amount of PccGCS tetrameric assemblies of PccGCS FeII−O2, as compared to PccGCS FeII, with concomitant decreases in the percentages of dimer and HMW. Addition of c-di-GMP (FeII−O2 vs FeII−O2 + c-di-GMP) lead to ∼13% decrease in tetramer and a shift to HMW (Table S1). Qualitative analysis of BpeGReg in the presence and absence of ligands yielded results similar to PccGCS (Figure 2E,F). In the absence of O2 or the presence of c-di-GMP, BpeGReg shifted away from tetrameric assemblies (which exhibit the greatest enzymatic activity), with an increase in dimeric assemblies. Unfortunately, BpeGReg is prone to aggregation in the presence 6646

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Biochemistry Table 1. Michaelis−Menten Kinetics of BpeGReg and PccGCS WT and I-Site Variants Measured for Various Ligation and Oligomerization States protein

oligomer a

BpeGReg WT BpeGReg WTa BpeGReg R364A BpeGReg R364A BpeGReg R364A BpeGReg R364A PccGCS WTa PccGCS WTa PccGCS R377A a

as purified as purified as purified as purified dimer tetramer as purified as purified as purified

ligation state II

Fe FeII−O2 FeII FeII−O2 FeII−O2 FeII−O2 FeII FeII−O2 FeII−O2

kcat (min−1)

KM (μM)

0.18 0.59 0.05 0.38 0.09 0.35 0.29 0.73 0.10

120 ± 11 57 ± 8 80 ± 19 50 ± 8 b b 62 ± 3 31 ± 6 33 ± 13

Reference 6. bNot determined.

binding kinetics within the globin domain. Isolated dimeric and tetrameric assemblies of BpeGReg R364A yielded intermediate rates for k2 (3.88, 5.30, and 6.72 s−1; for as purified, dimeric, and tetrameric BpeGReg R364A, respectively), suggesting that octamer/HMW species may be contributing to the rates observed in the mixture. While O2 dissociation rates of the octamer/HMW species were measured and exhibited slower k2 than dimer or tetramer, the low concentration of isolated octamers prohibited in depth comparison. Incubation of BpeGReg R364A variant with c-di-GMP did not cause significant changes in either rate, likely due to weak c-di-GMP binding and minimal changes to the protein structure/conformation. Measurement of O2 dissociation rates for PccGCS R377A (Figure 4, Table 2), which is a complex mixture of oligomeric states, resulted in increased slow (k1) and fast (k2) dissociation rates (1.7-fold and 1.4-fold, respectively, as compared to WT PccGCS), in contrast to the observed changes for BpeGReg WT and R364A.

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DISCUSSION The previous observation that both BpeGReg and PccGCS exist as mixtures of oligomeric assemblies that do not readily interconvert and have different catalytic activities6 suggested that GCS proteins might have evolved to control catalysis and downstream c-di-GMP signaling through modulation of oligomerization state equilibria. PccGCS consists of ∼2.75-fold more tetramer than BpeGReg (Figure 2D), which is consistent with the 2-fold greater kcat measured for PccGCS (∼2.3-fold greater kcat/KM) as tetrameric assemblies exhibit ∼4−5-fold greater cyclase activity than dimeric assemblies.6 Given that BpeGReg and PccGCS oligomers do not readily equilibrate, GCS proteins must sense a signal to shift to or away from various oligomeric states and thereby alter cyclase activity. To investigate the role of ligand binding in controlling GCS oligomer equilibria, oligomerization states were quantified in the presence and absence of O2 and c-di-GMP. As GCS proteins have been shown to serve as O2 sensors in vivo,12,17 O2 binding to or dissociation from the heme could trigger conformational changes and reequilibration of GCS oligomers. Additionally, as diguanylate cyclases with intact I-sites exhibit product inhibition,9,22,23,25 cdi-GMP binding potentially could shift the equilibrium away from tetrameric assemblies that exhibit higher catalytic activity. Comparison of representative traces of FeII and FeII−O2 PccGCS are shown in Figure 2A. Binding of O2 to the heme resulted in a shift toward tetrameric assemblies and away from octamer/HMW assemblies, with a ∼1.5-fold increase in the amount of tetrameric assemblies, which exhibit the highest

Figure 3. Michaelis−Menten kinetics. (A) Representative data for 3 μM BpeGReg WT (solid line) and R364A variant (dashed line). Inset shows vmax data at tested enzymes concentrations. (B) Representative data for 1.5 μM PccGCS WT (solid line) and R377A variant (dashed line). Inset shows vmax data at tested enzyme concentrations. (C) Comparison of BpeGReg R364A single oligomer enzyme kinetics under vmax conditions (500 μM GTP). Filled circles, tetramer; open squares, dimer.

catalytic activity, for FeII−O2 PccGCS as compared to FeII PccGCS (Figure 2C). This correlates well with the ∼2.5-fold increase in cyclase activity upon O2 binding, further supporting the role of higher order oligomers in catalysis. In addition, the relationship between catalysis and oligomerization state is solely due to O2 binding, as AGF of PccGCS in the presence of EcDocP (a phosphodiesterase used to eliminate product inhibition in 6647

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Biochemistry Table 2. BpeGReg and PccGCS WT and I-Site Variant O2 Dissociation Kineticsa protein b

BpeGReg BpeGRegb BpeGRegb BpeGReg BpeGReg R364A BpeGReg R364A BpeGReg R364A BpeGReg R364A BpeGReg R364A PccGCSb PccGCSb PccGCSb PccGCS PccGCS R377A PccGCS R377A a b

oligomer

c-di-GMP

k1 (s−1)

k2 (s−1)

% k1

% k2

as purified dimer tetramer as purified as purified as purified dimer tetramer HMW as purified dimer tetramer as purified as purified as purified

− − − + − + − − − − − − + − +

0.82 ± 0.01 1.33 ± 0.07 1.23 ± 0.01 0.95 ± 0.01 0.627 ± 0.007 0.614 ± 0.009 1.09 ± 0.06 1.20 ± 0.06 1.70 ± 0.09 0.56 ± 0.01 0.668 ± 0.018 0.641 ± 0.003 0.575 ± 0.007 0.97 ± 0.01 0.98 ± 0.02

6.30 ± 0.11 6.16 ± 0.97 7.50 ± 0.06 6.72 ± 0.24 3.88 ± 0.06 4.06 ± 0.06 5.30 ± 1.29 6.72 ± 0.99 5.06 ± 0.93 3.87 ± 0.08 4.68 ± 0.25 3.98 ± 0.03 4.56 ± 0.07 5.50 ± 0.12 5.60 ± 0.20

39.3 ± 2.2 40.7 ± 2.3 36.2 ± 0.9 37.9 ± 1.1 49.0 ± 1.2 48.3 ± 2.4 50.2 ± 4.8 43.8 ± 6.6 33.3 ± 9.6 56.1 ± 1.0 56.1 ± 0.8 56.3 ± 1.7 49.6 ± 1.5 44.4 ± 0.5 44.3 ± 0.8

60.7 ± 2.2 59.3 ± 2.3 63.8 ± 0.9 62.1 ± 1.1 51.0 ± 1.2 51.7 ± 2.4 49.8 ± 4.8 56.2 ± 6.6 66.7 ± 9.6 44.0 ± 1.9 43.9 ± 0.8 43.7 ± 1.7 50.4 ± 1.5 55.6 ± 0.5 55.7 ± 0.8

Rates were measured for as purified mixtures of oligomeric states and isolated single states, as well as with and without c-di-GMP bound. Reference 6.

Figure 4. O2 dissociation kinetics. (A) Representative stopped flow spectra for O2 dissociation from BpeGReg FeII−O2 R364A (as purified). (B) Raw data (black) and single exponential fit (red) for BpeGReg FeII−O2 R364A. Residuals (difference between actual data and fit) are shown in red above the plot. (C) Raw data (black) and double exponential fit (red) for BpeGReg R364A. Residuals (difference between actual data and fit) are shown in red above the plot. (D) Representative stopped flow spectra for PccGCS FeII−O2 R377A (as purified). (E) Raw data (black) and single exponential fit (red) for PccGCS R377A. Residuals (difference between actual data and fit) are shown in red above the plot. (F) Raw data (black) and double exponential fit (red) for PccGCS FeII−O2 R377A. Residuals (difference between actual data and fit) are shown in red above the plot.

to decreased enzymatic activity, supporting a mechanism to turn off GCS diguanylate cyclase activity when cellular c-di-GMP levels rise too high.22 Taken together, these studies explain previous data that demonstrated that O2 binding to the heme increased GCS catalytic activity;6 FeII−O2 PccGCS results in a shift to a higher percentage of tetrameric assemblies (Figure 2A,C), which have the highest cyclase activity. In contrast, FeII PccGCS results in a decrease in the percentage of tetrameric assemblies, as did binding of c-di-GMP to the I-site (Figure 2A,B,C). Furthermore,

enzyme assays) or magnesium chloride (included in enzyme assays) did not cause a shift in oligomeric state (Figure S4). In contrast, binding of c-di-GMP to PccGCS, which inhibits cyclase activity, resulted in a decrease (∼13%) in the percentage of tetrameric assemblies and a similar increase in octamer/HMW (Figure 2B,C). The shift away from tetramer in the presence of cdi-GMP was even more dramatic when PccGCS was isolated product-inhibited (∼0.4−0.8 mol of c-di-GMP/mol of PccGCS) following a 16-h induction (Figure S3). Binding of product to the I-site decreased the percentage of high-activity tetramer and lead 6648

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Biochemistry

ity.22,23,25−27,43 Previous studies demonstrated that mutation of the conserved Arg within the I-site RxxD motif in a Thermotoga maritima enzyme (tDGC) eliminated product inhibition and did not affect protein structure or catalytic activity.24,43 Surprisingly, the BpeGReg R364A variant was isolated as a mixture of monomer, dimer, tetramer, and octamer/HMW, while BpeGReg WT was not observed to form oligomeric species larger than tetramer (Figure 2G). In addition, PccGCS R377A was observed to consist of a large, complex distribution of oligomeric states, demonstrating a role for the conserved I-site in limiting accessible oligomerization states and suggesting that mutation of the I-site resulted in changes in conformation or flexibility. Despite the ∼2-fold increase in the percentage of high-activity tetramer for BpeGReg R364A, the kcat decreased ∼1.6-fold. Introduction of the I-site mutation into PccGCS, which resulted in larger disruption to oligomerization, resulted in an even larger decrease (∼7-fold) in catalytic activity (Table 1, Figure 3). However, the lack of change in KM for both proteins suggests that the structure near the substrate binding site was not significantly perturbed, as both proteins were able to bind substrate as tightly as WT. In contrast, the observed decreases in kcat suggest that the catalytic site, which is located at the interface between the two cyclase domains,22 is altered and is not optimally positioned or is too flexible and does not remain locked into the catalytic conformation long enough for effective catalysis to occur. The changes in accessible oligomerization states and catalytic activity following mutation within the I-site of BpeGReg and PccGCS were quite surprising (Figure 2D,G,H; Table 1) given that mutation of the homologous arginine within tDGC resulted in oligomerization profiles and catalytic activity that mirrored the nonproduct inhibited WT tDGC protein.24,43 Structural studies of tDGC identified an arginine residue (R115) outside of the Isite RxxD motif that interacts with the I-site arginine and bound c-di-GMP, locking the tDGC cyclase domain dimer into an inactive conformation.24 However, within BpeGReg and PccGCS, this position is filled by lysine and histidine, respectively (Figure 1). In addition, tDGC was found to have at least five more salt bridges than other crystallized DGC domains, helping to stabilize the structure.24 These differences may contribute to altered conformational dynamics within DGC domains, potentially leading to the differences observed for the GCS Isite variants. Furthermore, the studies described herein highlight a role for the I-site in controlling conformation/dynamics of diguanylate cyclase domains, in addition to serving as a productbinding site that results in cyclase inhibition. Given that mutation of the I-site affects both oligomerization and catalysis, the effects on BpeGReg and PccGCS sensor globin domains were investigated. The BpeGReg R364A I-site variant resulted in decreased O2 dissociation rates for both k1 and k2 (∼1.3-fold and ∼1.7-fold, respectively), yielding rates closer to those of PccGCS WT than BpeGReg WT (Table 2). As BpeGReg R364A is able to form octamer/HMW species, these data further highlight the roles of oligomerization state and the cyclase domain in controlling O2 binding kinetics within the globin domain. In contrast, PccGCS R377A exhibited increased O2 dissociation rates, as compared to PccGCS WT, likely due to the large mixture of oligomeric states that the PccGCS R377A variant can access (Figure 2H), suggesting substantially increased globin domain flexibility and heme pocket dynamics. Incubation of the BpeGReg R364A and PccGCS R377A variants with c-di-GMP did not cause significant changes in either rate, likely due to weak cdi-GMP binding. Taken together, c-di-GMP and I-site dependent changes in O2 dissociation rates for PccGCS and BpeGReg

qualitative analysis of BpeGReg mimics these trends, with BpeGReg FeII−O2 exhibiting more tetrameric assemblies than BpeGReg FeII and FeII−O2 + c-di-GMP. These data highlight a ligand-dependent mechanism for controlling GCS oligomerization state and activity, analogous to the oligomerization state changes observed for the diguanylate cyclases PleD and WspR.22,23,25−27 To our knowledge, these studies demonstrate the first O2-dependent regulation of diguanylate cyclase oligomerization and provide key insight as to how ligand binding to GCS proteins controls output domain activity. Although PleD and WspR exhibited the highest catalytic activity as dimers, with monomeric and tetrameric assemblies predominating in the catalytically “off” states,22,23,25,27 while tetrameric assemblies of PccGCS and BpeGReg exhibited the highest activity,6 these results extend our understanding of diguanylate cyclase activation, suggesting that coupling oligomerization state changes to cyclase activity may be a general mechanism to control c-di-GMP production. In addition, these results demonstrate that ligand-dependent changes in oligomerization state are a mechanism by which DGC-containing GCS proteins control cyclase activity. For GCS proteins that are coexpressed with binding partners, such as the E. coli GCS, EcDosC, and its in vivo partner EcDosP,29,33,34 the mechanism of transmitting the ligand-binding signal may involve their binding partner, potentially in similar conformation/oligomerization state changes. Previous studies demonstrated that BpeGReg and PccGCS WT proteins display biphasic O2 dissociation rates and that O2 dissociation rates differed between isolated GCS oligomeric states, suggesting that oligomerization affected conformations or hydrogen bonding patterns within the heme pocket.6 Biphasic O2 dissociation kinetics also have been observed for HemAT-Bs (GCS from B. subtilis, MCP output domain) and were attributed to different hydrogen bonding patterns between the distal pocket tyrosine and threonine, as well as open/closed conformations of the heme pocket.18,37 Furthermore, studies on the isolated globin domains of BpeGReg and PccGCS demonstrated that biphasic kinetics are correlated with globin domain dimerization and that the distal pocket serine is required for biphasic O2 dissociation.42 Therefore, to specifically probe the effects of shifting the oligomer equilibrium on O2 dissociation, PccGCS was incubated with c-di-GMP to shift the oligomer equilibrium away from tetrameric assemblies. The incubation with c-di-GMP and resultant shift in oligomer equilibrium resulted in an increase in the fast O2 dissociation rate (k2) to 4.56 s−1 (k2 = 4.68 s−1 and 3.98 s−1 for dimeric and tetrameric PccGCS, respectively; Table 2), supporting the AGF data that demonstrated addition of c-diGMP shifts the oligomer equilibrium away from tetrameric assemblies and toward dimeric assemblies (Figure 2B,C; Table S1). Very little change was observed upon addition of c-di-GMP to BpeGReg, likely due to the small percentage of tetramer (26%; Table S1) in the c-di-GMP-free protein. These data demonstrate that signaling within GCS proteins can occur both from Nterminus → C-terminus (O2-dependent changes in catalysis) and C-terminus → N-terminus (c-di-GMP-dependent changes in O2 binding). To further probe the role of the I-site in GCS oligomerization, variants of the conserved Arg were characterized. The effects of cdi-GMP binding to DGC domain I-sites previously have been probed using enzyme kinetics, binding assays, and X-ray crystallography. In general, incubation of DGC-containing proteins with c-di-GMP leads to binding at the I-site, often as an intercalated c-di-GMP dimer, and inhibits cyclase activ6649

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Biochemistry Author Contributions

(Table 2) support a strongly coupled interplay between the sensor globin and cyclase domains within GCS proteins, highlighting bidirectional transmission of ligand-binding information. While O2- and c-di-GMP-dependent changes in catalytic activity6,7,9,17,29 and oligomerization state for GCS proteins are modest, studies of GCS deletion strains of E. coli, B. pertussis, and S. putrefaciens demonstrate that these changes in the presence/ absence of ligands are sufficient to cause in vivo effects.9,17,44 For all three organisms, deletion of the respective GCS gene resulted in decreased aerobic biofilm formation and, in S. putrefaciens and E. coli, also decreased expression of key adhesin and curli genes, respectively.17,44 Furthermore, deletion of the P. carotovorum GCS gene resulted in O2-dependent changes in virulence factor excretion and motility, which yielded decreased aerobic virulence (Burns et al., manuscript under review). Taken together, these studies demonstrate that despite the modest changes in oligomerization equilibrium and catalytic activity upon O2 binding, changes in GCS activity and signaling in vivo are sufficient to alter bacterial phenotypes.

†

J.L.B. and S.R. contributed equally to this work.

Funding

The authors thank the National Science Foundation (Grant CHE 1352040), the University Research Committee of Emory University, and Emory University for financial support of this research. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS AGF, analytical gel filtration; BpeGReg, Bordetella pertussis globin coupled sensor; c-di-GMP, bis(3′,5′)-cyclic dimeric guanosine monophosphate; DGC, diguanylate cyclase; EcDosC, Escherichia coli globin coupled sensor; GCS, globin coupled sensor; HemDGC, Desulfotalea psychrophila globin coupled sensor; HMW, high molecular weight, oligomers larger than octamer; IPTG, isopropyl β-D-1-thiogalactopyranoside; I-site, diguanylate cyclase product-binding inhibitory site; MCP, methyl accepting chemotaxis protein; PccGCS, Pectobacterium carotovorum globin coupled sensor; SpDosD, Shewanella putrefaciens globin coupled sensor; tDGC, Thermotoga maritima diguanylate cyclase; WT, wild-type

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CONCLUSIONS In summary, O2 binding to the heme or c-di-GMP binding to the I-site of DGC-containing GCS proteins from B. pertussis and P. carotovorum alters the equilibrium between oligomeric states. Binding of O2 results in a shift to greater percentage of tetrameric assemblies, while c-di-GMP binding shifts the equilibrium away from tetramers. As oligomeric states exhibit different catalytic activity, with tetrameric assemblies exhibiting the highest cyclase activity, ligand binding to either the globin heme or I-site within the cyclase domain influences catalytic activity of the cyclase domain. Shifting the oligomer equilibrium through c-di-GMP binding to the I-site also results in changes within the globin domain, altering O2 dissociation kinetics and likely leading to rearrangements within the heme pocket. These studies demonstrate that changes in either the N-terminal globin or the C-terminal cyclase domain can lead to changes at the other terminus of the protein. Modulating the equilibrium between oligomeric states may be more broadly utilized within the GCS family as a mechanism to control enzymatic activity. In particular, as diguanylate cyclase domains require dimerization for activity, even subtle shifts in equilibria may be sufficient to elicit O2dependent responses in vivo.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b00526. Representative AGF peak fitting analyses, salt dependence of PccGCS oligomerization, AGF trace of PccGCS isolated following overnight induction, AGF of PccGCS in the absence and presence of EcDosP and MgCl2, and oligomer percentages and error quantified from AGF measurements (PDF)

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ACKNOWLEDGMENTS

The authors are grateful to Professor Toru Shimizu for the gift of the EcDosP plasmid and members of the Weinert group for helpful discussions.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 404-712-6865. ORCID

Emily E. Weinert: 0000-0002-4986-8682 6650

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DOI: 10.1021/acs.biochem.6b00526 Biochemistry 2016, 55, 6642−6651