Magnetic sensitivity of cryptochrome 4 from a migratory songbird.

Night-migratory songbirds are remarkably proficient navigators1. Flying alone and often over great distances, they use various directional cues including, crucially, a light-dependent magnetic compass2,3. The mechanism of this compass has been suggested to rely on the quantum spin dynamics of photoinduced radical pairs in cryptochrome flavoproteins located in the retinas of the birds4-7. Here we show that the photochemistry of cryptochrome 4 (CRY4) from the night-migratory European robin (Erithacus rubecula) is magnetically sensitive in vitro, and more so than CRY4 from two non-migratory bird species, chicken (Gallus gallus) and pigeon (Columba livia). Site-specific mutations of ErCRY4 reveal the roles of four successive flavin-tryptophan radical pairs in generating magnetic field effects and in stabilizing potential signalling states in a way that could enable sensing and signalling functions to be independently optimized in night-migratory birds.

Tissue-specific FAH deficiency alters sleep-wake patterns and results in chronic tyrosinemia in mice.

Fumarylacetoacetate hydrolase (FAH) is the last enzyme in tyrosine catabolism, and mutations in the FAH gene are associated with hereditary tyrosinemia type I (HT1 or TYRSN1) in humans. In a behavioral screen of N-ethyl-N-nitrosourea mutagenized mice we identified a mutant line which we named "swingshift" (swst, MGI:3611216) with a nonsynonymous point mutation (N68S) in Fah that caused age-dependent disruption of sleep-wake patterns. Mice homozygous for the mutation had an earlier onset of activity (several hours before lights off) and a reduction in total activity and body weight when compared with wild-type or heterozygous mice. Despite abnormal behavioral entrainment to light-dark cycles, there were no differences in the period or phase of the central clock in mutant mice, indicating a defect downstream of the suprachiasmatic nucleus. Interestingly, these behavioral phenotypes became milder as the mice grew older and were completely rescued by the administration of NTBC [2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione], an inhibitor of 4-hydroxyphenylpyruvate dioxygenase, which is upstream of FAH. Mechanistically, the swst mutation had no effect on the enzymatic activity of FAH, but rather promoted the degradation of the mutant protein. This led to reduced FAH protein levels and enzymatic activity in the liver and kidney (but not the brain or fibroblasts) of homozygous mice. In addition, plasma tyrosine-but not methionine, phenylalanine, or succinylacetone-increased in homozygous mice, suggesting that swst mutants provide a model of mild, chronic HT1.

Chemical and structural analysis of a photoactive vertebrate cryptochrome from pigeon.

Computational and biochemical studies implicate the blue-light sensor cryptochrome (CRY) as an endogenous light-dependent magnetosensor enabling migratory birds to navigate using the Earth's magnetic field. Validation of such a mechanism has been hampered by the absence of structures of vertebrate CRYs that have functional photochemistry. Here we present crystal structures of Columba livia (pigeon) CRY4 that reveal evolutionarily conserved modifications to a sequence of Trp residues (Trp-triad) required for CRY photoreduction. In ClCRY4, the Trp-triad chain is extended to include a fourth Trp (W369) and a Tyr (Y319) residue at the protein surface that imparts an unusually high quantum yield of photoreduction. These results are consistent with observations of night migratory behavior in animals at low light levels and could have implications for photochemical pathways allowing magnetosensing.

Formation of a repressive complex in the mammalian circadian clock is mediated by the secondary pocket of CRY1.

The basic helix-loop-helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ∼24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here we show that CRY1 binds directly to the PAS domain core of CLOCK:BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solution X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1.

Circadian Oscillations of NADH Redox State Using a Heterologous Metabolic Sensor in Mammalian Cells.

It is known that there are mechanistic links between circadian clocks and metabolic cycles. Reduced nicotinamide adenine dinucleotide (NADH) is a key metabolic cofactor in all living cells; however, it is not known whether levels of NADH oscillate or not. Here we employed REX, a bacterial NADH-binding protein, fused to the VP16 activator to convert intracellular endogenous redox balance into transcriptional readouts by a reporter gene in mammalian cells. EMSA results show that the DNA binding activity of both T- and S-REX::VP16 fusions is decreased with a reduced-to-oxidized cofactor ratio increase. Transient and stabilized cell lines bearing the REX::VP16 and the REX binding operator (ROP) exhibit two circadian luminescence cycles. Consistent with these results, NADH oscillations are observed in host cells, indicating REX can act as a NADH sensor to report intracellular dynamic redox homeostasis in mammalian cells in real time. NADH oscillations provide another metabolic signal for coupling the circadian clock and cellular metabolic states.

A heterodimeric complex of the LRR proteins LRIM1 and APL1C regulates complement-like immunity in Anopheles gambiae.

The leucine-rich repeat (LRR) proteins LRIM1 and APL1C control the function of the complement-like protein TEP1 in Anopheles mosquitoes. The molecular structure of LRIM1 and APL1C and the basis of their interaction with TEP1 represent a new type of innate immune complex. The LRIM1/APL1C complex specifically binds and solubilizes a cleaved form of TEP1 without an intact thioester bond. The LRIM1 and APL1C LRR domains have a large radius of curvature, glycosylated concave face, and a novel C-terminal capping motif. The LRIM1/APL1C complex is a heterodimer with a single intermolecular disulfide bond. The structure of the LRIM1/APL1C heterodimer reveals an interface between the two LRR domains and an extensive C-terminal coiled-coil domain. We propose that a cleaved form of TEP1 may act as a convertase for activation of other TEP1 molecules and that the LRIM1/APL1C heterodimer regulates formation of this TEP1 convertase.

A Drosophila pattern recognition receptor contains a peptidoglycan docking groove and unusual L,D-carboxypeptidase activity.

The Drosophila peptidoglycan recognition protein SA (PGRP-SA) is critically involved in sensing bacterial infection and activating the Toll signaling pathway, which induces the expression of specific antimicrobial peptide genes. We have determined the crystal structure of PGRP-SA to 2.2-A resolution and analyzed its peptidoglycan (PG) recognition and signaling activities. We found an extended surface groove in the structure of PGRP-SA, lined with residues that are highly diverse among different PGRPs. Mutational analysis identified it as a PG docking groove required for Toll signaling and showed that residue Ser158 is essential for both PG binding and Toll activation. Contrary to the general belief that PGRP-SA has lost enzyme function and serves primarily for PG sensing, we found that it possesses an intrinsic L,D-carboxypeptidase activity for diaminopimelic acid-type tetrapeptide PG fragments but not lysine-type PG fragments, and that Ser158 and His42 may participate in the hydrolytic activity. As L,D-configured peptide bonds exist only in prokaryotes, this work reveals a rare enzymatic activity in a eukaryotic protein known for sensing bacteria and provides a possible explanation of how PGRP-SA mediates Toll activation specifically in response to lysine-type PG.

Crystal structure of the heterodimeric CLOCK:BMAL1 transcriptional activator complex.

The circadian clock in mammals is driven by an autoregulatory transcriptional feedback mechanism that takes approximately 24 hours to complete. A key component of this mechanism is a heterodimeric transcriptional activator consisting of two basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) domain protein subunits, CLOCK and BMAL1. Here, we report the crystal structure of a complex containing the mouse CLOCK:BMAL1 bHLH-PAS domains at 2.3 Å resolution. The structure reveals an unusual asymmetric heterodimer with the three domains in each of the two subunits--bHLH, PAS-A, and PAS-B--tightly intertwined and involved in dimerization interactions, resulting in three distinct protein interfaces. Mutations that perturb the observed heterodimer interfaces affect the stability and activity of the CLOCK:BMAL1 complex as well as the periodicity of the circadian oscillator. The structure of the CLOCK:BMAL1 complex is a starting point for understanding at an atomic level the mechanism driving the mammalian circadian clock.

Secondary structure of Huntingtin amino-terminal region.

Huntington's disease is a genetic neurodegenerative disorder resulting from polyglutamine (polyQ) expansion (>36Q) within the first exon of Huntingtin (Htt) protein. We applied X-ray crystallography to determine the secondary structure of the first exon (EX1) of Htt17Q. The structure of Htt17Q-EX1 consists of an amino-terminal alpha helix, poly17Q region, and polyproline helix formed by the proline-rich region. The poly17Q region adopts multiple conformations in the structure, including alpha helix, random coil, and extended loop. The conformation of the poly17Q region is influenced by the conformation of neighboring protein regions, demonstrating the importance of the native protein context. We propose that the conformational flexibility of the polyQ region observed in our structure is a common characteristic of many amyloidogenic proteins. We further propose that the pathogenic polyQ expansion in the Htt protein increases the length of the random coil, which promotes aggregation and facilitates abnormal interactions with other proteins in cells.

Two mosquito LRR proteins function as complement control factors in the TEP1-mediated killing of Plasmodium.

Plasmodium development within Anopheles mosquitoes is a vulnerable step in the parasite transmission cycle, and targeting this step represents a promising strategy for malaria control. The thioester-containing complement-like protein TEP1 and two leucine-rich repeat (LRR) proteins, LRIM1 and APL1, have been identified as major mosquito factors that regulate parasite loads. Here, we show that LRIM1 and APL1 are required for binding of TEP1 to parasites. RNAi silencing of the LRR-encoding genes results in deposition of TEP1 on Anopheles tissues, thereby depleting TEP1 from circulation in the hemolymph and impeding its binding to Plasmodium. LRIM1 and APL1 not only stabilize circulating TEP1, they also stabilize each other prior to their interaction with TEP1. Our results indicate that three major antiparasitic factors in mosquitoes jointly function as a complement-like system in parasite killing, and they reveal a role for LRR proteins as complement control factors.

Structural basis for conserved complement factor-like function in the antimalarial protein TEP1.

Thioester-containing proteins (TEPs) are a major component of the innate immune response of insects to invasion by bacteria and protozoa. TEPs form a distinct clade of a superfamily that includes the pan-protease inhibitors alpha(2)-macroglobulins and vertebrate complement factors. The essential feature of these proteins is a sequestered thioester bond that, after cleavage in a protease-sensitive region of the protein, is activated and covalently binds to its target. Recently, TEP1 from the malarial vector Anopheles gambiae was shown to mediate recognition and killing of ookinetes from the malarial parasite Plasmodium berghei, a model for the human malarial parasite Plasmodium falciparum. Here, we present the crystal structure of the TEP1 isoform TEP1r. Although the overall protein fold of TEP1r resembles that of complement factor C3, the TEP1r domains are repositioned to stabilize the inactive conformation of the molecule (containing an intact thioester) in the absence of the anaphylotoxin domain, a central component of complement factors. The structure of TEP1r provides a molecular basis for the differences between TEP1 alleles TEP1r and TEP1s, which correlate with resistance of A. gambiae to infection by P. berghei.

Signal transduction pathway of TonB-dependent transporters.

Transcription of the ferric citrate import system is regulated by ferric citrate binding to the outer membrane transporter FecA. A signal indicating transporter occupancy is relayed across the outer membrane to energy-transducing and regulatory proteins embedded in the cytoplasmic membrane. Because transcriptional activation is not coupled to ferric citrate import, an allosteric mechanism underlies this complex signaling mechanism. Using evolution-based statistical analysis we have identified a sparse but structurally connected network of residues that links distant functional sites in FecA. Functional analyses of these positions confirm their involvement in the mechanism that regulates transcriptional activation in response to ferric citrate binding at the cell surface. This mechanism appears to be conserved and provides the structural basis for the allosteric signaling of TonB-dependent transporters.

Structure of tracheal cytotoxin in complex with a heterodimeric pattern-recognition receptor.

Tracheal cytotoxin (TCT), a naturally occurring fragment of Gram-negative peptidoglycan, is a potent elicitor of innate immune responses in Drosophila. It induces the heterodimerization of its recognition receptors, the peptidoglycan recognition proteins (PGRPs) LCa and LCx, which activates the immune deficiency pathway. The crystal structure at 2.1 angstrom resolution of TCT in complex with the ectodomains of PGRP-LCa and PGRP-LCx shows that TCT is bound to and presented by the LCx ectodomain for recognition by the LCa ectodomain; the latter lacks a canonical peptidoglycan-docking groove conserved in other PGRPs. The interface, revealed in atomic detail, between TCT and the receptor complex highlights the importance of the anhydro-containing disaccharide in bridging the two ectodomains together and the critical role of diaminopimelic acid as the specificity determinant for PGRP interaction.

NMR structures of the selenoproteins Sep15 and SelM reveal redox activity of a new thioredoxin-like family.

Selenium has significant health benefits, including potent cancer prevention activity and roles in immune function and the male reproductive system. Selenium-containing proteins, which incorporate this essential micronutrient as selenocysteine, are proposed to mediate the positive effects of dietary selenium. Presented here are the solution NMR structures of the selenoprotein SelM and an ortholog of the selenoprotein Sep15. These data reveal that Sep15 and SelM are structural homologs that establish a new thioredoxin-like protein family. The location of the active-site redox motifs within the fold together with the observed localized conformational changes after thiol-disulfide exchange and measured redox potential indicate that they have redox activity. In mammals, Sep15 expression is regulated by dietary selenium, and either decreased or increased expression of this selenoprotein alters redox homeostasis. A physiological role for Sep15 and SelM as thiol-disulfide oxidoreductases and their contribution to the quality control pathways of the endoplasmic reticulum are discussed.

Structure of the ectodomain of Drosophila peptidoglycan-recognition protein LCa suggests a molecular mechanism for pattern recognition.

The peptidoglycan-recognition protein LCa (PGRP-LCa) is a transmembrane receptor required for activation of the Drosophila immune deficiency pathway by monomeric Gram-negative peptidoglycan. We have determined the crystal structure of the ectodomain of PGRP-LCa at 2.5-A resolution and found two unique helical insertions in the LCa ectodomain that disrupt an otherwise L-shaped peptidoglycan-docking groove present in all other known PGRP structures. The deficient binding of PGRP-LCa to monomeric peptidoglycan was confirmed by biochemical pull-down assays. Recognition of monomeric peptidoglycan involves both PGRP-LCa and -LCx. We showed that association of the LCa and LCx ectodomains in vitro depends on monomeric peptidoglycan. The presence of a defective peptidoglycan-docking groove, while preserving a unique role in mediating monomeric peptidoglycan induction of immune response, suggests that PGRP-LCa recognizes the exposed structural features of a monomeric muropeptide when the latter is bound to and presented by the ectodomain of PGRP-LCx. Such features include N-acetyl glucosamine and the anhydro bond in the glycan of the muropeptide, which have been demonstrated to be critical for immune stimulatory activity.

A novel cysteine-rich domain of Sep15 mediates the interaction with UDP-glucose:glycoprotein glucosyltransferase.

Selenium is an essential trace element with potent cancer prevention activity in mammals. The 15-kDa selenoprotein (Sep15) has been implicated in the chemopreventive effect of dietary selenium. Although the precise function of Sep15 remains elusive, Sep15 co-purifies with UDP-glucose:glycoprotein glucosyltransferase (GT), an essential regulator of quality control mechanisms within the endoplasmic reticulum. Recent studies identified two GT and two Sep15 homologues in mammals. We characterize interactions between these protein families in this report. Sep15 and GT form a tight 1:1 complex, and these interactions are conserved between mammals and fruit flies. In mammalian cells, Sep15 co-immunoprecipitates with both GT isozymes. In contrast, a Sep15 homologue, designated selenoprotein M (SelM), does not form a complex with GT. Sequence analysis of members of the Sep15 family identified a novel N-terminal cysteine-rich domain in Sep15 that is absent in SelM. This domain contains six conserved cysteine residues that form two CxxC motifs that do not coordinate metal ions. If this domain is deleted or the cysteines are mutated, Sep15 no longer forms a complex with GT. Conversely, if the cysteine-rich domain of Sep15 is fused to the N-terminus of SelM, the resulting chimera is capable of binding GT. These data indicate that the cysteine-rich domain of Sep15 exclusively mediates protein-protein interactions with GT.

Tetramerization and ATP binding by a protein comprising the A, B, and C domains of rat synapsin I.

Synapsins are multidomain proteins that are critical for regulating neurotransmitter release in vertebrates. In the present study, two crystal structures of the C domain of rat synapsin I (rSynI-C) in complex with Ca(2+) and ATP reveal that this protein can form a tetramer and that a flexible loop (the "multifunctional loop") contacts bound ATP. Further experiments were carried out on a protein comprising the A, B, and C domains of rat synapsin I (rSynI-ABC). An ATP-stabilized tetramer of rSynI-ABC is observed during velocity sedimentation and size-exclusion chromatographic experiments. These hydrodynamic results also indicate that the A and B domains exist in an extended conformation. Calorimetric measurements of ATP binding to wild-type and mutant rSynI-ABC demonstrate that the multifunctional loop and a cross-tetramer contact are important for ATP binding. The evidence supports a view of synapsin I as an ATP-utilizing, tetrameric protein made up of monomers that have a flexible, extended N terminus.