Design of Fusion Proteins
YtvA comprises an N-terminal LOV sensor domain and a C-terminal STAS (sulfate transport antisigma factor antagonist) effector domain17
, joined by an α-helical linker sequence denoted Jα ()16
. In the dark, its LOV domain binds flavin mononucleotide (FMN) non-covalently. Blue light absorption promotes formation of a covalent bond between the flavin ring and the conserved cysteine 62 within the LOV domain18
. This light-activated state thermally decays to the ground, dark state with a time constant of roughly one hour in YtvA16
. Although the exact biochemical function of the STAS domain is not known, in response to blue light YtvA enhances transcription from promoters controlled by the general stress regulator σB 19
. The isolated LOV photosensor domain of YtvA adopts the common PAS domain fold and forms a tight dimer through a hydrophobic interface13
. In our crystal structure of YtvA, the Jα linker forms a C-terminal helix that extends from the core LOV domain. A similar quaternary structure arrangement was found for the heme-binding PAS domain of the oxygen sensor FixL from B. japonicum20,21
FixL is part of a two-component system that regulates the expression of proteins involved in microaerobic respiration, nitrate respiration and nitrogen fixation15
. Two PAS domains are linked to a histidine kinase which comprises phosphoacceptor (DHp) and catalytic (CA) subdomains (). In a two-step reaction, FixL first undergoes autophosphorylation at its conserved histidine 291 in the DHp subdomain and then transfers the phosphate moiety to its cognate, non-covalently-bound, response regulator FixJ14,22
. As in other two-component kinases, FixL also exhibits phosphatase activity23
. The biological response, i. e. activation of genes involved in nitrogen metabolism, depends on the concentration of phospho-FixJ which in turn reflects the net balance of counteracting kinase and phosphatase activities24
. FixL is water-soluble and forms a dimer; autophosphorylation is thought to occur in trans
as observed for related histidine kinases25
. Under aerobic conditions net kinase activity of FixL is strongly repressed to prevent futile activation of its target genes.
Although the PAS sensor domains of YtvA and FixL bind very different cofactors, FMN and heme, and respond to completely distinct physical and chemical stimuli, their structural similarity could reflect some functional correspondence. Thus they are good candidates for the design of novel proteins controlled by PAS sensor domains. In particular we asked: could the activity of FixL be removed from the control of oxygen and placed under the control of light by coupling its kinase domain to the YtvA LOV sensor domain? We generated such fusion proteins according to a structure-based sequence alignment between the PAS and LOV sensor domains of YtvA and FixL (). Structural information clearly defines the boundaries of the sensor domains and allows us to precisely align them to each other. To avoid disrupting folded domains, in all fusion proteins YtvA and FixL were linked within their common Jα linker region, which adopts a helical conformation in both isolated sensor domains (Fig. 1B, ).
In vitro Phosphorylation Assays of Fusion Constructs
Fusion Proteins Display Light-dependent Kinase Activity
The fusion protein YF1 covalently connects residues 1–127 of YtvA with 258–505 of FixL (). Incorporation of the FMN cofactor and the ability of YF1 to undergo the characteristic reversible LOV photoreaction were confirmed by absorption spectroscopy (Suppl. Fig. 2
). Sedimentation equilibrium centrifugation at protein concentrations between 0.5 and 7 μM showed that in solution YF1 forms a single species with an apparent molecular weight of (78.4 ± 10.4) kD corresponding to a dimer (data not shown; exp. monomer weight 42.1 kD). We estimate that its dissociation equilibrium constant is less than 0.5 μM.
We measured the autophosphorylation activity of YF1 by radiography using γ-32
P-ATP as a substrate. The initial reaction velocity in the dark of (0.080 ± 0.003) h−1
at 23 °C was decreased under saturating illumination with white light to (0.020 ± 0.001) h−1
. The autophosphorylation activity of the parent enzyme FixL is greatly stimulated by the presence of the response regulator FixJ even though FixJ does not directly participate in this reaction26
. In all subsequent experiments we therefore monitored enzyme activity and regulation in turnover assays in which multiple rounds of FixJ phosphorylation are catalyzed, whose kinetics reflect the net balance of counteracting kinase and phosphatase activities (). At the beginning of the reaction no phospho-FixJ is present and therefore the initial time-course of phosphorylation is dominated by the forward kinase reaction. We evaluated the initial velocity of phosphate incorporation into FixJ and report these values as kinase activities. In the dark, YF1 phosphorylated FixJ with an initial velocity of (56.4 ± 2.8) mol FixJ/(mol kinase·h) (). In the following, we drop the two mol terms and report turnover activities in units of h−1
. Under constant illumination net kinase activity was strongly suppressed to the point where it was too low to be quantified. Based on the minimum phosphorylation activity of 0.04 h−1
we can reliably measure in these turnover assays, we estimate that light absorption causes a more than 1000-fold reduction of net kinase activity in YF1.
Kinase activity of fusion kinase YF1.
In principle, any or all of the elementary steps in the overall reaction scheme could be regulated by light (). To address whether YF1 also possesses phosphatase activity, we performed kinase assays in the dark as before, but took aliquots after 7.5 and 30.5 min at which times appreciable amounts of phospho-FixJ had accumulated. Upon exposure to light, the amount of phospho-FixJ in these aliquots decreased sharply with initial velocities of (120 ± 20) and (140 ± 7) mol FixJ/(mol kinase·h), respectively (). The observation that these velocities depend only weakly on the initial amount of phospho-FixJ argues for a process that is enzyme-catalyzed by the YF1/FixJ complex; for uncatalyzed hydrolysis these velocities would be proportional to the initial concentration of phospho-FixJ. Furthermore, under similar reaction conditions phospho-FixJ is chemically quite stable; the lifetime of the closely related phospho-FixJ from Sinorhizobium meliloti
is on the order of two hours27
As a control, we determined the activity of the parent enzyme FixL under the same reaction conditions. In vivo
, FixL occurs in the ferrous state (FeII
); binding of oxygen to the ferrous deoxy state inhibits kinase activity. In vitro
, FixL is routinely assayed in its ferric (FeIII
) state which has activity similar to the ferrous deoxy form22
. The net kinase activity of ferric FixL can be greatly reduced by adding cyanide28
. Under our reaction conditions, ferric FixL phosphorylated FixJ with an initial velocity of (80.2 ± 3.6) h−1
which is faster by a factor of 1.4 ± 0.1 than the reaction catalyzed by YF1 in the dark (). Addition of cyanide led to an approximately 1000-fold decrease of net kinase activity of FixL to (0.086 ± 0.014) h−1
. These values are comparable with previous studies under similar reaction conditions that reported a maximum initial velocity of 81 h−1
and a more than 2700-fold reduction in the presence of cyanide28
We measured the net kinase activity of YF1 in the dark at varying substrate concentrations. Catalysis followed Michaelis-Menten kinetics with apparent KM
values of (33 ± 2) μM and (1.4 ± 0.4) μM for ATP and FixJ, respectively (Suppl. Table I
). In the case of FixL, the KM
values were (206 ± 13) μM for ATP and (1.6 ± 0.4) μM for FixJ which agree closely with values for the orthologous FixL from S. meliloti26
Kinase activity of YF1 has thus been placed under the control of light without incurring significant losses in kinase activity or alteration in substrate affinities. In the dark state, YF1 possesses net kinase activity closely similar to its parent FixL. In the light state, its phosphatase activity is greatly enhanced and YF1 is converted to a net phosphatase.
Light Inactivation is Linked to LOV Photocycle
To address whether the light dependence of YF1 activity is indeed mediated by its LOV domain, we conducted several control experiments.
First, we verified that the activity of the parent enzyme FixL is independent of light (not shown).
Second, we generated a point mutant of YF1 in which the active-site cysteine 62 in the LOV domain is replaced by alanine which abolishes the normal LOV photochemistry (Suppl. Fig. 2
). Net kinase activity of YF1 C62A was identical under dark ((52.9 ± 2.7) h−1
) and light conditions ((56.1 ± 2.8) h−1
) and corresponded to that noted above for YF1 in the dark ((56.4 ± 2.8) h−1
). Replacement of the phosphoacceptor histidine 291 within the DHp domain by alanine abolished phosphorylation of FixJ both in the dark and light.
Third, we explored whether light absorption might simply cause global unfolding and thus inactivation of YF1. Light absorption induces a 5–10% loss of the circular dichroism (CD) signal of YF1 at 208 nm, similar to observations for a plant phototropin LOV domain (Suppl. Fig. 3
. This indicates a partial loss or rearrangement of α-helical structure elements upon light absorption but rules out global unfolding of YF1. Global unfolding is also incompatible with the observations noted above, that the autophosphorylation activity of isolated YF1 is only moderately suppressed in the light and that the YF1/FixJ complex displays greatly enhanced phosphatase activity in the light.
Fourth, YF1 was illuminated through a (430 ± 10) nm interference filter which reduced light output to 240 μW. Using this illumination protocol, the fraction of YF1 in the dark state diminished in a first-order reaction with a time constant of (58.4 ± 1.0) s (, open circles). Net kinase activity of YF1 decayed approximately twice as fast, with a time constant of (27.1 ± 1.0) s (, closed symbols). Recovery of YF1 after photobleaching occurred with a complex time course over several hours, as indicated by absorption spectroscopy (). Approximately 70% of the absorption signal recovered with a time constant of (5900 ± 25) s, followed by a slower phase. Complex recovery kinetics have also been observed for natural LOV histidine kinases29
. The long recovery time enabled us to determine the kinase activity of YF1 as recovery proceeded. Net kinase activity was regenerated in a sigmoid time course (), and at 12 h after light absorption, activity had recovered to 95% of that of the dark state. The presence of a pronounced lag phase suggests that photorecovery proceeds through an intermediate state with little or no kinase activity.
Kinase activity of YF1 depends on LOV photocycle.
A minimal model to account for both recovery and photobleaching data is shown in . In this model, we assume that full kinase activity of YF1 requires both LOV domains to be in their dark state, and that after photobleaching, the two LOV monomers of YF1 independently relax to their dark state with the microscopic time constant τ. The value of τ of (10800 ± 1600) s is consistent with the recovery kinetics of the absorption signal after photobleaching of YF1 (). According to the model, the absorption signal should recover with an observable time constant τ/2 = (5400 ± 800) s which agrees well with the measured value of (5900 ± 25) s. The model also accounts for the observation that upon partial photobleaching, YF1 kinase activity diminishes twice as fast as the absorption signal (). Despite the simplicity of the model, it quantitatively explains the experimental data remarkably well. However, we cannot rule out other, more complex reaction schemes.
In summary, these experiments demonstrate that the effect of light on YF1 activity is mediated by its LOV domain, is fully reversible, and requires that YF1 be a dimer.
Kinase Activity and Regulation Depend on Properties of the Domain Linker
To address how the properties of the linker between the LOV sensor and kinase effector domains affect enzyme activity and regulation by light, we generated a number of protein variants which differ in that region. In a first series, we varied the site within the Jα helix at which the two domains are fused but retained the same linker length as in YF1. Variants YF2–4 successively derive more residues from FixL as indicated in and . Variant YF3 was inactive, but YF2 and YF4 showed light inactivation of net kinase activity as seen for YF1 albeit with quantitatively different activities in the dark of (67.9 ± 6.5) h−1
and (3.4 ± 0.1) h−1
, respectively (Suppl. Fig. 4
A second series of variants contains successive deletions of amino acids 274–277 at the C-terminus of the Jα linker sequence of YF1 (, ). Kinase activity in variants with 1, 2 or 4 residue deletions in the linker was below the detectable limit both in the dark and light (). Construct YF1Δ275–277 showed low activity of about 0.3 h−1 but regulation by light was abolished.
Figure 4 A. Activity of fusion kinase variants in turnover assays. Blue bars denote activity in the dark and orange bars under constant illumination. Asterisks indicate activities below the detectable limit of 0.04 h−1 (dashed line). The table lists changes (more ...)
In a third series of variants, YF1 I1–8, up to eight additional amino acids were inserted into the Jα linker region (). To reduce the chance of incorporating deleterious residues, the insertion site and the nature of the amino acids introduced were determined based on sequence comparison to Caulobacter crescentus
(Suppl. Fig. 5
). Insertion of 2, 3, 5 or 6 amino acids led to variants YF1 I2, YF1 I3, YF1 I5 and YF1 I6 that were inactive in the light and the dark (). Variant YF1 I7 had higher net kinase activity in the dark ((4.2 ± 0.2) h−1
) than in the light (< 0.04 h−1
), as in the parent YF1. Variant YF1 I4 had activity in the dark of (37.2 ± 1.7) h−1
comparable to YF1, but this activity was independent of light. Lastly, YF1 I1 and YF1 I8 with 1 or 8 extra amino acids showed inverse dependence on light. Although with much lower activity than YF1, both variants were more active in the light (around 0.1–0.3 h−1
) than in the dark (< 0.04 h−1
). That is, they were light-activated kinases.
Taken together, the deletion and insertion variants display a striking heptad periodicity of kinase activity and light regulation (). All pairs of fusion proteins that differ in their linker length by seven residues, as for example YF1 and YF1 I7, or YF1 I1 and YF1 I8, show remarkably similar activity patterns. Moreover, the length of the domain linker is an important design parameter that can be used to invert the regulatory behavior; insertion of one amino acid into the linker region of YF1 replaced net light-inactivated kinase activity by net light-activated kinase activity.
In Vivo Activity of Fusion Kinases
As discussed above, FixL activates expression of genes involved in nitrogen metabolism in an oxygen-dependent manner. Could we reengineer this regulatory network and use our fusion kinases to regulate gene expression in response to light in vivo?
The activity of fusion kinases in E. coli
was determined in β-galactosidase assays using a reporter construct which expresses LacZ under the control of the B. japonicum
promoter to which phospho-FixJ binds15
. In the absence of FixJ only low levels of β-galactosidase activity around 15 Miller Units/(mL·min) were observable (). Upon introduction of B. japonicum
FixJ alone into E. coli
, expression of the LacZ reporter was constitutively activated with β-galactosidase activities of around 3800 Miller Units/(mL·min). Constitutive activation of a related FixJ pathway has been ascribed to phosphorylation of FixJ either enzymatically by an endogenous E. coli
histidine kinase or non-enzymatically by intracellular acyl phosphates23
In vivo Assay of Fusion Kinase YF1
Constitutive activation of the FixJ pathway precludes measurements of kinase activity of our fusion kinases in E. coli
. However, our in vitro
experiments showed that light switches YF1 from net kinase to phosphatase activity (). Might the phosphatase activity of YF1 in its light state be sufficient to counteract constitutive phosphorylation of FixJ in E. coli
? Upon introduction of both YF1 and FixJ, we observed β-galactosidase activity of (2860 ± 360) Miller Units/(mL·min) in the dark (). In the light, β-galactosidase activity was suppressed by 70-fold to (42 ± 16) Miller Units/(mL·min). When YF1 was replaced by the photoinactive YF1 mutant C62A, β-galactosidase activity was independent of light (), consistent with the in vitro
kinase activity for this mutant (Suppl. Fig. 4
We conclude that the fusion kinase YF1 is active in vivo and regulates gene expression in a light-dependent manner, in accord with our in vitro results. Interestingly, in E. coli regulation of gene expression by light appears to be mediated mainly by the phosphatase activity of YF1.