Light-Regulated Synthesis of Cyclic-di-GMP by a Bidomain

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Light-regulated synthesis of cyclic-di-GMP by a bidomain construct of the cyanobacteriochrome Tlr0924 (SesA) without stable dimerization Matthew D. Blain-Hartung, Nathan Clarke Rockwell, and J. Clark Lagarias Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00734 • Publication Date (Web): 26 Oct 2017 Downloaded from http://pubs.acs.org on October 28, 2017

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Biochemistry

Light-regulated synthesis of cyclic-di-GMP by a bidomain construct of the cyanobacteriochrome Tlr0924 (SesA) without stable dimerization

Matthew Blain-Hartung, Nathan C. Rockwell, and J. Clark Lagarias* Department of Molecular and Cellular Biology, University of California, Davis, California 95616, United States *Corresponding Author: Tel: 530-752-1865; email: [email protected]

Running Title: Bidomain cyanobacteriochrome diguanylate cyclase

ACS Paragon Plus Environment

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ABSTRACT (250 words) Phytochromes and cyanobacteriochromes (CBCRs) use double bond photoisomerization of their linear tetrapyrrole (bilin) chromophores within cGMP-specific phosphodiesterases/Adenylyl cyclases/FhlA (GAF) domain-containing photosensory modules to regulate activity of Cterminal output domains. CBCRs exhibit much more diverse photocycles than phytochromes, and are often found in large modular proteins such as Tlr0924 (SesA), one of three blue light regulators of cell aggregation in the cyanobacterium Thermosynechococcus elongatus. Tlr0924 contains a single bilin-binding GAF domain adjacent to a C-terminal diguanylate cyclase (GGDEF) domain whose catalytic activity requires formation of a dimeric transition state presumably supported by a multi-domain extension at its N-terminus. To probe the structural basis of light-mediated signal propagation from the photosensory input domain to a signaling output domain for a representative CBCR, these studies explore the properties of a bidomain GAF-GGDEF construct of Tlr0924 (Tlr0924Δ) that retains light-regulated diguanylate cyclase activity. Surprisingly, CD spectroscopy and size exclusion chromatography data do not support formation of stable dimers in the either the blue-absorbing 15ZPb dark state or the green-absorbing 15E

Pg photoproduct state of Tlr0924Δ. Analysis of variants containing site-specific mutations

reveals that proper signal transmission requires both chromophorylation of the GAF domain and individual residues within the amphipathic linker region between GAF and GGDEF domains. Based on these data, we propose a model in which bilin binding and light signals are propagated from the GAF domain via the linker region to alter the equilibrium and interconversion dynamics between active and inactive conformations of the GGDEF domain to favor or disfavor formation of catalytic competent dimers.


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INTRODUCTION Photosensory proteins use light energy to trigger adaptive responses to light quality, direction, duration and intensity and are found in abundance in photosynthetic organisms. Cyanobacteria possess a large complement of photosensory proteins that use flavins, retinals, linear tetrapyrroles (bilins), and other chromophores.1, 2 Bilin-based photoreceptors of the phytochrome superfamily are the most spectrally diverse, providing full spectral coverage throughout the photosynthetically active spectral range.3-5 Members of this family share a conserved bilinbinding cGMP-specific phosphodiesterases/adenylyl cyclases/FhlA (GAF)1 domain and exhibit reversible, photochromic conversion between two photostates.6 Photoconversion is triggered by primary photoisomerization of the 15,16-double bond of the bilin chromophore upon light absorption. The GAF domain of phytochromes is part of a larger photosensory core module that includes a phytochrome-specific PHY domain.7,

8

In distantly related cyanobacteriochrome

(CBCR) photoreceptors, GAF domains alone are sufficient for chromophore binding, autocatalytic covalent ligation to the chromophore, and reversible photoconversion.6,

9

The

phytochrome and CBCR photosensory domains are associated with diverse C-terminal domains, including bacterial signaling modules such as two-component histidine kinases, methylaccepting chemotaxis proteins, and catalytic domains of enzymes associated with the synthesis or degradation of second messengers such as cyclic-di-GMP (c-di-GMP), cAMP or cGMP. CBCRs are promising tools for optogenetics and synthetic biology due to their flexibility and simplicity,10-13 with a two-domain construct featuring a light based input:output module being ideal to regulate a variety of circuits. The modular nature of CBCR GAF domains also holds the promise of generating a range of light responses for a given biological output, facilitating development of orthogonal multichannel systems. The diversity of CBCR


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photocycles is well documented, and tuning mechanisms underlying much of this diversity have been elucidated.14-24 However, the molecular mechanisms of signal propagation from GAF input domains to signaling output domains remains poorly understood. To date, only a few crystal structures have been solved for CBCR domains;25,

26

two-

domain structures are not yet available. Moreover, crystal structures of isolated fragments may not elucidate the structural changes that accompany photoactivation due to crystal lattice constraints and due to truncation necessary to yield diffractable crystals in one or both states. Solution NMR studies on CBCRs avoid the need for high-quality crystals and do not suffer from lattice constraints but are limited to single-domain constructs. NMR data for both photostates of CBCR GAF domains from two chemotaxis proteins, TePixJ from Thermosynechococcus elongatus and NpF2164g3 from Nostoc punctiforme, have both revealed light-induced changes of secondary structural elements.27,

28

Both CBCRs also exhibit light-induced changes in the

length of their C-terminal alpha helices, which has been proposed to facilitate dimerization of the C-terminally adjacent domain and signal activation.28 However, other light-induced changes were also detected for TePixJ and NpF2164g3, so it is unclear whether helix extension is a conserved signaling mechanism even in CBCRs with methyl chemotaxis output domains. Further work on this problem would require in vivo phototaxis complementation studies that are very difficult to perform.29 Development of general, flexible, and robust synthetic systems based on CBCRs would be aided by a detailed understanding of the molecular mechanisms coupling photoconversion of the photosensory input domain to modulation of the activity of the signaling output domain. In the present study, we use the CBCR Tlr0924 (SesA) from T. elongatus to probe signal propagation from its photoreceptor domain to its GGDEF output domain, whose diguanylate


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cyclase activity can be easily assessed in vitro and in vivo. A member of the DXCF subfamily of CBCRs, Tlr0924 exhibits a blue/green (B/G) photocycle for both its isolated CBCR GAF domain and a C-terminal GAF-GGDEF fragment (previously described as GAF-GGDEF Tlr0924,17 hereafter Tlr0924∆; Figure S1A) that are indistinguishable from that of full-length Tlr0924.14, 30 Tlr0924 is one of three CBCRs that modulate levels of c-di-GMP in T. elongatus and related Thermosynechococcus strains, and light-regulated synthesis of c-di-GMP has been demonstrated for the full-length protein in vitro.31,

32

Light-based regulation of GGDEF and c-di-GMP

phosphodiesterase (PDE) domains have also been described in larger phytochrome proteins as well as with blue light responsive LOV and BLUF domains, yet the characterization of a small CBCR-GGDEF light regulated module is of further interest for optogenetic applications33-38. We leverage these findings to develop Tlr0924∆ as a system for studying signal propagation in CBCRs. Our studies establish that light-regulated c-di-GMP synthesis by Tlr0924∆ is modulated by a flexible linker region between the two domains without formation of a stable dimer. These studies implicate the linker region as a key point for engineering CBCR-based systems for lightbased control of biological phenomena. MATERIALS AND METHODS Protein expression and spectral analysis: Tlr0924 was expressed as an intein-CBD fusion protein in Escherichia coli engineered to produce PCB using a dual plasmid system as described previously.30,

39

Protein was purified on a chitin column (NEB) in accordance with the

manufacturer’s directions. Absorption spectra for Tlr0924 and variants were acquired as described previously.30 For CD spectra, 100 µl Tlr0924 (A434 = 0.19 for the B-absorbing 15Z Pb state; ε434 for this preparation was estimated at 27,500 M-1 cm-1) was diluted to 1 ml with HPLCgrade water. Absorption spectra were taken to monitor photoconversion, and 300 µl aliquots


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were transferred to a short-path cuvette (Hellma 100-1-40) for acquisition of CD spectra (185320 nm) in an Applied Photophysics CD spectrometer. Duplicate spectra were averaged for each photostate and for a water/buffer blank to allow calculation of CD spectra for each photostate, but smoothing was not required under these conditions. For analysis of changes upon photoconversion, difference CD spectra were calculated as (15E – 15Z) to examine formation of secondary structure in the photoproduct and were visually compared to standard concentrationnormalized reference spectra for secondary structure types and to difference spectra manually calculated from the reference spectra. For comparison of one-domain and two-domain constructs, GAF-only and GGDEF-only CD spectra were approximately normalized for molar concentration using absorbance on the aromatic amino acid band and calculated extinction coefficients. We then calculated the sum of the resulting spectra in each photostate. Difference spectra were then calculated for (GAF+GGDEF) – Tlr0924∆ and were compared to the reference spectra and calculated difference spectra for changes in secondary structure. Variants of Tlr0924 were constructed via site-directed mutagenesis using QuikChange (Stratagene) and were expressed and purified as for wild-type Tlr0924 constructs. In vitro diguanylate cyclase (DGC) assay: The BCA assay (Pierce) was used to determine protein concentration. DGC assay buffer (0.5 M Tris-HCl [pH 7.6], 0.05 M NaCl, 0.01 M MgCl2, 0.5 mM EDTA) was prepared as described previously.40 Purified protein was added to DGC assay buffer to a final concentration of 5 µM and incubated for 15 min at the desired temperature under continuous exposure to saturating G or B light. Light conditions were achieved using LEE filters (#071 Tokyo Blue and #090 Dark Yellow Green, respectively) and a light fluence rate of 4-5 µmol m-2 s-1. Photoconversion was assessed by absorption spectroscopy prior to initiation of assay. The desired concentration of GTP (Thermo Fisher Scientific) was


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then added to initiate the reaction. Assays were terminated by immediately boiling for 5 min followed by centrifugation at 15,000 x g for 5 min. Boiling the reaction mixture did not affect cdi-GMP stability or levels. A 15 µl aliquot of supernatant was injected into an Agilent 1100 series HPLC and passed through a Phenomenex® Synergi 4 µm Fusion-RP 80 Å column and Security Guard Cartridge (C18, 4 x 2 mm). Column temperature was 40°C. HPLC conditions consisted of Buffer A: 10 mM NaOAc, 1% (v/v) CH3COOH; Buffer B: Acetonitrile, 1% (v/v) CH3COOH. Injection loop was 50 µl and flow rate was 0.6 ml/min. Gradient elution conditions consisted of the following: 100% A was used from 0-2 min, followed by a linear gradient from 100% A to 80% A: 20% B from 2-15 min. This was followed by 100% Buffer A from 15-23 min to flush the column. The internal standard of c-di-GMP (BioLog) eluted at 10.1 min. A standard curve calculation of c-di-GMP levels was constructed using area under peak values correlated with c-di-GMP concentrations of 0.5 to 400 µM. All area calculations were performed using Agilent ChemStation software. All initial rate measurements of Tlr0924, terminated at 15 min, were performed in triplicate. HPLC-SEC and SEC-MALS: The buffer used for HPLC-SEC assays was 40 mM Tris-acetate (pH 8.3), 1 mM EDTA, 150 mM NaCl. Run time was 40 min with an isocratic flow rate of 0.6 mL/min, using a ThermoScientific Ultimate 3000 HPLC and a GE Superdex™ 200 Increase 10/300 GL column. Purified Tlr0924 and variants (10 µL, 2.5 mg/ml) in either photostate were injected in a 25 µl loop. To test the effect of GTP on the elution behavior, the GTP analog GTPγS (100 µM final concentration) was added to the protein mixture prior to injection along with MgCl2 (0.01 M final concentration). Elution traces and peaks were analyzed using Chromeleon 7 software (Dionex). All runs were performed in dim light to avoid photoconversion. Dark reversion of Tlr0924 is known to take days,17 so the


15E

Pg state is stable

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over the timescale of this experiment. For SEC-MALS, runs were performed on an Agilent 1260 HPLC system equipped with a miniDawn TREOS multi-angle light scattering device and OptiLab t-rEX differential refractive index components (Wyatt Technology Corp.). Run duration was 60 min at 0.5 ml/min using SEC-HPLC buffer and sample preparation as described above. All SEC-MALS calculations were performed using ASTRA software (Wyatt Tech. Corp). Cell aggregation assays: Tlr0924 and variants were expressed in E. coli as described above, with samples continuously exposed to either B or G light after induction with arabinose and IPTG. To generate B or G light simultaneously in an illuminated culture shaker, white fluorescent light was filtered using LEE light filters (#071 Tokyo Blue and #090 Dark Yellow Green, respectively) wrapped around Pyrex 9820 glass culture tubes. The light fluence rate in the culture shaker was 4-5 µmol m-2 s-1. After 16 h incubation, cell cultures were immediately placed in a dark room without shaking. The initial OD600 was measured by pipetting 1 mL from the upper meniscus of the culture tube. Cultures were then allowed to sit in darkness for 15 min, after which 1 ml was again collected from the upper meniscus of the culture tube. The degree of aggregation was calculated as final OD600/initial OD600 (i.e., the ratio of O.D. loss) for each culture. Software predictions: Secondary structure predictions were performed using Phyre2 (http://www.sbg.bio.ic.ac.uk/).

Helical

predictions

were

performed

using

HeliQuest

(http://heliquest.ipmc.cnrs.fr). Alignment parameters: CBCR-GGDEF containing proteins were manually truncated to include only annotated C-terminal GAF and GGDEF domains. Resultant sequences were then aligned using MUSCLE (default settings) and viewed in JalView (jalview.org).


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RESULTS Tlr0924Δ DGC activity is regulated by blue and green light. Tlr0924Δ possesses a B/G photocycle that is indistinguishable from that of full length Tlr0924 (Figure 1), but can be obtained as purified holoprotein with much better yield. Interestingly, induction of Tlr0924∆ in E. coli produced a phenotype consistent with light-regulated DGC activity: cultures grown under B light filters exhibited a pronounced film of cells on the culture flask and cells settled more rapidly under B vs G light illumination (Figure 2A). Such films are a known response to c-diGMP in E. coli,41 suggesting that c-di-GMP production in the B-illuminated cultures triggered cell aggregation. To document this phenomenon, the optical density (O.D. 600 nm) of each culture was determined before and after a 15 min settling period. These measurements confirmed enhanced aggregation of the B-treated cultures containing the

15E

Pg form

relative to those of the G-treated cultures containing 15ZPb (Figure 2B).

DGC activity of purified Tlr0924∆ was next examined in vitro using an HPLC assay to measure c-di-GMP production (Figure 3A). Tlr0924Δ was incubated in assay buffer under B or G light, and GTP-dependent production of c-di-GMP was determined as a function of incubation time (Figure 3B). The rate of c-di-GMP production was larger for

15E

Pg than for

15Z

Pb, with

approximately linear product formation for the first 15 min with c-di-GMP formation


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approaching a maximum after approximately 40 min (Figure 3B). By contrast, no detectable product was observed when ATP was used instead of GTP (data not shown).

We used initial rate measurements of Tlr0924Δ activity to examine the kinetic basis for light activation. Data were analyzed using the Michaelis-Menten kinetics model. Light activation


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of Tlr0924Δ corresponded to an approximately nine-fold increase in Vmax and kcat values in the 15E

Pg state relative to the 15ZPb state (Figure 3C). Km (GTP) values were not significantly affected

by light (Table 1). A Km (GTP) of 13-14 µM was estimated using the Michaelis-Menten model. The diguanylate cyclase activity of Tlr0924∆ was lower in the

15E

Pg (~85% reduction) than that

observed with the full length protein, and had lower fold activation between photostates.31 This result supports the interpretation that the extra N-terminal CBS and PAS domains of full length Tlr0924 (Figure S1A) enhance catalytic activity possibly by favoring formation of homodimers. TABLE 1. Kinetic analysis of wild-type Tlr0924Δ Tlr0924Δ

Pg

Pb

Vmax (µmol/ s-1)

0.009 ± 0.0003

0.001 ± 0.0004

Km (µM GTP)

14.6 ± 3.0 (0.98)

13.1 ± 3.0 (0.98)

kcat (sec-1)

0.002

0.0002

kcat/Km (µmol/ sec-1)

0.0002

0.00002

Data was obtained under standard assay conditions at 37°C with 5 µM enzyme as described under Materials and Methods. R2 values for Km measurements are indicated in parentheses. We next used site-directed mutagenesis to examine several aspects of Tlr0924∆ function. We first examined the roles of the GGDEF active site and of chromophorylation. The E263A variant, lacking a conserved residue in the GGDEF active site, retained normal photoconversion (Figure S1B) but produced no c-di-GMP in either photostate (Figure 4A). By contrast, the C103A variant ablated chromophorylation and photoconversion (Figure S1C). This chromophore deficient variant also exhibited approximately half the DGC activity of the activated photoproduct state 15E(Pg) of wildtype Tlr0924Δ, and this activity did not vary upon exposure of B or G light as expected (Figure 4A).


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0.012 0.010

15E

0.008

15Z

200

A

Pg

Pb

c-di-GMP (uM)

µmol c-di-GMP sec -1

0.006 0.004 0.002 0

R252K/D255I D255I R252K wildtype

150

B

100 50

†GGDEF

E179A

E179R

P156A

L177E

L177A

L177I

L177P

(Δ L177)

L177 +AA

L177 +A

E263A

C103A

0

Tlr0924 WT

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0

50

100

150

200

250

time (min)

Figure 4. Comparative DGC activities of wild-type and variants of Tlr0924∆. (A) DGC activity of variants of Tlr0924∆ as compared to wild-type Tlr0924∆. GGDEF denotes GGDEF-only truncation. Total protein concentration for each construct is 5 μM. (B) Time course DGC plot comparing activity of mutations in the inhibition site (RxxD motif) as compared to the wild-type protein.

Many DGC domains exhibit product inhibition due to the presence of a conserved RXXD motif that comprises a c-di-GMP binding site.42 Tlr0924∆ possesses such a motif, so it seemed plausible that the loss of linearity after 15 min could arise due to such product inhibition. R252K and D255I variants were constructed based on the presence of equivalent substitutions in naturally occurring GGDEF domains that display no product inhibition.43, 44 Wildtype Tlr0924Δ yielded cdi-GMP product levels that reached a maximum near 60 min and remained unchanged up to 240 min under standard assay conditions (see above). In contrast, c-di-GMP product continued to increase after 60 min in R252K, D255I, and R252K/D255I variants (Figure 4B). The double R252K/D255I variant produced the most c-di-GMP over the 240-minute assay, accumulating nearly 10.5x the amount made by wildtype Tlr0924∆. All variants exhibited increasing c-diGMP production for at least 240 min in the

15E

Pg state, while product formation for R252K/D255I

and D255I variants was non-linear after 120 minutes. This data clearly implicates the presence of auto-inhibition in wild-type Tlr0924∆.


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Tlr0924 and its orthologs mediate c-di-GMP formation in cyanobacteria of the thermophilic genus Thermosynechococcus,31, 32 so we examined the properties of Tlr0924∆ as a function of temperature from 25°C to 55°C. DGC activity of Tlr0924Δ was robust, with specific effects on Vmax: activity at 55°C was nearly twice that measured at 25°C, and Vmax was 1.5-fold higher at 55°C than at 37°C. Temperature did not drastically affect Km (GTP) (Table 2). Overall, our data demonstrate clear B-activated and G-repressed DGC activity for the truncated bidomain construct Tlr0924∆ making this a good model system for examining light signal propagation for a CBCR-regulated diguanylate cyclase. TABLE 2. Temperature dependence of catalytic activity of wild-type Tlr0924Δ Tlr0924Δ

25°C

37°C

45°C

55°C

Vmax (µmol/ sec-1)

0.006 ± 0.0001

0.009 ± 0.0003

0.011 ± 0.0002

0.013 ± 0.020

Km (µM GTP)

13.0 ± 1.8 (0.99)

14.6 ± 3.0 (0.98)

15.3 ± 2.0 (0.99)

11.2 ± 2.2 (0.99)

kcat (sec-1)

0.001

0.002

0.002

0.003

kcat/Km (µmol/ sec-1)

0.0001

0.0002

0.0007

0.0010

Data was obtained under standard assay conditions with 5 µM enzyme at different temperatures as described under Materials and Methods. R2 values for Km measurements are indicated in parentheses

Changes in secondary structure upon light activation of Tlr0924Δ. One hypothesis for signal propagation in CBCRs entails light-dependent formation of a linker helix connecting the CBCR domain and its C-terminal neighbor.28 Helix formation then facilitates formation of a stable, signal-activated dimer. To test this model, we used CD spectroscopy to examine possible changes in helical content of Tlr0924Δ upon light activation. We first performed CD measurements on isolated GAF and GGDEF domains in the UVC region. As expected, the CD


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spectra of the GGDEF domain alone did not exhibit significant changes upon light treatment (Figure 5A). By contrast, light-induced changes in the isolated Tlr0924 CBCR domain were detected by CD (Figure 5B). While subtle, the observed CD differences upon photoconversion were more consistent with a strand-to-helix transition than a coil-to-helix transition (helix minus sheet vs. helix minus coil, Figure 5C). To interpret these results, we leveraged

published

solution

NMR

structures for TePixJ26 and solution NMR secondary

structure

assignments

for

NpF2164g3 in both photostates45 as models for changes in secondary structure that might occur in Tlr0924 upon photoconversion

(Figure

S2).

These

predictions were based on the assumption that light-induced changes in the CBCR domain of Tlr0924 would mirror those of the more closely related CBCR. For example, changes in the DXCF motif of TePixJ are assumed to be present in Tlr0924, while changes in the equivalent motif of NpF2164g3 are assumed to be absent (Figure S2); by contrast, changes in the C-terminal helix of Tlr0924 were modeled on changes in secondary structure of NpF2164g3.28. Based on these considerations, the observed CD spectral


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Biochemistry

measurements are consistent with a small light-dependent transition from β-strand to α-helix in the CBCR domain of Tlr0924 similar to that of TePixJ (Figure S2). Similar CD changes were observed upon photoconversion of Tlr0924∆ (Figure 6A), but the observed changes were even smaller than those of the isolated CBCR domain. Unfortunately, higher concentrations of Tlr0924∆ could not be used to enhance the signal-to-noise due to increased absorption at short wavelengths. We thus were not able to assign the Tlr0924∆ difference spectrum for photoconversion to a clear structural transition (Figure S3A) or secondary structural element (Figure S3B). However, the CD spectrum of Tlr0924∆ was not equivalent to the sum of the independent spectra for its constituent CBCR and GGDEF domains after correction for differences in concentration (Figure 6B, Figure S3C). We therefore derived a (calculated – observed) difference spectrum for each photostate to examine this discrepancy further. In both photostates, the difference spectra were in good agreement with the expected difference spectra for (helix – coil); that is, the experimental spectra contained more random coil and less α-helix than was expected (Figure 6C, Figure Figure 6. Characterization of Tlr0924∆ using CD spectroscopy. (A) CD spectra are shown for Tlr0924∆ in the B-absorbing 15Z dark state (blue trace) and G-absorbing 15E photoproduct (orange trace). (B) Concentrationcorrected CD spectra are shown for the GGDEF domain (rose trace) and CBCR domain (15Z dark state, dark blue trace). These spectra were used to calculate a predicted spectrum for Tlr0924∆ in the dark state, which did not match the observed spectrum. The residual difference, calculated as (calculated – observed), is shown in brick red. (C) The difference spectrum from panel B is compared to the indicated structural transitions. (D) The difference spectrum from panel B is compared to the indicated reference spectra for secondary structure elements.


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S3D). In view of the gap of 9 amino acids between the two one-domain constructs, it is conceivable that the presence of these residues would result in additional random coil. However, were that to be the only cause of this discrepancy, one would expect the CD spectra to report the presence of additional coil rather than a transition from helix to coil, and the observed discrepancy does not match this assignment (Figure 6D, Figure S3E). These results support the conclusion that the Tlr0924∆'s CBCR domain undergoes light-induced structural changes more similar to those of TePixJ than the formation of a linker helix observed in NpF2164g3. Photoactivation of Tlr0924∆ does not trigger stable dimerization. CD spectroscopy does not typically report changes in oligomerization state, the second stage in the proposed model for CBCR signal propagation.28 We therefore used size exclusion chromatography (SEC) to examine conformational heterogeneity in Tlr0924∆ upon photoactivation. Indeed, the elution profiles revealed dual conformers in both photostates with an injected sample concentration of 66 µM. The

15Z

Pb state possessed a

major species eluting at 23 min with a minor species eluting at 25 min, whereas the main peak of the

15E

Pg lit

state eluted at 25 min with a smaller peak eluting at 23 min (Figure 7A). Molecular

weight

calibration

indicated that the 23 min peak corresponded

to

an

apparent

molecular weight of 55 kDa, slightly larger than the expected MW of 40.6


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kDa, while the 25 min peak corresponded to an apparent molecular weight of 40 kDa (Figure S4A). Elution profiles for both photostates were also not significantly altered at a higher Tlr0924Δ protein concentration, i.e. 200 µM (Figure S4B). Since both estimates are smaller than that of a dimer, we interpret this data as indicating that photoactivation favors a more compact 'active' monomeric conformer. Moreover, addition of GTPγS did not change the elution behavior of both photostates of Tlr0924Δ (Figure S4C) and the catalytically inactive E263A active site variant elution behavior was similar to that of the wild type (Figure S4D). Additional analyses were performed using SEC-MALS-HPLC to more accurately estimate the molecular masses of the two species. Light scattering behavior of the major earlyeluting peak of Tlr0924Δ Pb dark state was consistent with the expected molecular weight of the Tlr0924∆ monomer (Figure S5A). However, we were neither able to estimate the masses of the late eluting Pb shoulder, nor those of the two peaks associated with the Pg lit state (Figure S5B). Molecular mass estimates for the two Pg conformers were not constant across both peaks and the early eluting conformer yielded a lower mass estimate than the late eluting, presumably more compact conformer (Figure S5B). Unfortunately, this problem could not be overcome by varying the ionic strength of the elution buffer in an attempt to better resolve the two peaks. Due to spectral overlap with the Pg state, the light scattering laser (658 nm) can generate a dynamic equilibrium between Pg and Pb states,30 which complicates MALS measurements of this species.

Mutagenesis of the linker region between GAF and GGDEF domains reveals a critical role in signal transfer. Algorithms for de novo analysis of secondary structure predict that the linker region between the GAF and GGDEF domain of Tlr0924 is likely to form a single α-helix, yet CD spectroscopy suggests that this is not the case. Changes in this region are potentially well


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positioned to modulate catalytic activity of the dimeric GGDEF active site,42, 46 so we performed site-directed and deletion mutagenesis experiments to test its role in signal propagation. We first examined DGC activity of the GGDEF-only variant (Figure S1A). In contrast to the nonchromophorylated “half-active” C103A variant of Tlr0924∆, the isolated GGDEF domain was catalytically inactive (Figure 4A). This suggested that the linker region and/or the CBCR domain are critical for diguanylate cyclase activity. Helical wheel projection identified an amphipathic surface adjacent to the N-terminus of the GGDEF domain of Tlr0924Δ (Figure S6A). This prediction raised the possibility that transient helix formation in this region, with concomitant transient dimerization, might still account for signal propagation. Were this the case, mutagenesis of residues on the hypothetical amphipathic surface could have different effects depending on the side of the surface, as observed in an artificial light-regulated histidine kinase system.46 To test this hypothesis, we began by targeting a conserved leucine residue (L177) on the hydrophobic surface of this amphipathic region. This leucine is conserved among putative CBCR-GGDEF proteins across cyanobacterial families (Figure S6B). Several variants incorporating substitutions for this residue (L177A, L177E, L177I, L177P, L177K) were characterized. All of these exhibited normal photoconversion (Figure S7), but drastically reduced DGC activities were seen in both photostates for all L177 variants (Figure 4A). Removal of Leu177 (ΔL177) or addition of one (L177+A) or two (L177+AA) Ala residues also nearly abolished DGC activity regardless of photostate (Figure 4A). By contrast, variant proteins incorporating substitutions for the conserved charged residue Glu179 on the hydrophilic face of the predicted amphipathic helix, did not ablate DGC activity (Figure S6B). The E179A variant exhibited normal activity in the activated photoproduct state yet higher basal activity in the dark state, whereas the


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E179R variant exhibited both higher basal activity yet lower lit state activity. A variant protein incorporating a P156A substitution N-terminal to the amphipathic region also exhibited drastically reduced DGC activity in both photostates (Figure 4A). We also examined the SEC-HPLC elution behavior of Tlr0924Δ variants. The C103A variant that lacks bound chromophore eluted as a broad peak between 23 and 25 min (Figure 7B). By comparison, the catalytically inactive P156A variant displayed earlier eluting peaks for both photostates; unlike wild type, the P156A photoproduct eluted slightly earlier than the dark state (Figure S4E). Substitutions for Leu177 resulted in a range of elution behaviors (Figure S4FI and 8C&D). Despite its lack of DGC activity, the elution behavior of Pb and Pg states of the L177I variant were similar to those of wild type. By contrast, L177A also exhibited increased elution time upon photoactivation, with the dark-adapted state's later elution time suggesting that its solution conformation differs from that of wild type (Figure 7C). Similar to the P156A variant, L177E, L177K, and L177P variants all eluted as single peaks regardless of photostate, as did the variant lacking Leu177 and variants with one or two added Ala residues (Figure 7D, Figure S4JL). With the exception of an increased proportion of the early-eluting species in both photostates, the catalytically active E179A variant retained elution behavior largely similar to wild type (Figure S4K). SEC-MALS-HPLC analysis of the Pg state of L177E variant was also consistent with a monomer of ~40 kDa (Figure S5C). As controls, we also examined the SEC-HPLC behavior of single domain GAF and GGDEF constructs. The isolated GAF domain did not exhibit changes in retention time upon photoconversion, despite the observed changes in secondary structure. Similarly, the construct lacking the photoactive GAF domain eluted as a light-insensitive monomer, in keeping with the loss of DGC activity (Figure S7). Taken together, these studies implicate the role of both GAF


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and linker regions for supporting DGC activity and indicate that both are needed for lightinduced changes in hydrodynamic properties.

DISCUSSION Our studies document the bidomain construct Tlr0924∆ to be a robust light-regulated DGC both in vivo and in vitro. Despite the dimeric nature of the DGC reaction, we find no evidence for stable dimerization of Tlr0924∆ in either the less active B-absorbing dark state or the activated G-absorbing photoproduct. It is thus clear that photoactivation facilitates formation of transient 'catalytically active' dimers, illustrating that signal propagation between domains is not a simple binary switch. Instead, activation is a quantitative process: photoproduct formation results in improved 'productive' dimerization in the presence of Mg-GTP (inferred from the higher turnover of the enzyme in the photoproduct state). Our studies show that dimerization is too transient to be observed by SEC or SEC-MALS assays in the absence of substrate. We cannot rule out that substrate binding might improve dimer formation, although we see no evidence of dimerization when the substrate analog GTP-γ-S was added to the protein sample and Mg-GTP substrate affinities are identical for both photostates. Taken together, our measurements support the conclusion that 'productive' dimer formation is enhanced by conversion to the lit state. Our studies also reveal a measurable change in hydrodynamic properties of Tlr0924Δ upon photoactivation which alters the equilibrium between a more flexible, early eluting monomer and a more compact, late eluting monomer (Figure 7A). Moreover, the C103A variant of Tlr0924∆ exhibits half-maximal DGC activity of the 15EPg state, and this activity did not vary upon exposure to either B or G light (Figure 4A). This variant cannot undergo covalent chromophorylation (Figure S1C), so it mimics the properties of the Tlr0924∆ apoprotein. We


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thus hypothesize that apo-Tlr0924∆ can readily sample both catalytically active and inactive conformations accounting for its intermediate activity between the dark and lit states (Figure 8A). We envisage that chromophore binding leads to formation of the 15Z B-absorbing dark state of the CBCR domain, which disfavors the active conformation of the DGC domain. Photoconversion shifts the equilibrium, favoring the active conformation that facilitates dimer formation. The absence of a stable dimer in Tlr0924∆ is at odds with the proposed general model for CBCR signaling proposed based on solution NMR studies of NpF2164g3,45 in which photoconversion was predicted to lead to formation of a single, stable linker helix that could stabilize dimer formation. However, characterization of one-domain constructs using CD spectroscopy suggests that the expected structural changes are taking place within the CBCR domain of Tlr0924∆ as reported for TePixJ.26 The isolated GGDEF domain exhibited no lightdependent structural changes, as expected, whereas the isolated CBCR domain exhibited the expected net change from β-strand to α-helix (see above). Therefore, it seems that the behavior of individual constituent domains of Tlr0924∆ are consistent with the proposed model. However, CD spectra for the two-domain Tlr0924∆ construct could not be explained as a simple sum of the two domains. Of course, nine residues in the linker region are not accounted for by the one-domain constructs. However, the discrepancy between the one-domain spectra and the two-domain spectra cannot be explained by these nine residues and the proposed model: were one to have a single linker helix in the Tlr0924∆ photoproduct state, these residues would provide extra α-helix content in that state and the difference spectrum between calculated and observed spectra would match that of α-helix. Instead, the (calculated – observed) difference spectrum is equivalent to (α-helix – random coil) for both photostates (Figure 5C-D and Figure


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S3D-E). Therefore, Tlr0924∆ actually has less α-helix and more random coil than expected based on the isolated CBCR and GGDEF domains, and this behavior is not changed by photoconversion. These experiments thus indicate that light-dependent formation of a single, long linker helix connecting the two domains of Tlr0924∆ does not occur.45 At first glance, these data seem to conflict with analysis of variant proteins containing substitutions in the linker region, which are readily interpreted in terms of a proposed amphipathic helix. Leu177 lies on the hydrophobic surface that would be expected to act as the dimerization interface, is essential for activity, and is essential for normal changes in hydrodynamic properties. Glu179, on the hydrophilic surface, is not essential for either activity or changes in hydrodynamic properties. Insertions or deletions at Leu177 disrupt activity, as would be expected for a helical linker region that undergoes coiled-coil oligomerization.42, 46-48 We believe this paradox can best be reconciled by assuming that helix formation occurs concomitant with formation of the transient dimeric catalytic complex (Figure 8B).

A

apoinact

apoact

PCB

PCB

PBinact

15Z

PGinact

15E

15Z

PBact

B hν G hν 15E

PGact

B 2 GTP

B hν G hν

cyc-di-GMP GG

incompetent monomer

competent monomer

transient active dimer

Figure 8. Signal propagation in Tlr0924∆. (A) Scheme for regulation of DGC activity by chromophorylation and photoconversion. (B) Scheme for regulation of DGC activity by the formation of transient active dimers upon photoconversion in the presence of substrate.


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In this view, photoconversion would alter the inter-domain configuration of Tlr0924∆ to facilitate formation of the linker helix, but the helical propensity of this region is apparently not sufficient to trigger helix formation and dimerization (at least in the absence of substrate). The observed changes in hydrodynamic properties of Tlr0924∆ upon photoconversion would indicate a more compact structure in the photoproduct, which could be further altered upon GTP binding, with the combination of photoconversion and substrate binding leading to transient dimerization. Catalysis would then lead to formation of c-di-GMP and breakdown of the transient dimer (Figure 8B). Although this model is clearly not yet proven, it is consistent with currently available data and can be tested using fluorescence assays suitable for detecting transient dimer formation in real time. Transient formation of a coiled-coil helical interface would also explain the surprising loss of function in the L177I variant, because these two residues are known to favor different oligomerization modes in coiled-coil interfaces.49 In conclusion, our studies also validate Tlr0924∆ as a useful system for developing CBCRs as tools for synthetic biology. Tlr0924∆ is notably smaller than full-length Tlr0924 (351 residues rather than 773) while retaining an identical B/G photocycle and similar light-regulated DGC activity. Moreover, light-regulated DGC activity of Tlr0924 can be scored in vitro, in vivo, and in vivo in heterologous systems such as E. coli. Although the specific activity of Tlr0924∆ is lower than that reported for full-length Tlr0924,31, 32 it is clear that Tlr0924∆ retains sufficient light regulation of DGC activity for effective light-regulated control of cell-cell adhesion in E. coli (Figure 2), even in the presence of the auto-inhibitory RXXD site. The presence of this autoinhibitory site also underscores the importance of a tightly regulated c-di-GMP network in T. elongatus. Indeed, Tlr0924 (TeSesA) previously was shown to work in concert with two other CBCRs, TeSesB and TeSesC, to regulate light-dependent cell aggregation in T. elongatus.32 The


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ability of Tlr0924 to hijack and induce native c-di-GMP dependent responses in E. coli demonstrates the potential for Tlr0924Δ as a small, streamlined module for in vivo synthetic biology applications in the absence of these other proteins. As c-di-GMP has been shown to bind to specific transcription factors and riboswitches to regulate downstream genes of interest,36, 50 we believe that Tlr0924's B/G DGC activity could be utilized in an optogenetic fashion; hence, molecular engineering of Tlr0924Δ should prove exceedingly useful for applications in any oxygenic photosynthetic species that can synthesize PCB. It is also possible to introduce this pathway into non-photosynthetic cells, allowing light-regulated c-di-GMP synthesis as a tool for studying immune responses to this bacterial second messenger.51-55 As more CBCR-GGDEF and CBCR-EAL domains are investigated, a suite of light-dependent DGCs and diguanylate phosphodiesterases could ultimately be developed to regulate synthesis and degradation of c-diGMP in live cells with different colors of light. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs. biochem.xxxxxx. AUTHOR INFORMATION Corresponding Author. Department of Molecular and Cellular Biology, University of California, Davis, CA 95616. Tel: 530-752-1865. E-mail: [email protected]. ORCID. John Clark Lagarias: 0000-0002-2093-0403 Author Contributions. M.B-H., N.C.R. and J.C.L. designed the experiments. M.B-H., N.C.R. performed the experiments and data analysis, and M.B-H. and J.C.L. wrote the manuscript.


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Funding. This work was supported by a grant from the Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Energy Sciences, Office of Science, United States Department of Energy (DOE DE-FG02-09ER16117). M.B-H. was in part supported by Grant Number T32-GM07377 from NIGMS-NIH. The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views of the DOE, NIGMS or NIH. Notes. The authors declare no competing financial interest. ABBREVIATIONS: B, blue light; BCA, bicinchoninic acid; CBCR, cyanobacteriochrome; c-di-GMP, cyclic-diGMP; CD, circular dichroism; DGC, diguanylate cyclase; EAL, guanylate phosphodiesterase domain named after characteristic sequence motif; G, green light; GAF (cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA); GGDEF, diguanylate cyclase domain named after characteristic sequence motif; SEC, size exclusion chromatography; SEC-MALS, SEC with multi-angle light scattering; Tlr0924∆, C-terminal CBCR-GGDEF construction construct. ACKNOWLEDGEMENT We acknowledge the help of Shelley S. Martin for expert technical support.


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[36] Ryu, M.-H., Kang, I.-H., Nelson, M. D., Jensen, T. M., Lyuksyutova, A. I., Siltberg-Liberles, J., Raizen, D. M., and Gomelsky, M. (2014) Engineering adenylate cyclases regulated by near-infrared window light, Proc. Natl. Acad. Sci. USA 111, 10167-10172. [37] Glantz, S. T., Carpenter, E. J., Melkonian, M., Gardner, K. H., Boyden, E. S., Wong, G. K., and Chow, B. Y. (2016) Functional and topological diversity of LOV domain photoreceptors, Proc. Natl. Acad. Sci. USA 113, E1442-1451. [38] Herrou, J., and Crosson, S. (2011) Function, structure and mechanism of bacterial photosensory LOV proteins, Nature Rev. Microbiol. 9, 713-723. [39] Gambetta, G. A., and Lagarias, J. C. (2001) Genetic engineering of phytochrome biosynthesis in bacteria, Proc. Natl. Acad. Sci. USA 98, 10566-10571. [40] Ryjenkov, D. A., Tarutina, M., Moskvin, O. V., and Gomelsky, M. (2005) Cyclic diguanylate is a ubiquitous signaling molecule in bacteria: insights into biochemistry of the GGDEF protein domain, J. Bacter. 187, 1792-1798. [41] Weber, H., Pesavento, C., Possling, A., Tischendorf, G., and Hengge, R. (2006) Cyclic-di-GMPmediated signalling within the σS network of Escherichia coli, Mol. Microbiol. 62, 1014-1034. [42] Paul, R., Weiser, S., Amiot, N. C., Chan, C., Schirmer, T., Giese, B., and Jenal, U. (2004) Cell cycledependent dynamic localization of a bacterial response regulator with a novel di-guanylate cyclase output domain, Genes Dev. 18, 715-727. [43] Ulijasz, A. T., Cornilescu, G., von Stetten, D., Cornilescu, C., Velazquez Escobar, F., Zhang, J., Stankey, R. J., Rivera, M., Hildebrandt, P., and Vierstra, R. D. (2009) Cyanochromes are blue/green light photoreversible photoreceptors defined by a stable double cysteine linkage to a phycoviolobilintype chromophore, J. Biol. Chem. 284, 29757-29772. [44] Römling, U., Galperin, M. Y., and Gomelsky, M. (2013) Cyclic di-GMP: the first 25 years of a universal bacterial second messenger, Microbiol. Mol. Biol. Rev. 77, 1-52. [45] Lim, S., Rockwell, N. C., Martin, S. S., Lagarias, J. C., and Ames, J. B. (2014) 1H, 15N, and 13C chemical shift assignments of cyanobacteriochrome NpF2164g3 in the photoproduct state, Biomol. NMR Assign. 8, 259. [46] Möglich, A., Ayers, R. A., and Moffat, K. (2009) Design and signaling mechanism of light-regulated histidine kinases, J. Mol. Biol. 385, 1433-1444. [47] Paul, R., Abel, S., Wassmann, P., Beck, A., Heerklotz, H., and Jenal, U. (2007) Activation of the diguanylate cyclase PleD by phosphorylation-mediated dimerization, J. Biol. Chem. 282, 2917029177. [48] Gourinchas, G., Etzl, S., Gobl, C., Vide, U., Madl, T., and Winkler, A. (2017) Long-range allosteric signaling in red light-regulated diguanylyl cyclases, Science Advances 3, e1602498. [49] Harbury, P. B., Kim, P. S., and Alber, T. (1994) Crystal structure of an isoleucine-zipper trimer, Nature 371, 80-83. [50] Sudarsan, N., Lee, E., Weinberg, Z., Moy, R., Kim, J., Link, K., and Breaker, R. (2008) Riboswitches in eubacteria sense the second messenger cyclic di-GMP, Science 321, 411-413. [51] Burdette, D. L., Monroe, K. M., Sotelo-Troha, K., Iwig, J. S., Eckert, B., Hyodo, M., Hayakawa, Y., and Vance, R. E. (2011) STING is a direct innate immune sensor of cyclic di-GMP, Nature 478, 515518. [52] Huang, Y.-H., Liu, X.-Y., Du, X.-X., Jiang, Z.-F., and Su, X.-D. (2012) The structural basis for the sensing and binding of cyclic di-GMP by STING, Nat. Struct. Mol. Biol. 19, 728-730. [53] Yin, Q., Tian, Y., Kabaleeswaran, V., Jiang, X., Tu, D., Eck, M. J., Chen, Z. J., and Wu, H. (2012) Cyclic di-GMP sensing via the innate immune signaling protein STING, Mol. Cell 46, 735-745. [54] Shu, C., Yi, G., Watts, T., Kao, C. C., and Li, P. (2012) Structure of STING bound to cyclic di-GMP reveals the mechanism of cyclic dinucleotide recognition by the immune system, Nat. Struct. Mol. Biol. 19, 722-724.


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Biochemistry

[55] Chandra, D., Quispe-Tintaya, W., Jahangir, A., Asafu-Adjei, D., Ramos, I., Sintim, H. O., Zhou, J., Hayakawa, Y., Karaolis, D. K., and Gravekamp, C. (2014) STING ligand c-di-GMP improves cancer vaccination against metastatic breast cancer, Cancer Immunol. Res. 2, 901-910.


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Biochemistry

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FIGURE LEGENDS Figure 1. The B/G photocycle of Tlr0924∆. Absorption spectra are shown for the B-absorbing 15Z dark state (Pb, blue trace) and G-absorbing 15E photoproduct (Pg, green trace) of Tlr0924∆. Inset, a domain diagram is shown for Tlr0924∆. Figure 2. Tlr0924∆ exhibits diguanylate cyclase activity in vivo. (A) E. coli cell aggregation after overnight expression of Tlr0924Δ and Tlr0924Δ(E263A) under G or B light. (B) O.D. ratio of Tlr0924Δ and Tlr0924Δ(E263A) after overnight induction and light exposure. Figure 3. Biochemical characterization of DGC activity. (A) RP-HPLC chromatogram of 15E

Pg and 15ZPb state Tlr0924Δ DGC assay results in a large GTP peak and a smaller peak at 10.1

min corresponding to c-di-GMP (indicated with arrows). (B) Time course DGC assay for Tlr0924Δ is used to determine initial rate time conditions. (C) Initial rate kinetic curves for DGC activity of Tlr0924Δ in Pg and Pb photostates fit to Michaelis-Menten model. Figure 4. Comparative DGC activities of wild-type and variants of Tlr0924∆. (A) DGC activity of variants of Tlr0924∆ as compared to wild-type Tlr0924∆. GGDEF denotes GGDEFonly truncation. Total protein concentration for each construct is 5 µM. (B) Time course DGC plot comparing activity of mutations in the inhibition site (RxxD motif) as compared to the wildtype protein. Figure 5. Characterization of isolated CBCR and GGDEF domains using CD spectroscopy. (A) CD spectra are shown for the isolated GGDEF domain of Tlr0924 before (blue trace) and after (orange trace) illumination with 400 nm light. Red trace, difference CD calculated as (before – after). (B) CD spectra are shown for the isolated CBCR domain of Tlr0924 in the B-


30

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Biochemistry

absorbing 15Z dark state (blue trace) and G-absorbing 15E photoproduct (orange trace). (C) Difference CD for photoconversion of the CBCR domain (red trace) was calculated as (15E – 15Z) and is compared to the indicated structural transitions. Figure 6. Characterization of Tlr0924∆ using CD spectroscopy. (A) CD spectra are shown for Tlr0924∆ in the B-absorbing 15Z dark state (blue trace) and G-absorbing 15E photoproduct (orange trace). (B) Concentration-corrected CD spectra are shown for the GGDEF domain (rose trace) and CBCR domain (15Z dark state, dark blue trace). These spectra were used to calculate a predicted spectrum for Tlr0924∆ in the dark state, which did not match the observed spectrum. The residual difference, calculated as (calculated – observed), is shown in brick red. (C) The difference spectrum from panel B is compared to the indicated structural transitions. (D) The difference spectrum from panel B is compared to the indicated reference spectra for secondary structure elements. Figure 7. Characterization of Tlr0924∆ variants using SEC. Elution profile of (A) Tlr0924Δ wildtype, (B) Tlr0924Δ(L177A), (C) Tlr0924Δ(C103A), and (D) Tlr0924Δ(L177E). Figure 8. Signal propagation in Tlr0924∆. (A) Scheme for regulation of DGC activity by chromophorylation and photoconversion. (B) Scheme for regulation of DGC activity by the formation of transient active dimers upon photoconversion in the presence of substrate.


31

Biochemistry

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Page 32 of 40

2 GTP

B hν G hν

cyc-di-GMP GG

incompetent monomer

competent monomer



Page 33 of 40

0.25 absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Biochemistry

0.20

Tlr0924∆ P

b

GAF

Pg

GGDEF

Cys

0.15 0.10 0.05 0.00 350 400 450 500 550 600 650 wavelength (nm)


Biochemistry

A

wild type

E263A

100

B

80

% O.D. loss

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55

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60 40 20 0

wild type

E263A


Page 35 of 40

A

relative absorbance

Tlr0924Δ

Pg

Pb 4

6 8 time (min)

10

12

12

c-di-GMP (μM)

10 8 6 4 2 0

B 0

5 10 15 20 25 30 35 40 time (min)

0.01 c-di-GMP (μmol sec -1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Pg

0.008 0.006

C

0.004 0.002 0

Pb 0

500 1000 1500 2000 2500 GTP (μM)


Biochemistry

0.012 0.010

15E

0.008

15Z

200

A

Pg

c-di-GMP (uM)

Pb

0.006 0.004 0.002 †GGDEF

E179R

E179A

P156A

L177E

L177A

L177P

(Δ L177)

L177 +AA

L177 +A

E263A

C103A

L177I


R252K/D255I D255I R252K wildtype

150 100

B

50 0

0 Tlr0924 WT

µmol c-di-GMP sec -1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0

50

100

150

time (min)

200

250

1 mdeg (∆CD)

40 20

GGDEF (time 0) GGDEF (+ 400 nm hν)

0

A

CD (mdeg)

-20

CBCR (15Z) CBCR (15E)

20 0

B

-20

CD (arb. units)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

CD (mdeg)

Page 37 of 40

helix - coil helix - sheet coil - sheet obs. CBCR ∆CD (15E – 15Z)

200

220 240 wavelength (nm)

C 260


Biochemistry

CD (mdeg)

22 11

CD (arb. units) CD (arb. units)

Tlr0924∆ (15Z) Tlr0924∆ (15E)

0

-11

A CBCR (15Z) GGDEF (time 0) Tlr0924∆ (calc. - obs., 15Z)

B helix - coil helix - sheet coil - sheet Tlr0924∆ (calc. - obs., 15Z)

C CD (arb. units)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 40

α helix β strand random coil β turn Tlr0924∆ (calc. - obs., 15Z)

D

200 220 240 wavelength (nm)

260


Page 39 of 40

Pb

20

9 6

C

0 20

0 20

16 14 12 10 8 6 4 2 0 20

24 26 time (min)

B

28

30

60

C103A

50

22

24

26

28

30

24 26 time (min)

D

L177A

28

30

L177E

40 30 20

Pb

10 22

Pg

10 5

22

Pb

15

3

absorbance

absorbance

12

A

25

Pg Tlr0924Δ absorbance

15

absorbance

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Biochemistry

0 20

22

time (min)


Pg

24 26 time (min)

28

30

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

apoinact

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apoact

PCB

PCB

15Z

PBinact

PBact

15Z

B hν 15E

G hν

PGinact

15E

PGact

B 2 GTP

B hν G hν

cyc-di-GMP GG

incompetent monomer

competent monomer



Light-Regulated Synthesis of Cyclic-di-GMP by a Bidomain

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