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Abstract Background This study evaluated the utility of immunization registries in identifying vaccine refusals among children. Among refusers, we studied their socioeconomic characteristics and health care utilization patterns. Methods Medical records were reviewed to validate refusal status in the immunization registries of two health plans. Racial, education, and income characteristics of children claiming refusal were collected based on the census tract of each child. Health care utilization was identified using both electronic medical record and insurance claims. Within the immunization registries of two HMOs in the study, some providers use refusal and medical contraindication interchangeably, and some providers tend to always use "ever refusal." Therefore, we combined medical contraindication and refusal together and treated them all as "refusal" in this study. Results The immunization registry, compared to chart review, had negative predictive values of 85–92% and 90–97% for 2- and 6-year olds, and positive predictive values of only 52–74% and 59–62% to identify vaccine refusals. Refusers were more likely to reside in well-educated, higher income areas than non-refusers. Refusers had not opted out of health care system and continued, although less frequently for the age 2 and under group, to use services. Conclusion Without enhancements to immunization registries, identifying children with immunization refusal would be time consuming. Since communities where refusers live are well educated, interventions should target these communities to communicate vaccine adverse events and consequences of vaccine preventable diseases.

Here, we present a protocol to quantify interactions among difficult-to-express proteins from Drosophila cells using the select western blot-free tagged-protein interaction (SWFTI) assay. We describe steps for plasmid design, cell plating, protein expression, and immunoprecipitation preparation. We then detail procedures for protein labeling, gel purification, and protein quantification. This protocol offers a fluorescence-based technique for rapid quantification of ectopically expressed proteins that are fused to SNAP and CLIP tags without the need for membrane transfer. For complete details on the use and execution of this protocol, please refer to Lin et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cryptochromes (CRYs), which are signaling proteins related to DNA photolyases, play pivotal roles in sensory responses throughout biology, including growth and development, metabolic regulation, circadian rhythm entrainment and geomagnetic field sensing. This review explores the evolutionary relationships and functional diversity of cryptochromes from the perspective of their molecular structures. In general, CRY biological activities derive from their core structural architecture, which is based on a Photolyase Homology Region (PHR) and a more variable and functionally specific Cryptochrome C-terminal Extension (CCE). The α/β and α-helical domains within the PHR bind FAD, modulate redox reactive residues, accommodate antenna cofactors, recognize small molecules and provide conformationally responsive interaction surfaces for a range of partners. CCEs add structural complexity and divergence, and in doing so, influence photoreceptor reactivity and tailor function. Primary and secondary pockets within the PHR bind myriad moieties and collaborate with the CCEs to tune recognition properties and propagate chemical changes to downstream partners. For some CRYs, changes in homo and hetero-oligomerization couple to light-induced conformational changes, for others, changes in posttranslational modifications couple to cascades of protein interactions with partners and effectors. The structural exploration of cryptochromes underscores how a broad family of signaling proteins with close relationship to light-dependent enzymes achieves a wide range of activities through conservation of key structural and chemical properties upon which function-specific features are elaborated., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 DeOliveira and Crane.)

Borrelia (or Borreliella ) burgdorferi , the causative agent of Lyme disease, is a motile and invasive zoonotic pathogen, adept at navigating between its arthropod vector and mammalian host. While motility and chemotaxis are well established as essential for its enzootic cycle, the function of methyl-accepting chemotaxis proteins (MCPs) in the infectious cycle of B. burgdorferi remains unclear. In this study, we demonstrate that MCP5, one of the most abundant MCPs in B. burgdorferi , is differentially expressed in response to environmental signals as well as at different stages of the pathogen's enzootic cycle. Specifically, the expression of mcp5 is regulated by the Hk1-Rrp1 and Rrp2-RpoN-RpoS pathways, which are critical for the spirochete's colonization of the tick vector and mammalian host, respectively. Infection experiments with an mcp5 mutant revealed that spirochetes lacking MCP5 could not establish infections in either C3H/HeN mice or Severe Combined Immunodeficiency (SCID) mice, which are defective in adaptive immunity, indicating the essential role of MCP5 in mammalian infection. However, the mcp5 mutant could establish infection and disseminate in NOD SCID Gamma (NSG) mice, which are deficient in both adaptive and most innate immune responses, suggesting a crucial role of MCP5 in evading host innate immunity. In the tick vector, the mcp5 mutants survived feeding but failed to transmit to mice, highlighting the importance of MCP5 in transmission. Our findings reveal that MCP5, regulated by the Rrp1 and Rrp2 pathways, is critical for the establishment of infection in mammalian hosts by evading host innate immunity and is important for the transmission of spirochetes from ticks to mammalian hosts, underscoring its potential as a target for intervention strategies.

Circadian clocks are composed of transcription-translation negative feedback loops that pace rhythms of gene expression to the diurnal cycle. In the filamentous fungus Neurospora crassa, the proteins F requency ( F RQ), the F RQ-interacting RNA helicase (FRH), and C asein-Kinase I (CK1) form the FFC complex that represses expression of genes activated by the white-collar complex (WCC). FRQ orchestrates key molecular interactions of the clock despite containing little predicted tertiary structure. Spin labeling and pulse-dipolar electron spin resonance spectroscopy provide domain-specific structural insights into the 989-residue intrinsically disordered FRQ and the FFC. FRQ contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. FRQ phosphorylation increases conformational flexibility and alters oligomeric state, but the changes in structure and dynamics are non-uniform. Full-length FRQ undergoes liquid-liquid phase separation (LLPS) to sequester FRH and CK1 and influence CK1 enzymatic activity. Although FRQ phosphorylation favors LLPS, LLPS feeds back to reduce FRQ phosphorylation by CK1 at higher temperatures. Live imaging of Neurospora hyphae reveals FRQ foci characteristic of condensates near the nuclear periphery. Analogous clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic clocks., Competing Interests: DT, NM, BB, SC, JD, AB, BC No competing interests declared, (© 2023, Tariq, Maurici et al.)

As an enzootic pathogen, the Lyme disease bacterium Borrelia burgdorferi possesses multiple copies of chemotaxis proteins, including two chemotaxis histidine kinases (CHK), CheA1 and CheA2. Our previous study showed that CheA2 is a genuine CHK that is required for chemotaxis; however, the role of CheA1 remains mysterious. This report first compares the structural features that differentiate CheA1 and CheA2 and then provides evidence to show that CheA1 is an atypical CHK that controls the virulence of B. burgdorferi through modulating the stability of RpoS, a key transcriptional regulator of the spirochete. First, microscopic analyses using green-fluorescence-protein (GFP) tags reveal that CheA1 has a unique and dynamic cellular localization. Second, loss-of-function studies indicate that CheA1 is not required for chemotaxis in vitro despite sharing a high sequence and structural similarity to its counterparts from other bacteria. Third, mouse infection studies using needle inoculations show that a deletion mutant of CheA1 (cheA1mut) is able to establish systemic infection in immune-deficient mice but fails to do so in immune-competent mice albeit the mutant can survive at the inoculation site for up to 28 days. Tick and mouse infection studies further demonstrate that CheA1 is dispensable for tick colonization and acquisition but essential for tick transmission. Lastly, mechanistic studies combining immunoblotting, protein turnover, mutagenesis, and RNA-seq analyses reveal that depletion of CheA1 affects RpoS stability, leading to reduced expression of several RpoS-regulated virulence factors (i.e., OspC, BBK32, and DbpA), likely due to dysregulated clpX and lon protease expression. Bulk RNA-seq analysis of infected mouse skin tissues further show that cheA1mut fails to elicit mouse tnf-α, il-10, il-1β, and ccl2 expression, four important cytokines for Lyme disease development and B. burgdorferi transmigration. Collectively, these results reveal a unique role and regulatory mechanism of CheA1 in modulating virulence factor expression and add new insights into understanding the regulatory network of B. burgdorferi., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Sze et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Spirochetes cause Lyme disease, leptospirosis, syphilis, and several other human illnesses. Unlike other bacteria, spirochete flagella are enclosed within the periplasmic space where the filaments distort and push the cell body by the action of the flagellar motors. We previously demonstrated that the oral pathogen Treponema denticola (Td) and Lyme disease pathogen Borreliella burgdorferi (Bb) form covalent lysinoalanine (Lal) cross-links between conserved cysteine and lysine residues of the FlgE protein that composes the flagellar hook. In Td, Lal is unnecessary for hook assembly but is required for motility, presumably due to the stabilizing effect of the cross-link. Herein, we extend these findings to other, representative spirochete species across the phylum. We confirm the presence of Lal cross-linked peptides in recombinant and in vivo-derived samples from Treponema spp., Borreliella spp., Brachyspira spp., and Leptospira spp. As was observed with Td, a mutant strain of Bb unable to form the cross-link has greatly impaired motility. FlgE from Leptospira spp. does not conserve the Lal-forming cysteine residue which is instead substituted by serine. Nevertheless, Leptospira interrogans FlgE also forms Lal, with several different Lal isoforms being detected between Ser-179 and Lys-145, Lys-148, and Lys-166, thereby highlighting species or order-specific differences within the phylum. Our data reveal that the Lal cross-link is a conserved and necessary posttranslational modification across the spirochete phylum and may thus represent an effective target for the development of spirochete-specific antimicrobials., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

Circadian rhythms influence many behaviours and diseases 1,2 . They arise from oscillations in gene expression caused by repressor proteins that directly inhibit transcription of their own genes. The fly circadian clock offers a valuable model for studying these processes, wherein Timeless (Tim) plays a critical role in mediating nuclear entry of the transcriptional repressor Period (Per) and the photoreceptor Cryptochrome (Cry) entrains the clock by triggering Tim degradation in light 2,3 . Here, through cryogenic electron microscopy of the Cry-Tim complex, we show how a light-sensing cryptochrome recognizes its target. Cry engages a continuous core of amino-terminal Tim armadillo repeats, resembling how photolyases recognize damaged DNA, and binds a C-terminal Tim helix, reminiscent of the interactions between light-insensitive cryptochromes and their partners in mammals. The structure highlights how the Cry flavin cofactor undergoes conformational changes that couple to large-scale rearrangements at the molecular interface, and how a phosphorylated segment in Tim may impact clock period by regulating the binding of Importin-α and the nuclear import of Tim-Per 4,5 . Moreover, the structure reveals that the N terminus of Tim inserts into the restructured Cry pocket to replace the autoinhibitory C-terminal tail released by light, thereby providing a possible explanation for how the long-short Tim polymorphism adapts flies to different climates 6,7 ., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

The link between cofactor binding and protein activity is well-established. However, how cofactor interactions modulate folding of large proteins remains unknown. We use optical tweezers, clustering and global fitting to dissect the folding mechanism of Drosophila cryptochrome (dCRY), a 542-residue protein that binds FAD, one of the most chemically and structurally complex cofactors in nature. We show that the first dCRY parts to fold are independent of FAD, but later steps are FAD-driven as the remaining polypeptide folds around the cofactor. FAD binds to largely unfolded intermediates, yet with association kinetics above the diffusion-limit. Interestingly, not all FAD moieties are required for folding: whereas the isoalloxazine ring linked to ribitol and one phosphate is sufficient to drive complete folding, the adenosine ring with phosphates only leads to partial folding. Lastly, we propose a dCRY folding model where regions that undergo conformational transitions during signal transduction are the last to fold., (© 2023. The Author(s).)

The Per-Arnt-Sim (PAS; named for the representative proteins: Period, Aryl hydrocarbon receptor nuclear translocator protein and Single-minded) domain of the dimeric Escherichia coli aerotaxis receptor Aer monitors cellular respiration through a redox-sensitive flavin adenine dinucleotide (FAD) cofactor. Conformational shifts in the PAS domain instigated by the oxidized FAD (FAD OX )/FAD anionic semiquinone (FAD ASQ ) redox couple traverse the HAMP (histidine kinases, adenylate cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and kinase control domains of the Aer dimer to regulate CheA kinase activity. The PAS domain of Aer is unstable and has not been previously purified. Here, residue substitutions that rescue FAD binding in an FAD binding-deficient full-length Aer variant were used in combination to stabilize the Aer PAS domain. We solved the 2.4 Å resolution crystal structure of this variant, Aer-PAS-GVV, and revealed a PAS fold that contains distinct features associated with FAD-based redox sensing, such as a close contact between the Arg115 side chain and N5 of the isoalloxazine ring and interactions of the flavin with the side chains of His53 and Asn85 that are poised to convey conformational signals from the cofactor to the protein surface. In addition, we determined the FAD ox /FAD ASQ formal potentials of Aer-PAS-GVV and full-length Aer reconstituted into nanodiscs. The Aer redox couple is remarkably low at -289.6 ± 0.4 mV. In conclusion, we propose a model for Aer energy sensing based on the low potential of Aer-PAS-FAD ox /FAD ASQ couple and the inability of Aer-PAS to bind to the fully reduced FAD hydroquinone., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Cryptochrome (CRY) entrains the fly circadian clock by binding to Timeless (TIM) in light. Undocking of a helical C-terminal tail (CTT) in response to photoreduction of the CRY flavin cofactor gates TIM recognition. We present a generally applicable select western-blot-free tagged-protein interaction (SWFTI) assay that allowed the quantification of CRY binding to TIM in dark and light. The assay was used to study CRY variants with residue substitutions in the flavin pocket and correlate their TIM affinities with CTT undocking, as measured by pulse-dipolar ESR spectroscopy and evaluated by molecular dynamics simulations. CRY variants with the CTT removed or undocked bound TIM constitutively, whereas those incapable of photoreduction bound TIM weakly. In response to the flavin redox state, two conserved histidine residues contributed to a robust on/off switch by mediating CTT interactions with the flavin pocket and TIM. Our approach provides an expeditious means to quantify the interactions of difficult-to-produce proteins., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Ltd.)

Electron transport through aromatic species (especially tryptophan and tyrosine) plays a central role in water splitting, redox signaling, oxidative damage protection, and bioenergetics. The cytochrome c peroxidase (CcP)-cytochrome c (Cc) complex (CcP:Cc) is used widely to study interprotein electron transfer (ET) mechanisms. Tryptophan 191 (Trp191) of CcP supports hole hopping charge recombination in the CcP:Cc complex. Experimental studies find that when Trp191 is substituted by tyrosine, phenylalanine, or redox-active aniline derivatives bound in the W191G cavity, enzymatic activity and charge recombination rates both decrease. Theoretical analysis of these CcP:Cc complexes finds that the ET kinetics depend strongly on the chemistry of the modified Trp site. The computed electronic couplings in the W191F and W191G species are orders of magnitude smaller than in the native protein, due largely to the absence of a hopping intermediate and the large tunneling distance. Small molecules bound in the W191G cavity are weakly coupled electronically to the Cc heme, and the structural disorder of the guest molecule in the binding pocket may contribute further to the lack of enzymatic activity. The couplings in W191Y are not substantially weakened compared to the native species, but the redox potential difference for tyrosine vs tryptophan oxidation accounts for the slower rate in the Tyr mutant. Thus, theoretical analysis explains why only the native Trp supports rapid hole hopping in the CcP:Cc complex. Favorable free energies and electronic couplings are essential for establishing an efficient hole hopping relay in this protein-protein complex.

Light-induction of an anionic semiquinone (SQ) flavin radical in Drosophila cryptochrome (dCRY) alters the dCRY conformation to promote binding and degradation of the circadian clock protein Timeless (TIM). Specific peptide ligation with sortase A attaches a nitroxide spin-probe to the dCRY C-terminal tail (CTT) while avoiding deleterious side reactions. Pulse dipolar electron-spin resonance spectroscopy from the CTT nitroxide to the SQ shows that flavin photoreduction shifts the CTT ~1 nm and increases its motion, without causing full displacement from the protein. dCRY engineered to form the neutral SQ serves as a dark-state proxy to reveal that the CTT remains docked when the flavin ring is reduced but uncharged. Substitutions of flavin-proximal His378 promote CTT undocking in the dark or diminish undocking in the light, consistent with molecular dynamics simulations and TIM degradation activity. The His378 variants inform on recognition motifs for dCRY cellular turnover and strategies for developing optogenetic tools.

The prokaryotic chemotaxis system is arguably the best-understood signaling pathway in biology. In all previously described species, chemoreceptors organize into a hexagonal (P6 symmetry) extended array. Here, we report an alternative symmetry (P2) of the chemotaxis apparatus that emerges from a strict linear organization of the histidine kinase CheA in Treponema denticola cells, which possesses arrays with the highest native curvature investigated thus far. Using cryo-ET, we reveal that Td chemoreceptor arrays assume an unusual arrangement of the supra-molecular protein assembly that has likely evolved to accommodate the high membrane curvature. The arrays have several atypical features, such as an extended dimerization domain of CheA and a variant CheW-CheR-like fusion protein that is critical for maintaining an ordered chemosensory apparatus. Furthermore, the previously characterized Td oxygen sensor ODP influences CheA ordering. These results suggest a greater diversity of the chemotaxis signaling system than previously thought.

Mammalian cryptochromes regulate sleep and metabolism as components of the circadian clock. In this issue of Cell Chemical Biology, Miller et al. (2020a) use phenotypic chemical screens to identify selective modulators of two cryptochrome isoforms. Binding specificity depends on conformational patterning of the ligand-binding pocket and a disordered C-terminal domain., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bacteria sense and respond to their environment through a highly conserved assembly of transmembrane chemoreceptors (MCPs), the histidine kinase CheA, and the coupling protein CheW, hereafter termed "the chemosensory array". In recent years, great strides have been made in understanding the architecture of the chemosensory array and how this assembly engenders sensitive and cooperative responses. Nonetheless, a central outstanding question surrounds how receptors modulate the activity of the CheA kinase, the enzymatic output of the sensory system. With a focus on recent advances, we summarize the current understanding of array structure and function to comment on the molecular mechanism by which CheA, receptors and CheW generate the high sensitivity, gain and dynamic range emblematic of bacterial chemotaxis. The complexity of the chemosensory arrays has motivated investigation with many different approaches. In particular, structural methods, genetics, cellular activity assays, nanodisc technology and cryo-electron tomography have provided advances that bridge length scales and connect molecular mechanism to cellular function. Given the high degree of component integration in the chemosensory arrays, we ultimately aim to understand how such networked molecular interactions generate a whole that is truly greater than the sum of its parts. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins., (Copyright © 2019. Published by Elsevier B.V.)

Many bacteria contain cytoplasmic chemoreceptors that lack sensor domains. Here, we demonstrate that such cytoplasmic receptors found in 8 different bacterial and archaeal phyla genetically couple to metalloproteins related to β-lactamases and nitric oxide reductases. We show that this oxygen-binding di-iron protein (ODP) acts as a sensor for chemotactic responses to both iron and oxygen in the human pathogen Treponema denticola ( Td ). The ODP di-iron site binds oxygen at high affinity to reversibly form an unusually stable μ-peroxo adduct. Crystal structures of ODP from Td and the thermophile Thermotoga maritima ( Tm ) in the Fe[III] 2 -O 2 2- , Zn[II], and apo states display differences in subunit association, conformation, and metal coordination that indicate potential mechanisms for sensing. In reconstituted systems, iron-peroxo ODP destabilizes the phosphorylated form of the receptor-coupled histidine kinase CheA, thereby providing a biochemical link between oxygen sensing and chemotaxis in diverse prokaryotes, including anaerobes of ancient origin., Competing Interests: The authors declare no conflict of interest.

Nitric oxide synthases (NOSs) are heme-based monooxygenases that convert l-Arg to l-citrulline and nitric oxide (NO), a key signaling molecule and cytotoxic agent in mammals. Bacteria also contain NOS proteins, but the role of NO production within these organisms, where understood, differs considerably from that of mammals. For example, a NOS protein in the marine cyanobacterium Synechococcus sp. PCC 7335 (syNOS) has recently been proposed to function in nitrogen assimilation from l-Arg. syNOS retains the oxygenase (NOS ox ) and reductase (NOS red ) domains present in mammalian NOS enzymes (mNOSs), but also contains an N-terminal globin domain (NOS g ) homologous to bacterial flavohemoglobin proteins. Herein, we show that syNOS functions as a dimer and produces NO from l-Arg and NADPH in a tetrahydrobiopterin (H 4 B)-dependent manner at levels similar to those produced by other NOSs but does not require Ca 2+ -calmodulin, which regulates NOS red -mediated NOS ox reduction in mNOSs. Unlike other bacterial NOSs, syNOS cannot function with tetrahydrofolate and requires high Ca 2+ levels (>200 μm) for its activation. NOS g converts NO to NO 3 - in the presence of O 2 and NADPH; however, NOS g did not protect Escherichia coli strains against nitrosative stress, even in a mutant devoid of NO-protective flavohemoglobin. We also found that syNOS does not have NOS activity in E. coli (which lacks H 4 B) and that the recombinant protein does not confer growth advantages on l-Arg as a nitrogen source. Our findings indicate that syNOS has both NOS and NO oxygenase activities, requires H 4 B, and may play a role in Ca 2+ -mediated signaling., (© 2019 Picciano and Crane.)

Pulsed dipolar electron spin resonance spectroscopy (PDS) is a powerful tool for measuring distances in solution-state macromolecules. Paramagnetic metal ions, such as Cu 2+ , are used as spin probes because they can report on metalloprotein features and can be spectroscopically distinguished from traditional nitroxide (NO)-based labels. Here, we demonstrate site-specific incorporation of Cu 2+ into non-metalloproteins through the use of a genetically encodable non-natural amino acid, 3-pyrazolyltyrosine (PyTyr). We first incorporate PyTyr in cyan fluorescent protein to measure Cu 2+ -to-NO distances and examine the effects of solvent conditions on Cu 2+ binding and protein aggregation. We then apply the method to characterize the complex formed by the histidine kinase CheA and its target response regulator CheY. The X-ray structure of CheY-PyTyr confirms Cu labeling at PyTyr but also reveals a secondary Cu site. Cu 2+ -to-NO and Cu 2+ -to-Cu 2+ PDS measurements of CheY-PyTyr with nitroxide-labeled CheA provide new insights into the conformational landscape of the phosphotransfer complex and have implications for kinase regulation.

A cognitively intensive companion service course has been introduced to the main fall general chemistry class at Cornell University. For years 2015 and 2016, priority students (those from groups under-represented and economically disadvantaged) show respectively improvement of +0.67 and +0.51 standard deviations in final course grade compared to priority students not in the program. Non-priority students show respectively a +0.66 and +0.62 standard deviation improvement. Progressive improvement (as measured by higher than expected Final Exam scores than what would have been expected solely from a given student's earlier Exam 1 score) demonstrates conclusively the service course's role in the enhanced outcomes. Progressive retention (as measured by the following year fall semester's organic chemistry exam scores compared to what would have been expected based on a given student's general chemistry final exam score) demonstrates that, on the average, the earlier observed progressive improvement is significantly retained in a chemistry course one year later. Preliminary retention statistics suggest a significant increase in first year to second year retention. A meta analysis of results from previously reported chemistry service courses indicate that such performance gains are difficult to achieve and hence common elements of the few effective programs may be of high value to the STEM education community., Competing Interests: The authors have declared that no competing interests exist.

The diarrheal pathogen Vibrio cholerae navigates complex environments using three chemosensory systems and 44-45 chemoreceptors. Chemosensory cluster II modulates chemotaxis, whereas clusters I and III have unknown functions. Ligands have been identified for only five V. cholerae chemoreceptors. Here, we report that the cluster III receptor, VcAer2, binds and responds to O 2 . VcAer2 is an ortholog of Pseudomonas aeruginosa Aer2 (PaAer2) but differs in that VcAer2 has two, rather than one, N-terminal PAS domain. We have determined that both PAS1 and PAS2 form homodimers and bind penta-coordinate b-type heme via an Eη-His residue. Heme binding to PAS1 required the entire PAS core, but receptor function also required the N-terminal cap. PAS2 functioned as an O 2 -sensor [ K d( O 2 ) , 19 μM], utilizing the same Iβ Trp (W276) as PaAer2 to stabilize O 2 . The crystal structure of PAS2-W276L was similar to that of PaAer2-PAS but resided in an active conformation mimicking the ligand-bound state, consistent with its signal-on phenotype. PAS1 also bound O 2 [ K d( O 2 ) , 12 μM], although O 2 binding was stabilized by either a Trp residue or Tyr residue. Moreover, PAS1 appeared to function as a signal modulator, regulating O 2 -mediated signaling from PAS2 and resulting in activation of the cluster III chemosensory pathway., (© 2018 John Wiley & Sons Ltd.)

Cryptochromes (CRYs) entrain the circadian clocks of plants and animals to light. Irradiation of the Drosophila cryptochrome (dCRY) causes reduction of an oxidized flavin cofactor by a chain of conserved tryptophan (Trp) residues. However, it is unclear how redox chemistry within the Trp chain couples to dCRY-mediated signaling. Here, we show that substitutions of four key Trp residues to redox-active tyrosine and redox-inactive phenylalanine tune the light sensitivity of dCRY photoreduction, conformational activation, cellular stability, and targeted degradation of the clock protein timeless (TIM). An essential surface Trp gates electron flow into the flavin cofactor, but can be relocated for enhanced photoactivation. Differential effects of Trp-mediated flavin photoreduction on cellular turnover of TIM and dCRY indicate that these activities are separated in time and space. Overall, the dCRY Trp chain has evolutionary importance for light sensing, and its manipulation has implications for optogenetic applications of CRYs., Competing Interests: The authors declare no conflict of interest.

In the Drosophila circadian clock, Period (PER) and Timeless (TIM) proteins inhibit Clock-mediated transcription of per and tim genes until PER is degraded by Doubletime/CK1 (DBT)-mediated phosphorylation, establishing a negative feedback loop. Multiple regulatory delays within this feedback loop ensure ~24 hr periodicity. Of these delays, the mechanisms that regulate delayed PER degradation (and Clock reactivation) remain unclear. Here we show that phosphorylation of certain DBT target sites within a central region of PER affect PER inhibition of Clock and the stability of the PER/TIM complex. Our results indicate that phosphorylation of PER residue S589 stabilizes and activates PER inhibitory function in the presence of TIM, but promotes PER degradation in its absence. The role of DBT in regulating PER activity, stabilization and degradation ensures that these events are chronologically and biochemically linked, and contributes to the timing of an essential delay that influences the period of the circadian clock., Competing Interests: DT, JO, GM, KD, BC, MY No competing interests declared, (© 2018, Top et al.)

Phosphorylation of CheY promotes association with the flagellar motor and ultimately controls the directional bias of the motor. However, biochemical studies of activated CheY-phosphate have been challenging due to the rapid hydrolysis of the aspartyl-phosphate in vitro. An inert analog of Tm CheY-phosphate, phosphono-CheY, was synthesized by chemical modification and purified by cation-exchange chromatography. Changes in HPLC retention times, chemical assays for phosphate and free thiol, and mass spectrometry experiments demonstrate modification of Cys54 with a phosphonomethyl group. Additionally, a crystal structure showed electron density for the phosphonomethyl group at Cys54, consistent with a modification at that position. Subsequent biochemical experiments confirmed that protein crystals were phosphono-CheY. Isothermal titration calorimetry and fluorescence polarization binding assays demonstrated that phosphono-CheY bound a peptide derived from FliM, a native partner of CheY-phosphate, with a dissociation constant of ∼29 µM, at least sixfold more tightly than unmodified CheY. Taken together these results suggest that Tm phosphono-CheY is a useful and unique analog of Tm CheY-phosphate., (© 2017 The Protein Society.)

HAMP domains are dimeric, four-helix bundles that transduce conformational signals in bacterial receptors. Genetic studies of the Escherichia coli serine receptor (Tsr) provide an opportunity to understand HAMP conformational behavior in terms of functional output. To increase its stability, the Tsr HAMP domain was spliced into a poly-HAMP unit from the Pseudomonas aeruginosa Aer2 receptor. Within the chimera, the Tsr HAMP undergoes a thermal melting transition at a temperature much lower than that of the Aer2 HAMP domains. Pulse-dipolar electron spin resonance spectroscopy and site-specific spin-labeling confirm that the Tsr HAMP maintains a four-helix bundle. Pulse-dipolar electron spin resonance spectroscopy was also used to study three well-characterized HAMP mutational phenotypes: those that cause flagella rotation that is counterclockwise (CCW) A and kinase-off; CCW B and also kinase-off; and, clockwise (CW) and kinase-on. Conformational properties of the three HAMP variants support a biphasic model of dynamic bundle stability, but also indicate distinct conformational changes within the helix bundle. Functional kinase-on (CW) and kinase-off (CCW A) states differ by concerted changes in the positions of spin-label sites at the base of the bundle. Opposite shifts in the subunit separation distances of neighboring residues at the C-termini of the α1 and α2 helices are consistent with a helix scissors motion or a gearbox rotational model of HAMP activation. In the drastic kinase-off lesion of CCW B, the α1 helices unfold and the α2 helices form a tight two-helix coiled-coil. The substitution of a critical residue in the Tsr N-terminal linker or control cable reduces conformational heterogeneity at the N-terminus of α1 but does not affect structure at the C-terminus of α2. Overall, the data suggest that transitions from on- to off-states involve decreased motional amplitudes of the Tsr HAMP coupled with helix rotations and movements toward a two-helix packing mode., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The interface between the membrane (MS) and cytoplasmic (C) rings of the bacterial flagellar motor couples torque generation to rotation within the membrane. The structure of the C-terminal helices of the integral membrane protein FliF (FliF C ) bound to the N terminal domain of the switch complex protein FliG (FliG N ) reveals that FliG N folds around FliF C to produce a topology that closely resembles both the middle and C-terminal domains of FliG. The interface is consistent with solution-state nuclear magnetic resonance, small-angle X-ray scattering, in vivo interaction studies, and cellular motility assays. Co-folding with FliF C induces substantial conformational changes in FliG N and suggests that FliF and FliG have the same stoichiometry within the rotor. Modeling the FliF C :FliG N complex into cryo-electron microscopy rotor density updates the architecture of the middle and upper switch complex and shows how domain shuffling of a conserved interaction module anchors the cytoplasmic rotor to the membrane., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Flagellated bacteria modulate their swimming behavior in response to environmental cues through the CheA/CheY signaling pathway. In addition to responding to external chemicals, bacteria also monitor internal conditions that reflect the availability of oxygen, light, and reducing equivalents, in a process termed "energy taxis." In Escherichia coli, the transmembrane receptor Aer is the primary energy sensor for motility. Genetic and physiological data suggest that Aer monitors the electron transport chain through the redox state of its FAD cofactor. However, direct biochemical data correlating FAD redox chemistry with CheA kinase activity have been lacking. Here, we test this hypothesis via functional reconstitution of Aer into nanodiscs. As purified, Aer contains fully oxidized FAD, which can be chemically reduced to the anionic semiquinone (ASQ). Oxidized Aer activates CheA, whereas ASQ Aer reversibly inhibits CheA. Under these conditions, Aer cannot be further reduced to the hydroquinone, in contrast to the proposed Aer signaling model. Pulse ESR spectroscopy of the ASQ corroborates a potential mechanism for signaling in that the resulting distance between the two flavin-binding PAS (Per-Arnt-Sim) domains implies that they tightly sandwich the signal-transducing HAMP domain in the kinase-off state. Aer appears to follow oligomerization patterns observed for related chemoreceptors, as higher loading of Aer dimers into nanodiscs increases kinase activity. These results provide a new methodological platform to study Aer function along with new mechanistic details into its signal transduction process., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pKa Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions., Competing Interests: The authors declare no conflict of interest.

In the Neurospora crassa circadian clock, a protein complex of frequency (FRQ), casein kinase 1a (CK1a), and the FRQ-interacting RNA Helicase (FRH) rhythmically represses gene expression by the white-collar complex (WCC). FRH crystal structures in several conformations and bound to ADP/RNA reveal differences between FRH and the yeast homolog Mtr4 that clarify the distinct role of FRH in the clock. The FRQ-interacting region at the FRH N-terminus has variable structure in the absence of FRQ A known mutation that disrupts circadian rhythms (R806H) resides in a positively charged surface of the KOW domain, far removed from the helicase core. We show that changes to other similarly located residues modulate interactions with the WCC and FRQ A V142G substitution near the N-terminus also alters FRQ and WCC binding to FRH, but produces an unusual short clock period. These data support the assertion that FRH helicase activity does not play an essential role in the clock, but rather FRH acts to mediate contacts among FRQ, CK1a and the WCC through interactions involving its N-terminus and KOW module., (© 2016 The Authors.)

Light-oxygen-voltage (LOV) receptors sense blue light through the photochemical generation of a covalent adduct between a flavin-nucleotide chromophore and a strictly conserved cysteine residue. Here we show that, after cysteine removal, the circadian-clock LOV-protein Vivid still undergoes light-induced dimerization and signalling because of flavin photoreduction to the neutral semiquinone (NSQ). Similarly, photoreduction of the engineered LOV histidine kinase YF1 to the NSQ modulates activity and downstream effects on gene expression. Signal transduction in both proteins hence hinges on flavin protonation, which is common to both the cysteinyl adduct and the NSQ. This general mechanism is also conserved by natural cysteine-less, LOV-like regulators that respond to chemical or photoreduction of their flavin cofactors. As LOV proteins can react to light even when devoid of the adduct-forming cysteine, modern LOV photoreceptors may have arisen from ancestral redox-active flavoproteins. The ability to tune LOV reactivity through photoreduction may have important implications for LOV mechanism and optogenetic applications.

Dynamics are hypothesized to play an important role in the transmission of signals across membranes by receptors. Bacterial chemoreceptors are long helical proteins that consist of a periplasmic ligand-binding domain; a transmembrane region; a cytoplasmic HAMP (histidine kinase, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) domain; and a kinase-control module (KCM). The KCM is further composed of adaptation, hinge, and protein interaction regions (PIRs), the latter of which binds the histidine kinase CheA and adaptor CheW. Fusions of the Escherichia coli aspartate receptor KCM to HAMP domains of defined structure (H1-Tar vs. H1-2-Tar) give opposite responses in phosphotransfer and cellular assays, despite similar binding to CheA and CheW. Pulsed dipolar ESR spectroscopy (PDS) of these isolated on and off dimeric effectors reveals that, in the kinase-on state, the HAMP is more conformationally destabilized compared with the PIR, whereas in the kinase-off state, the HAMP is more compact, and the PIR samples a greater breadth of conformations. On and off HAMP states produce different conformational effects at the KCM junction, but these differences decrease through the adaptation region and into the hinge only to return with the inverted relationship in the PIR. Continuous wave-ESR of the spin-labeled proteins confirms that broader PDS distance distributions correlate with increased rates of dynamics. Conformational breadth in the adaptation region changes with charge alterations caused by modification enzymes. Activating modifications broaden the HAMP conformational ensemble but correspondingly, compact the PIR. Thus, chemoreceptors behave as coupled units, in which dynamics in regions proximal and distal to the membrane change coherently but with opposite sign.

Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu(2+) binding site and defined cross-dimer copper-copper distance distributions. Importantly, we find that copper deficiency in these mutants promotes aggregation in a manner strikingly consistent with their clinical severities. G93 mutants seem to properly incorporate metal ions under physiological conditions when assisted by the copper chaperone but release copper under destabilizing conditions more readily than the WT enzyme. Altered intradimer flexibility in ALS mutants may cause differential metal retention and promote distinct aggregation trends observed for mutant proteins in vitro and in ALS patients. Combined biophysical and structural results test and link copper retention to the framework destabilization hypothesis as a unifying general mechanism for both SOD aggregation and ALS disease progression, with implications for disease severity and therapeutic intervention strategies.

We demonstrate the ability of pulsed dipolar electron spin resonance (ESR) spectroscopy (PDS) to report on the conformation of Cu-Zn superoxide dismutase (SOD1) through the sensitive measurement of dipolar interactions between inherent Cu(2+) ions. Although the extent and the anisotropy of the Cu ESR spectrum provides challenges for PDS, Ku-band (17.3 GHz) double electron-electron resonance and double-quantum coherence variants of PDS coupled with distance reconstruction methods recover Cu-Cu distances in good agreement with crystal structures. Moreover, Cu-PDS measurements expose distinct differences between the conformational properties of wild-type SOD1 and a single-residue variant (I149T) that leads to the disease amyotrophic lateral sclerosis (ALS). The I149T protein displays a broader Cu-Cu distance distribution within the SOD1 dimer compared to wild-type. In a nitroxide (NO)-labeled sample, distance distributions obtained from Cu-Cu, Cu-NO, and NO-NO separations reveal increased structural heterogeneity within the protein and a tendency for mutant dimers to associate. In contrast, perturbations caused by the ALS mutation are completely masked in the crystal structure of I149T. Thus, PDS readily detects alterations in metalloenzyme solution properties not easily deciphered by other methods and in doing so supports the notion that increased range of motion and associations of SOD1 ALS variants contribute to disease progression., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Entrainment of circadian rhythms in higher organisms relies on light-sensing proteins that communicate to cellular oscillators composed of delayed transcriptional feedback loops. The principal photoreceptor of the fly circadian clock, Drosophila cryptochrome (dCRY), contains a C-terminal tail (CTT) helix that binds beside a FAD cofactor and is essential for light signaling. Light reduces the dCRY FAD to an anionic semiquinone (ASQ) radical and increases CTT proteolytic susceptibility but does not lead to CTT chemical modification. Additional changes in proteolytic sensitivity and small-angle X-ray scattering define a conformational response of the protein to light that centers at the CTT but also involves regions remote from the flavin center. Reduction of the flavin is kinetically coupled to CTT rearrangement. Chemical reduction to either the ASQ or the fully reduced hydroquinone state produces the same conformational response as does light. The oscillator protein Timeless (TIM) contains a sequence similar to the CTT; the corresponding peptide binds dCRY in light and protects the flavin from oxidation. However, TIM mutants therein still undergo dCRY-mediated degradation. Thus, photoreduction to the ASQ releases the dCRY CTT and promotes binding to at least one region of TIM. Flavin reduction by either light or cellular reductants may be a general mechanism of CRY activation.

Charge recombination rate constants vary no more than 3-fold for interprotein ET in the Zn-substituted wild type (WT) cytochrome c peroxidase (CcP):cytochrome c (Cc) complex and in complexes with four mutants of the Cc protein (i.e., F82S, F82W, F82Y, and F82I), despite large differences in the ET distance. Theoretical analysis indicates that charge recombination for all complexes involves a combination of tunneling and hopping via Trp191. For three of the five structures (WT and F82S(W)), the protein favors hopping more than that in the other two structures that have longer heme → ZnP distances (F82Y(I)). Experimentally observed biexponential ET kinetics is explained by the complex locking in alternative coupling pathways, where the acceptor hole state is either primarily localized on ZnP (slow phase) or on Trp191 (fast phase). The large conformational differences between the CcP:Cc interface for the F82Y(I) mutants compared to that the WT and F82S(W) complexes are predicted to change the reorganization energies for the CcP:Cc ET reactions because of changes in solvent exposure and interprotein ET distances. Since the recombination reaction is likely to occur in the inverted Marcus regime, an increased reorganization energy compensates the decreased role for hopping recombination (and the longer transfer distance) in the F82Y(I) mutants. Taken together, coupling pathway and reorganization energy effects for the five protein complexes explain the observed insensitivity of recombination kinetics to donor-acceptor distance and docking pose and also reveals how hopping through aromatic residues can accelerate long-range ET.

Background: FliY is a flagellar rotor protein of the CheC phosphatase family., Results: The FliY structure resembles that of the rotor protein FliM but contains two active centers for CheY dephosphorylation., Conclusion: FliY incorporates properties of the FliM/FliN rotor proteins and the CheC/CheX phosphatases to serve multiple functions in the flagellar switch., Significance: FliY distinguishes flagellar architecture and function in different types of bacteria. Rotating flagella propel bacteria toward favorable environments. Sense of rotation is determined by the intracellular response regulator CheY, which when phosphorylated (CheY-P) interacts directly with the flagellar motor. In many different types of bacteria, the CheC/CheX/FliY (CXY) family of phosphatases terminates the CheY-P signal. Unlike CheC and CheX, FliY is localized in the flagellar switch complex, which also contains the stator-coupling protein FliG and the target of CheY-P, FliM. The 2.5 Å resolution crystal structure of the FliY catalytic domain from Thermotoga maritima bears strong resemblance to the middle domain of FliM. Regions of FliM that mediate contacts within the rotor compose the phosphatase active sites in FliY. Despite the similarity between FliY and FliM, FliY does not bind FliG and thus is unlikely to be a substitute for FliM in the center of the switch complex. Solution studies indicate that FliY dimerizes through its C-terminal domains, which resemble the Escherichia coli switch complex component FliN. FliY differs topologically from the E. coli chemotaxis phosphatase CheZ but appears to utilize similar structural motifs for CheY dephosphorylation in close analogy to CheX. Recognition properties and phosphatase activities of site-directed mutants identify two pseudosymmetric active sites in FliY (Glu(35)/Asn(38) and Glu(132)/Asn(135)), with the second site (Glu(132)/Asn(135)) being more active. A putative N-terminal CheY binding domain conserved with FliM is not required for binding CheY-P or phosphatase activity.

HAMP domains are signal relay modules in >26,000 receptors of bacteria, eukaryotes, and archaea that mediate processes involved in chemotaxis, pathogenesis, and biofilm formation. We identify two HAMP conformations distinguished by a four- to two-helix packing transition at the C-termini that send opposing signals in bacterial chemoreceptors. Crystal structures of signal-locked mutants establish the observed structure-to-function relationships. Pulsed dipolar electron spin resonance spectroscopy of spin-labeled soluble receptors active in cells verify that the crystallographically defined HAMP conformers are maintained in the receptors and influence the structure and activity of downstream domains accordingly. Mutation of HR2, a key residue for setting the HAMP conformation and generating an inhibitory signal, shifts HAMP structure and receptor output to an activating state. Another HR2 variant displays an inverted response with respect to ligand and demonstrates the fine energetic balance between "on" and "off" conformers. A DExG motif found in membrane proximal HAMP domains is shown to be critical for responses to extracellular ligand. Our findings directly correlate in vivo signaling with HAMP structure, stability, and dynamics to establish a comprehensive model for HAMP-mediated signal relay that consolidates existing views on how conformational signals propagate in receptors. Moreover, we have developed a rational means to manipulate HAMP structure and function that may prove useful in the engineering of bacterial taxis responses., Competing Interests: The authors have declared that no competing interests exist.

The CheA-CheW complex plays a key role in bacterial chemotaxis signal transduction by initiating phosphotransfer to response regulators via coupling to the chemoreceptors. CheA (P3-P4-P5 domains) and CheW from Thermotoga maritima were overexpressed in Escherichia coli and crystallized as a complex at 298 K using ammonium dihydrogen phosphate as a precipitant. X-ray diffraction data were collected to ~8 Å resolution at 100 K using synchrotron radiation. The crystal belonged to space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 184.2, b = 286.4, c = 327.7 Å. The asymmetric unit may contain six to ten CheA-CheW molecules.

Biologically important protein complexes often involve molecular interactions that are low affinity or transient. We apply pulsed dipolar electron spin resonance spectroscopy and site-directed spin labeling in what to our knowledge is a new approach to study aggregation and to identify regions on protein surfaces that participate in weak, but specific molecular interactions. As a test case, we have probed the self-association of the chemotaxis kinase CheA, which forms signaling clusters with chemoreceptors and the coupling protein CheW at the poles of bacterial cells. By measuring the intermolecular dipolar interactions sensed by spin-labels distributed over the protein surface, we show that the soluble CheA kinase aggregates to a small extent through interactions mediated by its regulatory (P5) domain. Direct dipolar distance measurements confirm that a hydrophobic surface at the periphery of P5 subdomain 2 associates CheA dimers in solution. This result is further supported by differential disulfide cross-linking from engineered cysteine reporter sites. We suggest that the periphery of P5 is an interaction site on CheA for other similar hydrophobic surfaces and plays an important role in structuring the signaling particle., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Chemoreceptor arrays are supramolecular transmembrane machines of unknown structure that allow bacteria to sense their surroundings and respond by chemotaxis. We have combined X-ray crystallography of purified proteins with electron cryotomography of native arrays inside cells to reveal the arrangement of the component transmembrane receptors, histidine kinases (CheA) and CheW coupling proteins. Trimers of receptor dimers lie at the vertices of a hexagonal lattice in a "two-facing-two" configuration surrounding a ring of alternating CheA regulatory domains (P5) and CheW couplers. Whereas the CheA kinase domains (P4) project downward below the ring, the CheA dimerization domains (P3) link neighboring rings to form an extended, stable array. This highly interconnected protein architecture underlies the remarkable sensitivity and cooperative nature of transmembrane signaling in bacterial chemotaxis.

The cryptochrome/photolyase (CRY/PL) family of photoreceptors mediates adaptive responses to ultraviolet and blue light exposure in all kingdoms of life. Whereas PLs function predominantly in DNA repair of cyclobutane pyrimidine dimers (CPDs) and 6-4 photolesions caused by ultraviolet radiation, CRYs transduce signals important for growth, development, magnetosensitivity and circadian clocks. Despite these diverse functions, PLs/CRYs preserve a common structural fold, a dependence on flavin adenine dinucleotide (FAD) and an internal photoactivation mechanism. However, members of the CRY/PL family differ in the substrates recognized (protein or DNA), photochemical reactions catalysed and involvement of an antenna cofactor. It is largely unknown how the animal CRYs that regulate circadian rhythms act on their substrates. CRYs contain a variable carboxy-terminal tail that appends the conserved PL homology domain (PHD) and is important for function. Here, we report a 2.3-Å resolution crystal structure of Drosophila CRY with an intact C terminus. The C-terminal helix docks in the analogous groove that binds DNA substrates in PLs. Conserved Trp 536 juts into the CRY catalytic centre to mimic PL recognition of DNA photolesions. The FAD anionic semiquinone found in the crystals assumes a conformation to facilitate restructuring of the tail helix. These results help reconcile the diverse functions of the CRY/PL family by demonstrating how conserved protein architecture and photochemistry can be elaborated into a range of light-driven functions.

Rotation and switching of the bacterial flagellum depends on a large rotor-mounted protein assembly composed of the proteins FliG, FliM and FliN, with FliG most directly involved in rotation. The crystal structure of a complex between the central domains of FliG and FliM, in conjunction with several biochemical and molecular-genetic experiments, reveals the arrangement of the FliG and FliM proteins in the rotor. A stoichiometric mismatch between FliG (26 subunits) and FliM (34 subunits) is explained in terms of two distinct positions for FliM: one where it binds the FliG central domain and another where it binds the FliG C-terminal domain. This architecture provides a structural framework for addressing the mechanisms of motor rotation and direction switching and for unifying the large body of data on motor performance. Recently proposed alternative models of rotor assembly, based on a subunit contact observed in crystals, are not supported by experiment.

The chemotaxis histidine kinase CheA assembles into a dimer in which the P3 dimerization domain forms a four-helix bundle by the parallel association of two α-helical hairpins from each subunit. Ligand occupancy of the chemoreceptor regulates signal transduction by controlling the autophosphorylation activity of CheA. Autophosphorylation of CheA occurs in trans, i.e. one subunit phosphorylates the other. The P3 domain of CheA from Escherichia coli has been overexpressed in E. coli and crystallized at 298 K using PEG as a precipitant. X-ray diffraction data to 2.80 Å resolution have been collected at 100 K using synchrotron radiation. The crystal belonged to space group P1, with unit-cell parameters a = 59.271, b = 67.674, c = 82.815 Å, α = 77.568, β = 86.073, γ = 64.436°. The asymmetric unit may contain up to ten dimeric units of P3 four-helix bundles.

The CheW protein plays a key role in bacterial chemotaxis signal transduction by coupling CheA to chemotaxis receptors. CheW from Thermotoga maritima has been overexpressed in Escherichia coli and crystallized at 298 K using ammonium sulfate as a salt precipitant. X-ray diffraction data have been collected to 3.10 Å resolution at 100 K using synchrotron radiation. The crystal belonged to space group P6(3), with unit-cell parameters a = b = 61.265, c = 361.045 Å. The asymmetric unit may contain four to six CheW molecules.

Homologs to mammalian nitric oxide synthases are found in many mostly Gram-positive bacteria. In some genera such as bacilli, and staphylococci, these enzymes produce protects against oxidative damage, this effect has now been shown to provide an advantage against antibiotics that kill by increasing cellular levels of reactive oxygen species.

HAMP domains are widespread prokaryotic signaling modules found as single domains or poly-HAMP chains in both transmembrane and soluble proteins. The crystal structure of a three-unit poly-HAMP chain from the Pseudomonas aeruginosa soluble receptor Aer2 defines a universal parallel four-helix bundle architecture for diverse HAMP domains. Two contiguous domains integrate to form a concatenated di-HAMP structure. The three HAMP domains display two distinct conformations that differ by changes in helical register, crossing angle, and rotation. These conformations are stabilized by different subsets of conserved residues. Known signals delivered to HAMP would be expected to switch the relative stability of the two conformations and the position of a coiled-coil phase stutter at the junction with downstream helices. We propose that the two conformations represent opposing HAMP signaling states and suggest a signaling mechanism whereby HAMP domains interconvert between the two states, which alternate down a poly-HAMP chain., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Deinococcus radiodurans (Dr) withstands desiccation, reactive oxygen species, and doses of radiation that would be lethal to most organisms. Deletion of a gene encoding a homolog of mammalian nitric oxide synthase (NOS) severely compromises the recovery of Dr from ultraviolet (UV) radiation damage. The Deltanos defect can be complemented with recombinant NOS, rescued by exogenous nitric oxide (NO) and mimicked in the wild-type strain with an NO scavenging compound. UV radiation induces both upregulation of the nos gene and cellular NO production on similar time scales. Growth recovery does not depend on NO being present during UV irradiation, but rather can be manifested by NO addition hours after exposure. Surprisingly, nos deletion does not increase sensitivity to oxidative damage, and hydrogen peroxide does not induce nos expression. However, NOS-derived NO upregulates transcription of obgE, a gene involved in bacterial growth proliferation and stress response. Overexpression of the ObgE GTPase in the Deltanos background substantially alleviates the growth defect after radiation damage. Thus, NO acts as a signal for the transcriptional regulation of growth in D. radiodurans.