Autokinase Activity Research Articles

Dephosphorylation of the core clock protein KaiC in the cyanobacterial KaiABC circadian oscillator proceeds via an ATP synthase mechanism.

The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro from three proteins, KaiA, KaiB, and KaiC in the presence of ATP, to tick in a temperature-compensated manner. KaiC, the central cog of this oscillator, forms a homohexamer with 12 ATP molecules bound between its N- and C-terminal domains and exhibits unusual properties. Both the N-terminal (CI) and C-terminal (CII) domains harbor ATPase activity, and the subunit interfaces between CII domains are the sites of autokinase and autophosphatase activities. Hydrolysis of ATP correlates with phosphorylation at threonine and serine sites across subunits in an orchestrated manner, such that first T432 and then S431 are phosphorylated, followed by dephosphorylation of these residues in the same order. Although structural work has provided insight into the mechanisms of ATPase and kinase, the location and mechanism of the phosphatase have remained enigmatic. From the available experimental data based on a range of approaches, including KaiC crystal structures and small-angle X-ray scattering models, metal ion dependence, site-directed mutagenesis (i.e., E318, the general base), and measurements of the associated clock periods, phosphorylation patterns, and dephosphorylation courses as well as a lack of sequence motifs in KaiC that are typically associated with known phosphatases, we hypothesized that KaiCII makes use of the same active site for phosphorylation and dephosphorlyation. We observed that wild-type KaiC (wt-KaiC) exhibits an ATP synthase activity that is significantly reduced in the T432A/S431A mutant. We interpret the first observation as evidence that KaiCII is a phosphotransferase instead of a phosphatase and the second that the enzyme is capable of generating ATP, both from ADP and P(i) (in a reversal of the ATPase reaction) and from ADP and P-T432/P-S431 (dephosphorylation). This new concept regarding the mechanism of dephosphorylation is also supported by the strikingly similar makeups of the active sites at the interfaces between α/β heterodimers of F1-ATPase and between monomeric subunits in the KaiCII hexamer. Several KaiCII residues play a critical role in the relative activities of kinase and ATP synthase, among them R385, which stabilizes the compact form and helps kinase action reach a plateau, and T426, a short-lived phosphorylation site that promotes and affects the order of dephosphorylation.

Insights into the regulation of eukaryotic elongation factor 2 kinase and the interplay between its domains

eEF2K (eukaryotic elongation factor 2 kinase) is a Ca2+/CaM (calmodulin)-dependent protein kinase which regulates the translation elongation machinery. eEF2K belongs to the small group of so-called ‘α-kinases’ which are distinct from the main eukaryotic protein kinase superfamily. In addition to the α-kinase catalytic domain, other domains have been identified in eEF2K: a CaM-binding region, N-terminal to the kinase domain; a C-terminal region containing several predicted α-helices (resembling SEL1 domains); and a probably rather unstructured ‘linker’ region connecting them. In the present paper, we demonstrate: (i) that several highly conserved residues, implicated in binding ATP or metal ions, are critical for eEF2K activity; (ii) that Ca2+/CaM enhance the ability of eEF2K to bind to ATP, providing the first insight into the allosteric control of eEF2K; (iii) that the CaM-binding/α-kinase domain of eEF2K itself possesses autokinase activity, but is unable to phosphorylate substrates in trans; (iv) that phosphorylation of these substrates requires the SEL1-like domains of eEF2K; and (v) that highly conserved residues in the C-terminal tip of eEF2K are essential for the phosphorylation of eEF2, but not a peptide substrate. On the basis of these findings, we propose a model for the functional organization and control of eEF2K.

Study of the TmoS/TmoT two‐component system: towards the functional characterization of the family of TodS/TodT like systems

SummaryThe two‐component system TmoS/TmoT controls the expression of the toluene‐4‐monooxygenase pathway in Pseudomonas mendocina RK1 via modulation of PtmoX activity. The TmoS/TmoT system belongs to the family of TodS/TodT like proteins. The sensor kinase TmoS is a 108 kDa protein composed of seven different domains. Using isothermal titration calorimetry we show that purified TmoS binds a wide range of aromatic compounds with high affinities. Tightest ligand binding was observed for toluene (KD = 150 nM), which corresponds to the highest affinity measured between an effector and a sensor kinase. Other compounds with affinities in the nanomolar range include benzene, the 3 xylene isomers, styrene, nitrobenzene or p‐chlorotoluene. We demonstrate that only part of the ligands that bind to TmoS increase protein autophosphorylation in vitro and consequently pathway expression in vivo. These compounds are referred to as agonists. Other TmoS ligands, termed antagonists, failed to increase TmoS autophosphorylation, which resulted in their incapacity to stimulate gene expression in vivo. We also show that TmoS saturated with different agonists differs in their autokinase activities. The effector screening of gene expression showed that promoter activity of PtmoX and PtodX (controlled by the TodS/TodT system) is mediated by the same set of 22 compounds. The common structural feature of these compounds is the presence of a single aromatic ring. Among these ligands, toluene was the most potent inducer of both promoter activities. Information on the TmoS/TmoT and TodS/TodT system combined with a sequence analysis of family members permits to identify distinct features that define this protein family.

Signal Transduction through CsrRS Confers an Invasive Phenotype in Group A Streptococcus

The CsrRS (or CovRS) two component system controls expression of up to 15% of the genome of group A Streptococcus (GAS). While some studies have suggested that the sensor histidine kinase CsrS responds to membrane perturbations as a result of various environmental stresses, other data have implicated the human antimicrobial peptide LL-37 and extracellular Mg2+ as specific signals. We now report that Mg2+ and LL-37 have opposite effects on expression of multiple genes that are activated or repressed by the transcriptional regulator CsrR. Using a GAS isolate representative of the recently emerged and widely disseminated M1T1 clone implicated in severe invasive disease, we found marked up-regulation by CsrRS of multiple virulence factors including pyrogenic exotoxin A, DNase Sda1, streptolysin O, and the hyaluronic acid capsular polysaccharide, among others. Topology and surface protein labeling studies indicated that CsrS is associated with the bacterial cell membrane and has a surface-exposed extracellular domain accessible to environmental ligands. Replacement of a cluster of three acidic amino acids with uncharged residues in the extracellular domain of CsrS abrogated LL-37 signaling and conferred a hyporesponsive phenotype consistent with tonic activation of CsrS autokinase activity, an effect that could be overridden by mutation of the CsrS active site histidine. Both loss- and gain-of-function mutations of a conserved site in the receiver domain of CsrR established an essential role for lysine 102 in CsrS-to-CsrR signal transduction. These results provide strong evidence that Mg2+ and LL-37 are specific signals that function by altering CsrS autokinase activity and downstream phosphotransfer to CsrR to modulate its activity as a transcriptional regulator. The representation of multiple antiphagocytic and cytotoxic factors in the CsrRS regulon together with results of in vitro phagocytic killing assays support the hypothesis that CsrRS mediates conversion of GAS from a colonizing to an invasive phenotype in response to signaling by host LL-37.

Nitric oxide dioxygenation reaction in DevS and the initial response to nitric oxide in Mycobacterium tuberculosis.

DevS and DosT from Mycobacterium tuberculosis (MTB) are paralogous heme-based sensor kinases that respond to hypoxia and to low concentrations of nitric oxide (NO). Both proteins work with the response regulator DevR as a two-component regulatory system to induce the dormancy regulon in MTB. While DevS and DosT are inactive when dioxygen is bound to the heme Fe(II) at their sensor domain, autokinase activity is observed in their heme Fe(II)-NO counterparts. To date, the conversion between active and inactive states and the reactivity of the heme-oxy complex toward NO have not been investigated. Here, we use stopped-flow UV-vis spectroscopy and rapid freeze quench resonance Raman spectroscopy to probe these reactions in DevS. Our data reveal that the heme-O(2) complex of DevS reacts efficiently with NO to produce nitrate and the oxidized Fe(III) heme through an NO dioxygenation reaction that parallels the catalytic reactions of bacterial flavohemoglobin and truncated hemoglobins. Autophosphorylation activity assays show that the Fe(III) heme state of DevS remains inactive but exhibits a high affinity for NO and forms an Fe(III)-NO complex that is readily reduced by ascorbate, a mild reducing agent. On the basis of these results, we conclude that upon exposure to low NO concentrations, the inactive oxy-heme complex of DevS is rapidly converted to the Fe(II)-NO complex in the reducing environment of living cells and triggers the initiation of dormancy.

New paradigm in ethylene signaling

The membrane protein ETHYLENE INSENSITIVE2 (EIN2), which is supposed to act between the soluble serine/threonine kinase CTR1 and the EIN3/EIL family of transcription factors, is a central and most critical element of the ethylene signaling pathway in Arabidopsis. In a recent study, we have identified that EIN2 interacts tightly with all members of the Arabidopsis ethylene receptor family – proteins that mark the starting point of the signaling pathway. Our studies show consistently that the kinase domain of the receptors is essential for the formation of the EIN2-receptor complex. Furthermore, mutational analysis demonstrates that phosphorylation is a key mechanism in controlling the interaction of EIN2 and the ethylene receptors. Interaction studies in the presence of the ethylene agonist cyanide revealed a causal link between hormone binding and complex formation. In the presence of the plant hormone agonist the auto-kinase activity of the receptors is inhibited and the non-phosphorylated kinase domain of the receptors binds tightly to the carboxyl-terminal domain of EIN2. In the absence of cyanide inhibition of the auto-kinase activity is relieved and complex formation with the phosphorylated kinase domain of the receptors is reduced. Our data suggest a novel model on the integration of EIN2 in the ethylene signaling pathway.

Phosphorylation of a 65 kDa cytoplasmic protein by the Escherichia coli YeaG kinase

The Escherichia coli YeaG cytoplasmic protein, which is endowed with autokinase and casein kinase activities, has been shown to phosphorylate specifically a 65-kDa cytoplasmic protein. This protein cannot be phosphorylated in an extract from the yeaG mutant, but it can be phosphorylated in an extract from the parental strain or in mutant extract supplemented with purified YeaG. This phosphoprotein displays a pH of 6.8 in 2D gels. Stationary phase induction of YeaG correlates with a significant growth defect of the yeaG mutant at the end of the exponential phase.

Conserved mechanism for sensor phosphatase control of two-component signaling revealed in the nitrate sensor NarX

Two-component signal transduction mediates a wide range of phenotypes in microbes and plants. The sensor transmitter module controls the phosphorylation state of the cognate-response-regulator receiver domain. Whereas the two-component autokinase and phosphotransfer reactions are well-understood, the mechanism by which sensors accelerate the rate of phospho-response regulator dephosphorylation, termed "transmitter phosphatase activity," is unknown. We identified a conserved DxxxQ motif adjacent to the phospho-accepting His residue in the HisKA_3 subfamily of two-component sensors. We used site-specific mutagenesis to make substitutions for these conserved Gln and Asp residues in the nitrate-responsive NarX sensor and analyzed function both in vivo and in vitro. Results show that the Gln residue is critical for transmitter phosphatase activity, but is not essential for autokinase or phosphotransfer activities. The documented role of an amide moiety in phosphoryl group hydrolysis suggests an analogous catalytic function for this Gln residue in HisKA_3 members. Results also indicate that the Asp residue is important for both autokinase and transmitter phosphatase activities. Furthermore, we noted that sensors of the HisKA subfamily exhibit an analogous E/DxxT/N motif, the conserved Thr residue of which is critical for transmitter phosphatase activity of the EnvZ sensor. Thus, two-component sensors likely use similar mechanisms for receiver domain dephosphorylation.

Bacterial Sensor Kinases: Diversity in the Recognition of Environmental Signals

Bacteria sense and respond to a wide range of physical and chemical signals. Central to sensing and responding to these signals are two-component systems, which have a sensor histidine kinase (SK) and a response regulator (RR) as basic components. Here we review the different molecular mechanisms by which these signals are integrated and modulate the phosphorylation state of SKs. Apart from the basic mechanism, which consists of signal recognition by the SK that leads to an alteration of its autokinase activity and subsequently a change in the RR phosphorylation state, a variety of alternative modes have evolved. The biochemical data available on SKs, particularly their molecular interactions with signals, nucleotides, and their cognate RRs, are also reviewed.

A hybrid two-component system protein from Azospirillum brasilense Sp7 was involved in chemotaxis

We here report the sequence and functional analysis of org35 of Azospirillum brasilense Sp7, which was originally identified to be able to interact with NifA in yeast-two-hybrid system. The org35 encodes a hybrid two-component system protein, including N-terminal PAS domains, a histidine kinase (HPK) domain and a response regulator (RR) domain in C-terminal. To determine the function of the Org35, a deletion–insertion mutant in PAS domain [named Sp7353] and a complemental strain Sp7353C were constructed. The mutant had reduced chemotaxis ability compared to that of wild-type, and the complemental strain was similar to the wild-type strain. These data suggested that the A. brasilense org35 played a key role in chemotaxis. Variants containing different domains of the org35 were expressed, and the functions of these domains were studied in vitro. Phosphorylation assays in vitro demonstrated that the HPK domain of Org35 possessed the autokinase activity and that the phosphorylated HPK was able to transfer phosphate groups to the RR domain. The result indicated Org35 was a phosphorylation-communicating protein.

Characterization of a DNA damage‐inducible membrane protein kinase from Deinococcus radiodurans and its role in bacterial radioresistance and DNA strand break repair

Deinococcus radiodurans mutant lacking pyrroloquinoline-quinone (PQQ) synthesis shows sensitivity to γ-rays and impairment of DNA double strand break repair. The genome of this bacterium encodes five putative proteins having multiple PQQ binding motifs. The deletion mutants of corresponding genes were generated, and their response to DNA damage was monitored. Only the Δdr2518 mutant exhibited higher sensitivity to DNA damage. Survival of these cells decreased by 3-log cycle both at 6 kGy γ-rays and 1200 Jm(-2) UV (254 nm) radiation, and 2.5-log cycle upon 14 days desiccation at 5% humidity. The Δdr2518 mutant showed complete inhibition of DSB repair until 24 h PIR and disappearance of a few phosphoproteins. The Δdr2518pqqE:cat double mutant showed γ-ray sensitivity similar to Δdr2518 indicating functional interaction of these genes in D. radiodurans. DR2518 contains a eukaryotic type Ser/Thr kinase domain and structural topology suggesting stress responsive transmembrane protein. Its autokinase activity in solution was stimulated by nearly threefold with PQQ and twofold with linear DNA, but not with circular plasmid DNA. More than 15-fold increase in dr2518 transcription and several-fold enhanced in vivo phosphorylation of DR2518 were observed in response to γ irradiation. These results suggest that DR2518 as a DNA damage-responsive protein kinase plays an important role in radiation resistance and DNA strand break repair in D. radiodurans.

New Insight in Ethylene Signaling: Autokinase Activity of ETR1 Modulates the Interaction of Receptors and EIN2

Ethylene insensitive 2 (EIN2), an integral membrane protein of the ER network, has been identified as the central regulator of the ethylene signaling pathway. Still, the mechanism by which the ethylene signal is transferred from the receptors to EIN2 has not been solved yet. Here, we show that protein phosphorylation is a key mechanism to control the interaction of EIN2 and the receptors. In vivo and in vitro fluorescence studies reveal that the kinase domain of the receptors is essential for the interaction. Cyanide, an ethylene agonist, which is known to reduce auto-phosphorylation of the ethylene receptor ethylene resistant 1 (ETR1) or a mutation in the kinase domain of ETR1 that prevents auto-phosphorylation (H353A), increases the affinity of the receptors for EIN2. On the other hand, mimicking permanent auto-phosphorylation of ETR1 as in the mutant H353E releases the EIN2-ETR1 interaction from the control by the plant hormone. Based on our data, we propose a novel model on the integration of EIN2 in the ethylene signaling cascade.

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Abstract 2680: Nonautoinhibitory properties of the C-terminal helix of the mitogen- and stress-activated protein kinase 1

Abstract Mitogen- and stress-activated protein kinase 1 (MSK1) is a growth factor-stimulated serine/threonine kinase that is important in the regulation of gene transcription and proinflammatory cytokine stimulation. MSK1 is a dual kinase possessing two distinct kinase domains linked by a hydrophobic region. The C-terminal kinase domain (CTD MSK1) is an important regulatory domain activated by ERKs and p38 mitogen-activated protein kinases (MAPKs) in response to various cellular stimuli. The activity of full length MSK1 is controlled by multiple phosphorylation sites and is mediated through stimulation of the C-terminal kinase domain. We have determined the crystal structure of the isolated active CTD MSK1 (residues 414-738) in apo- form and in complex with the non-hydrolyzable ATP analogue AMP-PNP at 2.0 and 2.5 Å resolution, respectively. The structure showed that, despite overall similarity with the p90 ribosomal S6 kinase 2 (RSK2) and the MAPK-activated protein kinase 2 (MK2), the CTD MSK1 is unexpectedly not autoinhibited by the C-terminal αL-helix. The helix does not form hydrogen bonds with a substrate-binding loop and nearby helices and lacks putative “inhibitory” residues responsible for the interaction with the kinase core. CTD MSK1 undergoes active autophosphorylation in vitro as shown by luminescent assay, and mutation of two residues, H712K and F719Y, located on the αL-helix does not convert it to RSK2-like autoinhibitory state. The protein was phosphorylated at Thr581 in the T-activation loop, and a T581A mutant showed significantly impaired autokinase activity. The “full length” C-terminal domain of MSK1 (residues 414-802) possessed weaker autophosphorylation activity compared with the truncated fragment (residues 414-738), which lacks the MAPK-docking site. Overexpression of active truncated CTD MSK1 (residues 414-738) in JB6 cells resulted in neoplastic transformation in response to EGF or TPA stimulation in a manner similar to that induced by the full length MSK1. On the other hand, the “full length” CTD (residues 414-802) suppressed colony formation ex vivo. This results indicate that the extreme C-terminal tail (residues 739-802), which encloses the ERK/p38 docking site, inhibits CTD MSK1 kinase activity. Using surface plasmon resonance (SPR) binding experiments, we showed that the C-terminal end-deleted construct does not interact with ERK1 in vitro, but the “full length” CTD MSK1 binds with ERK1 in a dose-dependent manner with a KD ∼ 70 nM. Similar binding results were obtained for interaction with ERK2 and p38. The SPR data indicate that the αL-helix is not involved in the interaction with MAP kinases. The intrinsic non-autoinhibitory C-terminal αL-helix feature suggests that the CTD has a αL-helix-independent activation mechanism, and the MAPK-docking site may regulate CTD MSK1 activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2680.

Regulation of the Ppr Histidine Kinase by Light-Induced Interactions between Its Photoactive Yellow Protein and Bacteriophytochrome Domains

Ppr is a unique bacteriophytochrome that bleaches rather than forming a far-red-shifted Pfr state upon red light activation. Ppr is also unusual in that it has a blue light photoreceptor domain, PYP, which is N-terminally fused to the bacteriophytochrome domain (Bph). When both photoreceptors are activated by light, the fast phase of Bph recovery (1 min lifetime) corresponds to the formation of an intramolecular long-lived complex between the activated PYP domain and the Bph domain (lifetime of 2-3 days). Since this state is unusually long-lived as compared to other intermediates in the photocycle of both PYP and Bph, we interpret this as formation of a metastable complex between activated PYP and Bph domains that takes days to relax. In the metastable complex, the PYP domain is locked in its activated UV absorbing state and the Bph domain is in a slightly red-shifted state (from 701 to 702 nm), which is photochemically inactive to red or white light. The amount of metastable complex formed increases with the degree of prior activation of PYP, reaching a maximum of 50% when PYP is fully activated compared to 0% when no PYP is activated. The saturation of complex formation at 50% is believed to be due to light-induced heterogeneity within the Ppr dimer. UV irradiation (365 nm) of the metastable complex state photoreverses the activated PYP and the red-shifted Bph to the initial dark state within seconds. We therefore postulate that Ppr functions as a UV-red light sensor and describe the different Ppr states that can be obtained depending on the light quality. Both red and white light upregulate the autokinase activity, while it is downregulated in the dark. The physiological state of Ppr is most likely a mixture of three different states, dark, metastable complex, and red light-activated, with fractional populations whose amounts depend on the light quality of the environment and that regulate the extent of phosphorylation by the kinase.

Combining Genomic Information with Molecular Dynamics Simulation to Model Two-Component Signal-Transduction Systems

Bacteria, archaea and some Eukaryotes employ so-called two-component signal transduction systems (TCS) as a means of adaptation to cellular and environmental stimuli. Typically, these systems feature a membrane bound sensor histidine kinase (SK), which modulates its autokinase activity in response to the stimulus and a transcription factor/response regulator (RR), which accepts a phosphoryl group from the SK and in turn mediates a cellular response. The phosphoryl-transfer requires the formation of a SK/RR complex ruled by transient interaction. This and other transient protein complexes involved in signal transduction are difficult to resolve by experimental means, such as X-ray crystallography or NMR, as evidenced by a lack of structural representatives for many such systems in the protein database PDB. The presented work demonstrates how theoretical methods can close this gap. A genomic direct coupling analysis extracts protein-protein interaction contacts from protein sequence databases. This information is integrated with experimentally determined structures of the unbound proteins in molecular dynamical simulation to understand protein docking and predict the structure of the protein complex. The reliability of this approach is demonstrated by achieving crystal resolution accuracy when reconstituting the known sporulation phosphotransferase complex between Spo0B and Spo0F, which is related to the TCS phsphotransfer complex. We introduce a structural model for the complex of TCS proteins TM0853 with TM0468, consistent with all available experimental data.

Role of Mitogen-Activated Protein Kinase Sty1 in Regulation of Eukaryotic Initiation Factor 2α Kinases in Response to Environmental Stress in Schizosaccharomyces pombe

The mitogen-activated protein kinase (MAPK) Sty1 is essential for the regulation of transcriptional responses that promote cell survival in response to different types of environmental stimuli in Schizosaccharomyces pombe. In fission yeast, three distinct eukaryotic initiation factor 2alpha (eIF2alpha) kinases, two mammalian HRI-related protein kinases (Hri1 and Hri2) and the Gcn2 ortholog, regulate protein synthesis in response to cellular stress conditions. In this study, we demonstrate that both Hri1 and Hri2 exhibited an autokinase activity, specifically phosphorylated eIF2alpha, and functionally replaced the endogenous Saccharomyces cerevisiae Gcn2. We further show that Gcn2, but not Hri1 or Hri2, is activated early after exposure to hydrogen peroxide and methyl methanesulfonate (MMS). Cells lacking Gcn2 exhibit a later activation of Hri2. The activated MAPK Sty1 negatively regulates Gcn2 and Hri2 activities under oxidative stress but not in response to MMS. In contrast, Hri2 is the primary activated eIF2alpha kinase in response to heat shock. In this case, the activation of Sty1 appears to be transitory and does not contribute to the modulation of the eIF2alpha kinase stress pathway. In strains lacking Hri2, a type 2A protein phosphatase is activated soon after heat shock to reduce eIF2alpha phosphorylation. Finally, the MAPK Sty1, but not the eIF2alpha kinases, is essential for survival upon oxidative stress or heat shock, but not upon MMS treatment. These findings point to a regulatory coordination between the Sty1 MAPK and eIF2alpha kinase pathways for a particular range of stress responses.

Structural Insight into Partner Specificity and Phosphoryl Transfer in Two-Component Signal Transduction

The chief mechanism used by bacteria for sensing their environment is based on two conserved proteins: a sensor histidine kinase (HK) and an effector response regulator (RR). The signal transduction process involves highly conserved domains of both proteins that mediate autokinase, phosphotransfer, and phosphatase activities whose output is a finely tuned RR phosphorylation level. Here, we report the structure of the complex between the entire cytoplasmic portion of Thermotoga maritima class I HK853 and its cognate, RR468, as well as the structure of the isolated RR468, both free and BeF(3)(-) bound. Our results provide insight into partner specificity in two-component systems, recognition of the phosphorylation state of each partner, and the catalytic mechanism of the phosphatase reaction. Biochemical analysis shows that the HK853-catalyzed autokinase reaction proceeds by a cis autophosphorylation mechanism within the HK subunit. The results suggest a model for the signal transduction mechanism in two-component systems.

Structural plasticity and catalysis regulation of a thermosensor histidine kinase

Temperature sensing is essential for the survival of living cells. A major challenge is to understand how a biological thermometer processes thermal information to optimize cellular functions. Using structural and biochemical approaches, we show that the thermosensitive histidine kinase, DesK, from Bacillus subtilis is cold-activated through specific interhelical rearrangements in its central four-helix bundle domain. As revealed by the crystal structures of DesK in different functional states, the plasticity of this helical domain influences the catalytic activities of the protein, either by modifying the mobility of the ATP-binding domains for autokinase activity or by modulating binding of the cognate response regulator to sustain the phosphotransferase and phosphatase activities. The structural and biochemical data suggest a model in which the transmembrane sensor domain of DesK promotes these structural changes through conformational signals transmitted by the membrane-connecting two-helical coiled-coil, ultimately controlling the alternation between output autokinase and phosphatase activities. The structural comparison of the different DesK variants indicates that incoming signals can take the form of helix rotations and asymmetric helical bends similar to those reported for other sensing systems, suggesting that a similar switching mechanism could be operational in a wide range of sensor histidine kinases.

Comparative Photochemistry of Animal Type 1 and Type 4 Cryptochromes

Cryptochromes (CRYs) are blue-light photoreceptors with known or presumed functions in light-dependent and light-independent gene regulation in plants and animals. Although the photochemistry of plant CRYs has been studied in some detail, the photochemical behavior of animal cryptochromes remains poorly defined in part because it has been difficult to purify animal CRYs with their flavin cofactors. Here we describe the purification of type 4 CRYs of zebrafish and chicken as recombinant proteins with full flavin complement and compare the spectroscopic properties of type 4 and type 1 CRYs. In addition, we analyzed photoinduced proteolytic degradation of both types of CRYs in vivo in heterologous systems. We find that even though both types of CRYs contain stoichiometric flavin, type 1 CRY is proteolytically degraded by a light-initiated reaction in Drosophila S2, zebrafish Z3, and human HEK293T cell lines, but zebrafish CRY4 (type 4) is not. In vivo degradation of type 1 CRYs does not require continuous illumination, and a single light flash of 1 ms duration leads to degradation of about 80% of Drosophila CRY in 60 min. Finally, we demonstrate that in contrast to animal type 2 CRYs and Arabidopsis CRY1 neither insect type 1 nor type 4 CRYs have autokinase activities.